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United States Patent Application
20040029266
Kind Code
A1
Barbera-Guillem, Emilio
February 12, 2004
Cell and tissue culture device
Abstract
A cell culture device (100, 830) incorporating confronting planar anterior and posterior shells or walls (110, 140, 834, 836) that are joined about peripheral edges to define a media reservoir or cistern (170, 850). At least one of the shells and walls and edges is optionally formed with an aperture or respirator (180, 873). At least one fluid transfer port (220, 870) with a resealable elastomeric septum (230, 872) compatible for use with a small needle or needleless connector or pipetter tip (T, T') is preferably formed in least one of the shells and walls (110, 140, 834, 836) and edges and that is in fluid communication with the media reservoir or cistern or chamber (170, 850). The device (100, 830) also includes at least one gas valvule (320, 875) that is formed in one or more of the shells and walls (110, 140, 834, 836) and edges and is in fluid communication with the media reservoir (170, 850) to vent gas from and supply gas to the reservoir (170, 850). The at least one gas valvule (320, 875) is preferably hydrophobic and is configured to pass only sterile air and to prevent liquid flow. In various embodiments, the preferred cell culture device (100, 830) that minimizes non-media containing headspace and that defines an internal surface (115, 145) having an area that bounds an internal volume whereby the ratio between the volume and the surface area is approximately between 100 microliters per square centimeter and 1000 microliters per square centimeter.
Inventors:
Barbera-Guillem; Emilio
(Powell, OH)
Correspondence Name and Address:
P.O. Box 710
SEAN M. CASEY CO., LPA
New Albany
OH
43054-0710
US
Series Code:
216554
Filed:
August 9, 2002
U.S. Current Class:
435/297.5;
435/304.1
U.S. Class at Publication:
435/297.5;
435/304.1
Intern'l Class:
C12M 003/00
Claims
I claim:
1. A cell culture device, comprising: generally planar anterior and posterior shells arranged in a confronting relationship and joined by respective opposing dextral and sinistral longitudinal peripheral edges, and opposing superior and inferior peripheral lateral edges, the shells and edges having a surface area defining a media reservoir, at least one of the anterior and posterior shells and edges being formed with at least one circumfluent periphery and defining at least one aperture; at least one gas permeable membrane sealing the at least one aperture and joined to the periphery; at least one fluid transfer port formed in least one of the shells and edges and in fluid communication with the media reservoir; and at least one gas valvule formed in at least one of the shells and edges and in fluid communication with the media reservoir.
2. The cell according to claim 1, wherein the surface area of the membrane is approximately between 1% and 10% of the surface area of the media reservoir.
3. The cell culture device according to claim 1, wherein the surface area of the membrane is approximately between 1.5% and 5% of the surface area of the media reservoir.
4. The cell culture device according to claim 1, wherein the at least one gas valvule is hydrophobic and adapted to prevent liquid flow therethrough.
5. The cell culture device according to claim 1, wherein the media reservoir is adapted to receive approximately between 20 milliliters and 140 milliliters of a fluid mixture.
6. The cell culture device according to claim 1, wherein the media reservoir is adapted to receive at least about 25 milliliters of a fluid mixture.
7. The cell culture device according to claim 1, wherein the joint formed between the respective lateral, superior, and inferior peripheral edges is formed as releasably hermetically sealed joint.
8. The cell culture device according to claim 1, wherein the media reservoir is formed to have a lateral dimension between the laterally opposed longitudinal edges of approximately between 6.5 centimeters and 9
centimeters, a longitudinal dimension between opposing superior and inferior lateral edges of approximately between 11 and 13 centimeters, and a dimension between interior surfaces of the anterior and posterior shells of approximately between 2 millimeters and 6 millimeters.
9. The cell culture device according to claim 1, wherein at least one of the anterior and posterior shells are formed from a thermoplastic material that is substantially transparent.
10. The cell culture device according to claim 9, wherein at least one of the shells is formed from a thermoplastic material to have a pigment adapted to filter photonic energy outside the range of between approximately 500 and 600 nanometers.
11. The cell culture device according to claim 9, wherein at least one of the shells is formed from a thermoplastic material having a pigment adapted to filter photonic energy outside the range of between approximately 550 and 570 nanometers.
12. The cell culture device according to claim 1, wherein the at least one gas permeable membrane is formed from a sheet material to have a thickness approximately between 0.09 and 0.14 millimeters.
13. The cell culture device according to claim 1, wherein the media reservoir defines in internal surface area and the at least one gas permeable membrane has a second surface area approximately between 1.5% and 10% of the internal surface area.
14. The cell culture device according to claim 1, wherein the at least one fluid transfer port communicates fluid with the media reservoir through a siphon lock lumen formed with at least one fluid path that bends through at least one angle of approximately between 45 and 135 degrees of arc.
15. The cell culture device according to claim 1, wherein the at least one fluid transfer port incorporates a resealable elastomeric septum adapted to releasably receive a means to communicate a fluid through the port.
16. The cell culture device according to claim 1, wherein the at least one gas valvule communicates gas with the media reservoir through a second siphon lock lumen formed with at least one fluid path that bends through at least one angle of approximately between 45 and 135 degrees of arc.
17. The cell culture device according to claim 1, wherein the at least one gas valvule incorporates a filtration element adapted to pass gaseous molecules and to prevent the passage of particles having an average diametrical dimension of approximately between 0.1 and 0.3 microns.
18. The cell culture device according to claim 17, wherein the filtration element incorporates a hydrophobic material.
19. The cell culture device according to claim 17, wherein the filtration element is an assembly of at least 2 layers with a first layer being adapted to prevent the passage of particles having an average diametral dimension of at least between about 80 to 120 microns, and a second layer being adapted to prevent the passage of particles having an average diametral dimension of at least between about 0.1 to 0.3 microns.
20. The cell culture device according to claim 17, wherein the filtration element is a hybrid filter medium having a filtration property wherein the size of the particles that are filtered changes across a cross-section of the filter medium such that at a first exterior surface the medium is adapted to prevent the passage of particles having an average diametral dimension of at least between about 80 to 120 microns, and whereby at a second opposite exterior surface the medium is adapted to prevent the passage of particles having an average diametral dimension of at least between about 0.1 to 0.3 microns.
21. The cell culture device according to claim 1, wherein the media reservoir is defined by an internal surface area of the shells and edges that bounds an internal volume whereby the ratio between the volume and the surface area is approximately between 100 microliters per square centimeter and 1000 microliters per square centimeter.
22. The cell culture device according to claim 1, wherein the media reservoir is defined by a plurality of internal surfaces of the shells and edges wherein substantially all of the surfaces are adapted to support growth of cells.
23. A cell culture device, comprising: a reservoir formed from generally transparent confronting anterior and posterior walls joined about respectively opposed superior and inferior peripheral lateral edges, and respective laterally opposed peripheral longitudinal edges, the walls and edges defining an interior cistern; at least one aperture formed in a least one of the walls and edges and sealed with a gas permeable membrane and in gaseous communication with the cistern; at least one injection and aspiration port formed in at least one of the walls and edges and in fluid communication with the cistern; and at least one pressure relief valvule formed in at least one of the walls and edges and in gaseous communication with the cistern, the valvule being operative to equalize pressure within the cistern to ambient atmospheric pressure as fluid is communicated through the at least one port.
24. The cell culture device according to claim 23, wherein the surface area of the membrane is approximately between 1% and 10% of the surface area of the cistern.
25. The cell culture device according to claim 23, wherein the surface area of the membrane is approximately between 1.5% and 5% of the surface area of the cistern.
26. The cell culture device according to claim 23, wherein the at least one valvule is hydrophobic and adapted to prevent fluid flow therethrough.
27. The cell culture device according to claim 23, wherein the cistern is adapted to receive approximately between 20 milliliters and 140
milliliters of a fluid mixture.
28. The cell culture device according to claim 23, wherein the cistern is adapted to receive at least about 25 milliliters of a fluid mixture.
29. The cell culture device according to claim 23, wherein the joint formed between the respective lateral, superior, and inferior peripheral edges is formed as releasably hermetically sealed joint.
30. The cell culture device according to claim 23, wherein the media reservoir is formed to have a lateral dimension between the opposing longitudinal edges of at least approximately between 6 centimeters and 9
centimeters, a longitudinal dimension between opposing superior and inferior lateral edges of at least approximately between 11 and 13
centimeters, and a dimension between interior surfaces of the anterior and posterior shells of at least approximately between 2 millimeters and 6 millimeters.
31. The cell culture device according to claim 23, wherein at least one of the anterior and posterior shells are formed from a thermoplastic material that is substantially transparent.
32. The cell culture device according to claim 31, wherein at least one of the shells is formed from a thermoplastic material to have a pigment adapted to filter photonic energy outside the range of between approximately 500 and 600 nanometers.
33. The cell culture device according to claim 31, wherein at least one of the shells is formed from a thermoplastic material having a pigment adapted to filter photonic energy outside the range of between approximately 550 and 570 nanometers.
34. The cell culture device according to claim 23, wherein the at least one gas permeable membrane is formed from a sheet material to have a thickness approximately between 0.09 and 0.14 millimeters.
35. The cell culture device according to claim 23, wherein the cistern defines in internal surface area and the at least one gas permeable membrane has a second surface area approximately between 1.5% and 10% of the internal surface area.
36. The cell culture device according to claim 23, wherein the at least one injection and aspiration port communicates fluid with the cistern through a lumen formed with at least one fluid path that bends through at least one angle of approximately between 45 and 135 degrees of arc.
37. The cell culture device according to claim 23, wherein the at least one injection and aspiration port incorporates a resealable elastomeric septum adapted to releasably receive a means to communicate a fluid through the port.
38. The cell culture device according to claim 23, wherein the at least one pressure relief valvule communicates gas with the cistern through a second lumen formed with at least one fluid path that bends through at least one angle of approximately between 45 and 135 degrees of arc.
39. The cell culture device according to claim 23, wherein the at least one pressure relief valvule incorporates a filtration element adapted to pass gas and to inhibit liquid flow and to prevent the passage of particles having an average diametrical dimension of approximately between 0.1 and 0.3 microns.
40. The cell culture device according to claim 39, wherein the filtration element incorporates a hydrophobic material.
41. The cell culture device according to claim 39, wherein the filtration element is an assembly of at least 2 layers with a first layer being adapted to prevent the passage of particles having an average diametral dimension of at least between about 80 to 120 microns, and a second layer being adapted to prevent the passage of particles having an average diametral dimension of at least between about 0.1 to 0.3 microns.
42. The cell culture device according to claim 39, wherein the filtration element is a hybrid filter medium having a filtration property wherein the size of the particles that are filtered changes across a cross-section of the filter medium such that at a first exterior surface the medium is adapted to prevent the passage of particles having an average diametral dimension of at least between about 80 to 120 microns, and whereby at a second opposite exterior surface the medium is adapted to prevent the passage of particles having an average diametral dimension of at least between about 0.1 to 0.3 microns.
43. The cell culture device according to claim 23, wherein the cistern is defined by an internal surface area of the wall and edges that bounds an internal volume whereby the ratio between the volume and the surface area is approximately between 100 microliters per square centimeter and 1000
microliters per square centimeter.
44. The cell culture device according to claim 23, wherein the cistern is defined by a plurality of internal surfaces of the walls and edges wherein substantially all of the surfaces are adapted to support growth of cells.
45. A cell culture device, comprising: a reservoir formed from generally transparent confronting anterior and posterior walls joined about respectively longitudinally opposed superior and inferior peripheral lateral edges, and respective laterally opposed peripheral longitudinal edges, the walls and edges defining an interior cistern; at least one injection and aspiration port formed in at least one of the walls and edges and in fluid communication with the cistern; and at least one pressure relief valvule formed in at least one of the walls and edges and in gaseous communication with the cistern, the valvule being operative to equalize pressure within the cistern to ambient atmospheric pressure as fluid is communicated through the at least one port.
46. The cell culture device according to claim 45, wherein the at least one valvule is hydrophobic and adapted to prevent fluid flow therethrough.
47. The cell culture device according to claim 45, wherein the cistern is adapted to receive approximately between 20 milliliters and 140
milliliters of a fluid mixture.
48. The cell culture device according to claim 45, wherein the cistern is adapted to receive at least about 25 milliliters of a fluid mixture.
49. The cell culture device according to claim 45, wherein the joint formed between the respective lateral, superior, and inferior peripheral edges is formed as releasably hermetically scaled joint.
50. The cell culture device according to claim 45, wherein the media reservoir is formed to have a lateral dimension between the opposing lateral edges of at least approximately between 6 centimeters and 9
centimeters, a longitudinal dimension between opposing superior and inferior edges of at least approximately between 11 and 13 centimeters, and a dimension between interior surfaces of the anterior and posterior shells of at least approximately between 2 millimeters and 6 millimeters.
51. The cell culture device according to claim 45, wherein at least one of the anterior and posterior shells are formed from a thermoplastic material that is substantially transparent.
52. The cell culture device according to claim 51, wherein at least one of the shells is formed from a thermoplastic material to have a pigment adapted to filter photonic energy outside the range of between approximately 500 and 600 nanometers.
53. The cell culture device according to claim 51, wherein at least one of the shells is formed from a thermoplastic material having a pigment adapted to filter photonic energy outside the range of between approximately 550 and 570 nanometers.
54. The cell culture device according to claim 45, wherein the at least one gas permeable membrane is formed from a sheet material to have a thickness approximately between 0.09 and 0.14 millimeters.
55. The cell culture device according to claim 45, wherein the at least one injection and aspiration port communicates fluid with the cistern through a lumen formed with at least one fluid path that bends through at least one angle of approximately between 45 and 135 degrees of arc.
56. The cell culture device according to claim 45, wherein the at least one injection and aspiration port incorporates a resealable elastomeric septum adapted to releasably receive a means to communicate a fluid through the port.
57. The cell culture device according to claim 45, wherein the at least one pressure relief valvule communicates gas with the cistern through a second lumen formed with at least one fluid path that bends through at least one angle of approximately between 45 and 135 degrees of arc.
58. The cell culture device according to claim 45, wherein the at least one pressure relief valvule incorporates a filtration element adapted to pass gas and to inhibit liquid flow and to prevent the passage of particles having an average diametrical dimension of approximately between 0.1 and 0.3 microns.
59. The cell culture device according to claim 58, wherein the filtration element incorporates a hydrophobic material.
60. The cell culture device according to claim 58, wherein the filtration element is an assembly of at least 2 layers with a first layer being adapted to prevent the passage of particles having an average diametral dimension of at least between about 80 to 120 microns, and a second layer being adapted to prevent the passage of particles having an average diametral dimension of at least between about 0.1 to 0.3 microns.
61. The cell culture device according to claim 58, wherein the filtration element is a hybrid filter medium having a filtration property wherein the size of the particles that are filtered changes across a cross-section of the filter medium such that at a first exterior surface the medium is adapted to prevent the passage of particles having an average diametral dimension of at least between about 80 to 120 microns, and whereby at a second opposite exterior surface the medium is adapted to prevent the passage of particles having an average diametral dimension of at least between about 0.1 to 0.3 microns.
62. The cell culture device according to claim 45, wherein the cistern is defined by an internal surface area of the wall and edges that bounds an internal volume whereby the ratio between the volume and the surface area is approximately between 100 microliters per square centimeter and 1000
microliters per square centimeter.
63. The cell culture device according to claim 45, wherein the cistern is defined by a plurality of internal surfaces of the walls and edges wherein substantially all of the surfaces are adapted to support growth of cells.
64. The cell culture device according to claim 45, wherein the anterior and posterior walls and the laterally opposed peripheral longitudinal edges form a body having a superior body edge and an inferior body edge, and wherein the superior and inferior peripheral lateral edges are further formed on respective superior and inferior end manifolds adapted to be respectively engaged with the superior and inferior body edges to further define the cistern
65. The cell culture device according to claim 45, further comprising: an insulative and protective container defining at least one interior cavity for receiving at least one cell culture device and wherein the container incorporates a means for controlling the temperature of the device.
Description
TECHNICAL FIELD
[0001] This invention relates to a device adapted for use in maintaining and culturing biological cells in a medium. More specifically, the invention relates to an apparatus adapted to maintain and propagate prokaryotic, eukaryotic, hybrid, and artificial cells in a scientific research, laboratory, or clinical setting.
BACKGROUND OF THE INVENTION
[0002] In the last few decades, the biological sciences have exploded in what has often been called the molecular revolution. A particular emphasis of modern biology is molecular biology, which is the study of the molecular building blocks and products of cells and sub-cellular structures and the relationships of those individual molecules to each other. Molecular biology encompasses such diverse fields of study as genetics, immunology, microbiology, cell biology, cell signaling, protein biochemistry, and a multitude of others. While molecular biology continues to focus on progressively and more discretely defined subject matter, the field is often hampered by problems associated with the ability to maintain and to propagate biological cells.
[0003] In much the same way that the diverse array of life as we know it can be placed into discrete classes, such biological cells of interest to molecular biology can be classified in broad terms as either prokaryotes or eukaryotes. Prokaryotes, a classification that includes principally archaebacteria and bacteria, are often referred to by those with skill in the art as simply bacteria. These prokaryotes, or bacteria, are usually single cells substantially capable of living free of associations with other cells. Such cells reproduce asexually, most often by binary fission. It is estimated that only a very small percentage of the bacteria that exist in nature can currently be grown or cultured in a laboratory, perhaps less than one percent. However, many of those bacteria, or prokaryotes, that can be grown in a lab tend to be relatively easy to grow and to propagate so long as the basic nutritional requirements of the cells of interest are supplied. Bacteria are also inclined to be hardy and resistant to environmental stresses such as transient peaks and troughs of nutrient availability, sub and supra optimal temperatures, harsh chemical agents present in the environment, and other less than desirable variations in environmental parameters.
[0004] Such bacteria have a significant impact on human existence and culture. For example, bacteria can cause disease in humans, crops, and livestock. Some bacteria naturally produce clinically desirable antibiotics and various therapeutic agents, while still other bacteria may be engineered to produce commercially valuable vaccines, insulin, growth hormones for humans and livestock, and products suitable for use in other economically and scientifically significant applications. Bacteria are both an ingredient in and a producer of food products such as yogurt and sauerkraut. Prokaryotes have even played a role in shaping the course of human history the black plague that redrew the geopolitical landscape of Europe was caused by a bacterium.
[0005] In spite of their relevance and importance to both human society in general and to molecular biology in particular, individuals with ordinary skill in the art generally reserve the terms cell culture and tissue culture for the maintenance and propagation of eukaryotic cells. Eukaryotes normally live in the multi-cellular arrangements such as plants, animals and mushrooms, although some eukaryotes such as the yeasts and the protozoa are usually single-celled. Eukaryotic cells can be capable of sexual reproduction, asexual reproduction, or both. Compared to the prokaryotes, eukaryotic cells tend to have more stringent nutritional needs and frequently require more precise and stable physical, biochemical, and thermal environments. When a eukaryotic cell is removed from an organism and placed into an appropriate nutrient medium, the cell will usually grow and divide by mitosis for only a few generations. Even if all environmental and nutritional conditions are ideal, such cells will lose viability in relatively short order. Such a eukaryotic cell culture is known to those skilled in the art as a primary cell culture. In contrast to primary cell cultures, some other eukaryotic cells and especially those cells derived from tumors or cancerous tissue will continue to grow and divide without unexpected or significant degradation or anomalous deterioration for as long as environmental and nutritional requirements are permissive for growth. Persons with skill in the art often refer to this type of eukaryotic cell culture as a cell line, permanent cell line, or as an immortalized cell line. Some of the cell lines studied today have been propagated in laboratories around the world for more than three decades.
[0006] A wide variety of eukaryotic cells and cell lines are of great interest and importance to modern molecular biology. Insecticides and herbicides, invaluable tools used to provide adequate food supplies for human populations, are typically engineered for and evaluated in eukaryotic cells from insects and plants. Eukaryotes help to feed people even more directly; virtually everything on a dinner plate is, was, or derived from a eukaryote. We not only consume but also are consumed by eukaryotes; malaria, an affliction that has killed more people throughout history than any other disease, is caused by a protozoan and is transmitted by an insect, both of which are eukaryotes. Eukaryotic cells can also help to cure diseases. Studies of both hereditary and communicable diseases often make extensive use of plant, animal, and human cells, all of which are eukaryotic cells. Vaccines and other therapeutics are normally tested in primary cell cultures and in immortalized cell lines long before they are evaluated in clinical trials. Some eukaryotes have even more direct clinical applications, antibiotics such as streptomycin and penicillin are natural products of eukaryotic cells. Many areas of eukaryotic cell investigation have the potential to provide enormous benefit to humanity. Examples of such areas of investigation include the interaction of human cells with pathogens, the cellular response to toxins, the development of treatments and cures for cancers and tumors, the regulation of the immune system, and the details of cell to cell signaling. Comprehension of how a single cell may be triggered to proliferate and then differentiate into an entire tissue or organ could translate into powerful new treatments for cancers, organ and heart diseases, and many other human maladies.
[0007] While living cells are normally classified as either eukaryotes or prokaryotes, a virus is yet another general type of biological agent not considered to be a cell by many persons skilled in the art. The viruses are unable to grow or propagate on their own in a nutrient medium because they depend upon a host cell to provide the biochemical machinery required for propagation of the virus. Those skilled in the art often refer to these types of parasites as obligate intracellular parasites. Some viruses can infect a wide range of cells and cell types, for example, influenza can infect respiratory tract cells of various waterfowl, seals, pigs, and humans. Other viruses may infect only one or a few specific cell types in a single species of host. For illustrative purposes, the Human Immunodeficiency Virus (HIV) will infect the T cells and macrophages of humans and will normally not, with limited exceptions, infect any other cell from humans or any other species. Nearly every cell identified to date, whether prokaryotic or eukaryotic, is a host to at least one virus, making viruses one of the most well represented biological agents on the planet.
[0008] Some viruses cause disease, such as HIV and the influenza virus mentioned herein. Others have little or no effect on their host cell. Still others can be beneficial; for example, the variegation popular in many types of tulips is caused by infection of the tulip plant with a virus. As another example of the potential utility of viruses, the vaccinia and other viruses have been used as vehicles to deliver vaccine to plants and animals. For these and other medical and commercial reasons, viruses are also of great interest to modern molecular biology. While viruses cannot be cultured per se, viruses may be propagated in appropriate eukaryotic or prokaryotic host cells.
[0009] In addition to viruses, eukaryotes, and prokaryotes, there are other known biological agents that do not fit into this classification system. Prions, for example, are naked infectious proteins that cause such animal diseases as Bovine Spongiform Encephalopathy (B.S.E., Scrapie, or "Mad Cow Disease") and the human diseases Kuru and Creutzfeldt-Jakob disease. It should be understood that, while useful, the herein-described eukaryotic/prokaryotic classification system is a construct of the human mind that is designed to categorize and organize a myriad of data collected over thousands of years of human culture and science. It is a descriptive rather than a prescriptive system. To illustrate this point, consider chloroplasts, a sub-cellular organelle found in many plant and algal cells. It is believed by many of those skilled in the art that chloroplasts are derived from an ancient, free-living prokaryote. Chloroplasts are semiautonomous organelles that have their own genetic information distinct from that of the host plant or algal cell and that govern much of their own reproduction via organelle division. The present classification system sees the question of whether eukaryotic plant and algal cells that possess such organelles be considered eukaryotes or prokaryotes. Perhaps such cells should be classified as quasi-prokaryotic.
[0010] Even if nature had not provided cells and biological agents that do not fit easily into extant classification systems, humankind certainly has. For example, bacterial genes are commonly expressed in plant and animal cells, and vice versa. As another example, cells such as hybridomas are engineered by fusing dissimilar cells into a resulting hybrid cell. Regardless of the true nature of a cell or biological agent, be it a prokaryote, a eukaryote, a hybrid of the two, or even a yet to be discovered cell type, cell culture should be understood to be the deliberate growth and propagation of a particular cell or cell line of interest. This purpose may include any one or several of the following applications: 1) the harvest of the cells themselves to be used in some application, such as the growth and purification of the yeast cells that are combined with flour and water to make bread; 2) to reap some useful compound elaborated by the cells, such as the purification of human insulin from recombinant bacteria; 3) to harvest some cellular component such as membranes, antibodies, enzymes, and the like to be used for some subsequent application or purpose; 4) the evaluation or monitoring of some cellular process under various conditions, such as the response of cells to sudden changes in temperature or pH; 5) to assay, monitor, or study the cellular response to a pathogen, chemical, therapeutic, or other agent or condition; 6) to provide a sufficient number of appropriate cells to propagate a virus or other intracellular parasite; 7) any other circumstance in which cells, cellular products or biological agents are used, needed, desired, or involved.
[0011] There are undoubtedly far fewer eukaryotes in the world than there are viruses and bacteria. Nevertheless, eukaryotic cell biology occupies a significant portion of the focus of modern molecular biology because humans and their pets, livestock, and crops are all eukaryotes. Prokaryotes and viruses are also studied extensively, but frequently in the context of their impact on eukaryotes. With few exceptions, the raw material of modern molecular biology must be harvested from and evaluated in living cells, often in eukaryotic cells. Those with skill in the art have long recognized various problems central to this application in the field of molecular biology. For example, cells exquisitely adapted to life inside of and as a part of a living creature must be maintained and propagated with reasonably high yields in a laboratory or clinical setting: 1) without contamination; 2) without the loss of desirable traits; and 3) without the acquisition of undesirable traits.
[0012] Many specific additional issues arise from these and other core problems. First, a cell or cell line of interest often requires a substrate upon which to adhere during growth. Second, cells require regular exposure to or immersion in some form of a solid, liquid, gaseous, trans-phase, or multi-phase medium and or media that supplies nutrients and growth factors, and which media and or medium is also adapted to remove any potentially damaging waste products either by diluting them or during removal and replenishment of the medium and or media. A relatively small number of the cells of interest can be grown, but the geometry of the ratio of the surface area covered or immersed by the available medium to the volume of that medium makes higher cell yields cumbersome and unwieldy using present-day technology, methods, and equipment.
[0013] An additional issue in maintaining and growing cells is gas diffusion. During growth, cells must be exposed to precisely controlled and periodically replenished amounts of N.sub.2, CO.sub.2, O.sub.2, and other gasses. The proper ratio of appropriate gasses can be either mechanically introduced into the nutrient medium on a regular basis or must passively diffuse into the nutrient medium via a phase boundary. Where the latter method is employed, the cell culture flask or multiple-well plate is usually placed into a substantially sealed compartment or container. This compartment or container is frequently referred to by those skilled in the art as an incubator, which is often maintained in a controlled environment selected to have a predetermined temperature, humidity, and gaseous composition.
[0014] Another and even more pervasive issue that continues to vex prior art devices is that of contamination, either with an undesirable cell such as a ubiquitous and hardy bacterium or fungus or with some other undesirable contaminant. Although the cells of interest must be exposed to an initial supply of media and gas and possibly to periodic replenishments of the same, it is critical to grow only the cells of interest without introducing undesirable contaminants. Since most living and non-living surfaces contain viruses, bacteria, fungi, and the like, it is often difficult to establish and maintain a cell culture without introducing undesirable contaminants. Such undesirable contaminants can cause a multitude of deleterious and costly effects. Contaminating cells can kill or injure the cells of interest by producing toxins or antibiotics. Undesirable cells can significantly reduce growth yields of the cells of interest by consuming nutrients and growth factors intended for the cells of interest. Even if the undesirable contaminants do not directly harm the cells of interest, they can contaminate any products being elaborated by the cells of interest. Such contamination can skew the results of any testing performed upon the cells of interest by producing unexpected or unknown substances or by producing markedly less than anticipated substances or a superabundance of anticipated substances.
[0015] For the purpose of explicating the field of the invention and background of the art, the terms cell(s), cell culture(s), culture(s), primary cell culture(s), cell line(s), and immortalized cell line(s) should be understood to refer to those cells of interest and their progeny that are maintained and propagated. Additionally, the terms culture, cell culture, and cell culturing may also be understood to refer to the process or technique of such cell maintenance and propagation for the reasons discussed herein or for any other purpose. Those having skill in the art may also use the term "tissue culture" in lieu of such terms, although this term is customarily restricted to the culture of eukaryotic cells derived from the tissue of higher, multi-cellular organisms. The cells of interest in cell culture are frequently but not necessarily eukaryotic cells. The terms undesirable cell and contaminant(s) should be understood to refer to unwanted or contaminating cells, viruses, prions, or other similarly undesirable or unwanted chemicals, compositions, elements, biological agents, components, or constituents. The terms medium and nutrient medium (media-plural) should be understood to refer to the solid, liquid, gaseous, trans-phase, or multi-phase medium that may supply nutrients, growth factors, trace elements, salts, buffering capacity, or any other element or component required or desirable to support the survival, growth, and or propagation of the cells and tissues of interest and their progeny.
[0016] The difficulties inherent in growing the cells of interest in a laboratory, research, or clinical setting may be better appreciated by considering one well-established and readily available cell culture technique. This approach to cell culture is to inoculate the cells into an appropriate medium and place the inoculated medium into a sterile vessel. If the sterile vessel includes a single compartment, then those with skill in the art customarily refer to it as a tissue culture flask, a cell culture flask, or a flask. While there are many exceptions to the general rule, the general rule is that there are two types of flasks: flasks for culturing eukaryotes and flasks for culturing prokaryotes. The former type of flask being preferably adapted to incorporate an atmosphere external to the media and or to exchange the gases either directly with the media contained in the flask or with such an atmosphere with an external replenishment source of gas. The latter type of flask is often referred to in its classical configuration by those skilled in the art as an Erlenmeyer flask and is most commonly adapted to culture prokaryotic cells and related tissues, materials, and substances with or without an atmosphere because such cells can be aerobic and also may be anaerobic such that they can be cultured without exposure to an external atmosphere for gas exchange. Examples of such cells include without limitation non-adherent tumor cells, bacteria, and hybridomas, to name a few. While either type flask can be adapted to support cell culture of cells that must attach to a surface to grow or which can grow unattached or in suspension, more customarily, the eukaryotic culture flasks have internal surfaces that are adapted or treated specifically for either attached or suspended cell growth applications. Most commonly, the prokaryotic cell culture flasks are adapted for unattached or suspended cell growth applications.
[0017] An illustration of a cell culture flask with some of these elements and others is found in U.S. Pat. No. 6,114,165 to Cai et al. The cell culture flask is typically a rectangular cube defining an interior space that is to be used for cell culture. Both the top and the bottom surfaces or dimensions of the cell culture flask preferably have substantially more surface area than any one of the four sides. In operation, the bottom of the cell culture flask is kept approximately horizontal and an opening to the cell culture flask is formed as a substantially vertical aperture located on one of the four sides of the flask. A cap is often removably affixed to the opening of the flask.
[0018] The sterile vessel may also have a plurality of compartments. In this arrangement, the vessel is then frequently referred to by persons skilled in the art as a multiple-well plate, with each compartment of the vessel defining a well formed in the plate. In this configuration there are four side walls that project upwardly from and substantially perpendicular to an approximately rectangular base member. Other walls project upwardly from the base member and attach to each other and to side walls to define the plurality of compartments or wells. A lid is often included in such devices, which lid is typically slightly longer and wider than the base member. The lid device functions by resting against and on top of the multiple-well plate, to enclose said multiple-well plate. An example of a multiple-well plate that incorporates some of these as well as other features is shown in U.S. Pat. No. 4,349,632 to Lyman et al.
[0019] In use, after cells are inoculated into the cell culture vessel, the cells of interest generally adhere to the bottom of the flask or well and propagate. While cells can be cultured in many types of flasks, cells do not grow or propagate equally well on all types of materials that are used to fabricate such culture flasks or vessels. As a result, considerable attention has been devoted to the investigation of various materials that have been developed and tested to ascertain their efficacy for the wide range of cell culture applications. Over the past many decades, many types of materials have become generally accepted by those skilled in the art as being preferred for use as flasks and for multiple-well plates. Such materials are most commonly selected from the group of materials that includes glass, ceramics, metals, thermoset and elastomer monomers and polymers, and polymeric thermoplastics including, for further purposes of illustration but not for purposes of limitation, thermoplastic materials selected from any of a variety of commercially available and suitable materials including acetal resins, delrin, fluorocarbons, polyesters, polyester elastomers, metallocenes, polyamides, nylon, polyvinyl chloride, polybutadienes, silicone resins, ABS (acrylonitrile, butadiene, styrene), polycarbonate, polypropylene, liquid crystal polymers, alloys and combinations and mixtures and composites thereof and reinforced alloys and combinations and mixtures and composites thereof.
[0020] While many configurations of cell culture devices, flasks, and vessels exist, most commonly, it is the bottom surface of the cell culture vessel where adherent-type cells are grown. Preferably, the bottom surface is kept in a substantially horizontal position during incubation and cell growth and is usually covered by a layer of the preferred nutrient medium. The medium is configured to supply necessary nutrients and growth factors to the cells. The rest of the internal volume of the flask or the well is, for eukaryotic culture applications, adapted to establish a volume space for the supply of gasses needed for growth and for the expulsion or diffusion of waste gasses that are the by-product of cell culture.
[0021] Those skilled in the art have come to investigate and understand many principles that guide the understanding of gas exchange during incubation between the external atmosphere or source of gasses, and the gasses or atmosphere contained in such a volume or head space in the flask, the media contained in the flask, and the cells that are either attached to a surface of the flask or that are unattached to any surface and suspended during growth in the media. Within and between the external atmosphere and the atmosphere contained in the head or volume space of the flask, the exchange and movement of gaseous or vaporous substances or the gasses is controlled by the random diffusive Brownian motion of the gas molecules, which is also affected by the temperature of the constituent gasses, and is further influenced by the kinetic energy of the gases which is parameterized by the molecular weights of the respective constituent gasses and many other parameters including, for example without limitation, the relative solubilities, concentrations, and partial pressures of such gasses.
[0022] With respect to the exchange and movement of vapors and gasses between the head or volume space in the flasks above the media, and the media, the rates of diffusion across the gas-liquid boundary at the surface of the media is a function of the preceding parameters as well as the solubilities, concentrations, temperature, and partial pressures of the gasses external to the media and those dissolved, absorbed, and otherwise present in and mixed with the media. The exchange and diffusion of gasses within the media is similarly affected by each of the preceding parameters, as well as by the formulation of the media, the type of cells being cultured, and by the potential energy inherent in the molecular structure of the media, which can further increase or decrease the kinetic or Brownian diffusion rates of gasses in the media and between the media and cells.
[0023] In sum, such gasses can efficiently and passively diffuse into the liquid medium because of the large surface area to volume ratio contemplated by the various cell culture devices, flasks, and vessels illustrated herein. In the majority of prior art devices, such gas exchange can only be accomplished with well-characterized and predictable results by establishing a large boundary interface between the liquid surface of the media and the head space or volume contained above to the surface. Even so, the vapors and gasses maintained in such head or volume space must be monitored and, depending upon the particularly application, removed and or replenished periodically so as to maintain preferably amounts of desired gases, such as diatomic oxygen and carbon dioxide, and or vapors, such as water.
[0024] The proper concentration of carbon dioxide can typically and preferably be about 5%, which is nearly twice that present in the Earth's ambient atmosphere, and which if deficient in an atmosphere proximate to a cell culture, can result in over diffusion of carbon dioxide out of the media and catastrophic over alkalinization of the cell culture media. Similarly, if the vapor pressure of water in or the relative humidity of the atmosphere proximate to the cell culture media falls to low, the media can quickly become catastrophically dehydrated. In contrast, over humidification and or failure to maintain proper water vapor pressures and temperatures of the culture can result in fog formation, which prevents visualization and imaging of the cell culture. This same over humidification issue can also result in condensation, which can create contamination pathways and or escape of culture materials from the flask or vessel.
[0025] Although this technology has been in use for some time to maintain and propagate cells, it is replete with the noted problems and other technical difficulties. Such past attempts at improving the art of cell culture remains severely hampered by many issues and problems and is although widely in use, very limited in the scope of its efficacy and applicability, and is generally unsuited for the purposes of the more highly refined, very precise, and high yield techniques, methods, and applications undertaken by modern biotechnologists. One significant restriction is that of limited growth yield.
[0026] For further example, when using previously described cell and tissue culture devices, flasks, vessels, and similar hardware and related techniques, the layer of the nutrient medium contained therein in which the cells are immersed must be relatively shallow, for example between about 3 to about 20 millimeters deep, for efficient gas diffusion and other reasons as explained herein. For added example, when using a standard T-75 cell culture flask, which can hold a total volume of about 75 milliliters, is preferred to use a media volume of about 25
milliliters for cell and tissue culture, which results in a media depth of about 3 millimeters when the flask is placed on its side as normally used for incubation.
[0027] With this configuration and arrangement of media and flask, unless the media is properly titrated with the proper concentrations of constituents for the particular application, and incubated under precisely controlled temperature, humidity, and related conditions, without carefully synchronously-controlled media replenishment, the cells being cultured may quickly exhaust the nutrients and growth factors supplied by this relatively small volume and depth of the liquid media. More significantly, toxic waste products that are a natural byproduct of cell growth and metabolism can quickly accumulate and kill or injure the desirable cells. In order to overcome these limitations, the old or spent liquid medium must regularly be removed and replaced with an approximately equivalent volume of fresh liquid. Each successive manipulation increases the chance of contaminating the cells of interest.
[0028] Compounding the problem of limited growth yield is the issue of available surface area. In molecular biology, the cells of interest are frequently derived from human, plant, or animal samples. Such a cell generally requires a substrate upon which to grow and is known to those skilled in the art as an adherent cell. These cells will often continue to grow and to divide until all of the available surface area provided by the flask or the well is occupied, a condition often known to those skilled in the art as confluence. After confluence, the cells will usually stop growing, a growth pattern in many instances referred to as contact inhibition. Such contact inhibited cells will often not grow, however, if the initial cell density is inadequate. That is, if there is too much surface area for the number of Cells in the inoculum, the cells will not readily propagate. In other words, the cells must be cultured initially in generally smaller flasks or wells and then, following confluence, the cells and their progeny must either be harvested immediately or be transferred to progressively larger flasks or wells. The cells may also be harvested from a given flask or well and then re-seeded into a plurality of new flasks or wells, a process known to those having ordinary skill in the art as splitting cells.
[0029] Before transferring cells to a new flask or well, adherent cells are removed from the substrate. This removal is typically by mechanical scraping or by chemical or enzymatic detachment of the cells from the substrate. Non-adherent cells are usually separated from the spent liquid medium by centrifugation before transfer to a new flask. This cycle of inoculation, harvest, and re-inoculation is performed serially in most applications until an adequate number of cells have been obtained. This procedure is cumbersome and labor intensive, is wasteful of supplies and media, and is prone to contamination because of the requisite frequency of manipulation.
[0030] Another shortcoming of this type of cell culture is that of efficient and effective gas exchange. As already noted herein, the requirement for a shallow layer of the nutrient medium for efficient gas exchange places a limit on the supply of nutrients and growth factors available to the cells, which also limits the growth yield of these cells. This shallow layer of nutrient medium is also prone to relatively rapid evaporation, since much cell culture is conducted at elevated temperatures of approximately 37 degrees Celsius. Even small amounts of evaporation can alter the concentration of, for example, metabolites, cofactors, salts, waste products, or growth factors in the nutrient medium, leading to non-permissive conditions and possibly to cell death. If the layer of nutrient medium is deepened to overcome these limitations, gas exchange is impeded and the cells may suffer from sub-optimal, non-permissive, or even lethal levels of CO.sub.2, N.sub.2, O.sub.2, and other gasses.
[0031] This type of cell culture also results in a large volume of wasted space inside of the flask or the multiple-well plate, a space commonly known to those with skill in the art as headspace. This is inefficient because only a small fraction of the space occupied by the flasks or multiple-well plates is actually being used for cell culture, the rest is the headspace. Additionally, atmospheric levels of O.sub.2 and CO.sub.2, for example, are not conducive to growth of the cells and therefore the flask or the multiple-well plate must be placed unsealed into an artificially maintained atmosphere. The incubator is often used to establish this artificially maintained atmosphere. The flask or the multiple-well plate is not sealed when placed inside such an artificial atmosphere, as a seal would hinder the diffusion of fresh gasses into the headspace and diffusion of consumed gasses out of the headspace. The lack of an adequate seal increases the likelihood of contamination of the cells with undesirable cells.
[0032] Some attempts have been made to overcome the limitations of the herein-described technology by increasing the available surface area of the flasks and wells and by reducing the likelihood of contamination during manipulation. Among many of the already described elements of the prior art and others, O'Connell et al. in U.S. Pat. No. 5,272,084 teach the use of ridges or grooves in the substrate to increase the surface area available for cell culture. The proposed increase in available surface area should cause a proportional increase in growth yield, but the increase in surface area and yield is relatively modest because there are several problems attendant with the proposed approach to increased yields. Most prominently, the ridges or grooves influence the cells being cultured to propagate and grow unevenly with unpredictable results across the surface area. More specifically, depending upon the types of cells or tissues being cultured, the cells can be seen to aggregate in the valleys and be sparsely populated at the crests of the ridges or grooves. As to practical operational limitations, the cells are difficult to remove by either mechanical or chemical techniques for purposes of harvest, splitting, and or transfer. Even if chemical release techniques are used in combination with tapping and or scraping removal methods, the cells that have aggregated in the valleys still tend to be resistant to release. Additionally, the incorporation of the ridges and grooves in device such as the '084-type flask create optical aberrations in the walls of the vessel that preclude visual observations, analysis, and imaging. Even more importantly, such devices also create difficult to characterize and unpredictable results in terms growth rates and yields of anticipated and expected by products. These problems are compounded by the fact that there is little improvement made to the minimization of damage to cells during release and removal. In fact, those skilled in the art have reported that use of such devices as that disclosed in the '084
reference can result in destruction of up to approximately 30% or more of any cell or tissue culture that has been cultivated. Even if more refined chemical release and tapping techniques are employed to preserve the molecular integrity of the exterior cell walls, for example where the cellular surface receptors are of primary interest to the operator, much of the culture is lost because of the resistance to release of those portions of the culture that have aggregated in the valleys between the crests of the grooves and ridges of the proposed '084 apparatus.
[0033] The '165 patent to Cai et al., in addition to disclosing the elements already discussed herein, further teaches the use of a wide, oblong opening in lieu of the standard narrow, screw-top openings taught by the '084 patent and others, which suggests improved cell removal capabilities but which fails to address the noted pitfalls. Further to this type of proposed culture device, one of the elements taught by U.S. Pat. No. 5,523,236 to Nuzzo is the use of a hinged closure apparatus to be attached to the opening of the cell culture flask. The incorporation of either the wide, oblong opening or of the hinged apparatus may reduce the risk of contamination during a particular manipulation, but it does little to reduce the requisite frequency of manipulation that is an underlying cause of the contamination. Furthermore, the '165, '084 and '236 patents fail to address many of the other shortcomings or limitations of the prior art such as the need to lessen the excess of wasted volume manifest in the headspace.
[0034] Other attempts that have been made to overcome the difficulties of maintaining and propagating cells. Some of these attempts at improvement are now described for the purposes of illustration. U.S. Pat. No. 5,010,013 to Serkes et al., for example, discloses a roller bottle technique and device. One portion of the '013 patent instructs in the use of a cell culture flask that is substantially cylindrical. The cells and a relatively shallow layer of liquid medium are placed inside of the roller bottle flask, and the flask is then rotated about its longitudinal axis. Since the entire inner surface area of the roller bottle flask is exposed to the liquid medium at some frequency, this approach of the '013
reference can significantly increase the surface area available as a substrate for growth, which can also increase growth yields. However, attendant with the increase in surface of the Serkes et al. type devices is a drastic increase in the air or gas volume that is established in the head space with the cylinder. This essentially wasted volume does little to minimize the footprint of the device and in fact results in the requirement for larger incubation spaces and more lab bench space to accommodate the larger sizes contemplated by Serkes et al. and similarly configured devices.
[0035] The '013 reference, which teaches among other elements the use of corrugation or ridges to increase available surface area, has the same drawbacks noted herein in connection with similar technologies, including especially the difficulties imposed on the operator trying to mechanically scrape adherent cells from the walls of the roller bottle. Furthermore, the rate of rotation in such an arrangement has been noted by those skilled in the art to be critical to cell propagation. If the roller bottle turns too slowly, portions of the interior surface area will receive inadequate supplies of nutrients or even become dehydrated and the cells will be distressed and or die. If the roller bottle turns too rapidly, the shearing forces present in the resulting fluid media flow can physically distress the cells causing lysis and detachment of adherent cells from the substrate. Furthermore, the constant mixing of gas and liquid inside of the roller bottle may result in frothing or bubbling. Frothing can denature proteins associated with the cells, thereby killing or injuring the cells. Frothing can also denature proteins found in the liquid medium, thereby destroying the very cell products that are to be studied or used.
[0036] Another attempt to overcome the deficiencies of the prior art involves the use of semi-permeable or selectively permeable membranes to supply fresh gas or nutrients or to remove waste or desirable metabolic end products. This technology can be seen in, for example, U.S. Pat. No. 6,043,079 to Leighton and U.S. Pat. No. 6,329,195 B1 to Pfaller. The '079
patent teaches, among other things, the use of a membrane in which the cell culture is sealed and to which the cells of interest adhere. When a cell culture device so constructed is immersed in a nutrient medium, preferably a liquid medium, nutrients diffuse into the cell culture and waste products diffuse out of the cell culture. One of the components taught by the '195 patent is the use of an additional gas permeable, liquid impermeable membrane for the diffusion of fresh and waste gasses. The use of semi-permeable and selectively permeable membranes reduces or eliminates the requirement to frequently access the interior of such a sealed cell culture flask and more closely mimics the in vivo conditions preferred by some cell types. However, cells have widely disparate requirements for nutrients, cofactors, pH, gasses, and the like. A bath of nutrient medium appropriate for one cell or cell line may not support another cell line. Since the cell culture devices described by the '079
and '195 patents are immersed in or placed into contact with the pool of nutrient medium, this technology hinders or even precludes the simultaneous culture of cells or cell lines with different or incompatible needs. Furthermore, while this membranous device reduces the risk of contamination by repeated entry into the flask to supply fresh medium and to remove spent medium, this technology requires that the exterior of the cell culture flask to be handled aseptically too. A contaminant on the exterior of such a cell culture flask can contaminate the pool of nutrient medium that it contacts. The membrane described by the '079 and '195 references, as well as other references in the art is generally impermeable to such contaminating cells, but contaminating cells in the pool of nutrient medium can consume nutrients intended for the cells of interest and may elaborate potentially damaging or lethal products to which the membrane is permeable. This contamination can potentially damage the cells of interest or alter them such that they are less useful or useless for their intended purpose. The need to aseptically handle the exterior of such a cell culture flask places an additional burden on the operator. Furthermore, many of these cell culture devices do not appear to contemplate and are not readily adapted to the high yield cell culture needed in many biotechnology and molecular biology applications.
[0037] Some examples of the prior art appear in some respects to contemplate high density cell culture and even suggest some attempts that may avoid some of the shortcomings found in using semipermeable or selectively permeable membranes for purposes of gas exchange and replenishment. For example, international Patent Cooperation Treaty (PCT) Publication WO 00/56870, published Sep. 28, 2000 to Barbera-Guillem, (hereafter also referred to as "the '870 device") teaches among other elements the use of two such membranes sealed to a plastic frame such that the membranes and frame define an interior chamber. In operation, a technician suspends cells of interest in a nutrient medium and then injects the cell suspension into the device via an access port. The cells adhere to the membrane and gas exchange with the cells takes place across the membranes. A technician may remove spent medium and add fresh medium as needed using a needle introduced through a resealable septum.
[0038] One of many significant limitations of the '870 device is the that it appears to establish an internal positive pressure as the operator or technician injects suspended cells or fresh medium into the device, which injection compresses the preexisting volume of air and builds pressure inside the sealed chamber. Without venting to release excess pressure, the interior chamber pressure may become sufficiently high to burst or rupture the membrane, thereby ruining the device, destroying the cells of interest as well as any potentially valuable or important cell products, as well as contaminating the surrounding environment. Even if the membrane remains intact, pressures slightly above atmospheric pressure may be sufficient to lyse or damage relatively fragile cells and components thereof.
[0039] In the best of all possible circumstances, it appears from the proposed '870 apparatus that the cells and tissues being cultured are under pressure above ambient atmospheric pressure. Thus, it is also further apparent that the contemplated membranes of the '870 could rupture with only minor abrasion in normal use or from impact with a sharp instrument or edge since the contents are under pressure and the membranes are described as being only thin polymeric films. For a number of reasons, it also further appears that the cells or tissues could be physically damaged during chemical release and removal or aspiration through the resealable septum taught in the '870 reference.
[0040] Initially, the shearing forces in the flowing liquid media encountered by the cells during withdrawal or aspiration through the proposed needle of the '870 reference, which in embodiments available from the assignee corporation can be as long as about 10 to 12
millimeters or more, when compounded with the unavoidable change in pressure, may have dire consequences--most notably breach of the cell walls causing complete lysis of the cells of interest. Next, it appears that the '870 device, when used in the dual confronting membrane configuration illustrated will experience collapse of the membranes against one another as the media is withdrawn, which thereby results in any cell culture contained therein, whether attached to the membranes or in suspension in the media, being crushed against the collapsing membranes.
[0041] Third, since the cells cultured in a device according to the '870
reference may, during infusions and aspirations, be exposed to a pressure that is much greater than and a vacuum that is much less than the ambient atmospheric pressure. Harvesting the cells through a needle lumen subjects the cells, whether it be the cells passing through the needle lumen or being left behind as media is withdrawn, to possibly harsh rapid pressurization and decompression. Even if various techniques are employed to mitigate such effects, such as incremental aspiration and injection of air to minimize decompressive effects, the cells may be exposed to repeated compression and decompression, which can shock the cells. Moreover, if air is injected periodically during withdrawal, such air must be sterile, which adds further complexity and added steps to the process.
[0042] The rapid decompression effect, also known to many people as "the bends," is also experienced by individuals diving in water who rise too quickly after operating at depths below sea level and under corresponding pressures above ambient atmospheric sea level pressure. With more specific reference the device taught by the '870 and related references, the gases dissolved in the media and the cells contained in the '870, are under pressure. When the pressure of the media and the cells is reduced during removal, the dissolved gases expand and bubble, which creates the bends or rapid decompression effect that destroys the cells.
[0043] The rapid decompression effects noted herein may be exacerbated as to those cells that are actually removed. Those skilled in the art of fluid flow dynamics can appreciate that when any particles of fluid are accelerated to have a velocity that is different from its initial or nominal velocity prior to such acceleration, which velocity could be zero, then the particles experience a net drop in the pressure associated with the volume proximate to the particles. More generally and with respect to the device of the '870 reference, the cells that may be withdrawn through the needle lumen will experience an additional pressure drop while being accelerated and withdrawn through the needle lumen. Thus, it can be further understood by those knowledgeable in the related arts that that compounded pressure drop experienced by such cells will only further induce breach of the walls of the cells. If this lethal combination of compounded pressure drops does not damage the cells moving through the needle lumen, then upon exiting the needle lumen, the reintroduction of what may be standard atmospheric sea level pressure may finally rupture the cell structure, which may have at least been weakened by the earlier rapid decompression effects. Accordingly, those skilled in the art of cell culture techniques and devices may be able to appreciate that such devices, like that contemplated by the '870 and related references, do little to improve the state of the art of cell culture devices and methods.
[0044] Additionally, devices like those described by the '870 reference, can require technicians to remove adherent cells from their substrate, the membranes of the '870 device, before harvest by either chemical or mechanical methods. To accomplish this mechanically with the '870 device, the technician must physically break into the device apart or cut the membranes to expose the cells of interest, and then to scrape the membranes. This process increases the risk of contamination of the cells of interest, can physically damage the cells, and may expose the technician to possibly biologically harmful cells or by products. These risks are more pronounced because the '870 device can operate during incubation under a pressure above atmospheric such that the membranes may rupture in an uncontrolled manner due to the sudden pressure release experienced when scoring or cutting the membranes. In fact, the very type of small diameter needle contemplated for use with inoculation and or injection of cells and media into and aspiration of same from the device of the '870 can present an enormous threat of puncture of the membranes and subsequent rupture, especially in the hands of an untrained technician. Further, even if chemical release means are employed to harvest cells from the membranes without breaking the device or cutting the membranes, the '870 device appears to be very limited in its capability to withstand the extraordinary loads and forces encountered during centrifugation subsequent to cell release, since the membranes of the '870 device are necessarily thin and may rupture when subjected to such forces.
[0045] As with many other prior art attempts, the devices described in the PCT WO 00/56870 reference to Barbera-Guillem also suffer from other shortcomings, including the inability to mitigate dehydration in environments having unsuitable or less than optimum humidity control capabilities. In fact, the device contemplated by Barbera-Guillem et al. must receive fluid replenishment as often as or nearly as often as other prior art devices so that the proper or desirable cell culture hydration can be maintained. Each instance wherein replenishment is required is an additional instance when infections can be inadvertently introduced or when other similarly debilitating mishaps can occur.
[0046] There are several additional examples of the prior art that are related to the herein-captioned reference PCT WO 00/56870. For example, international Patent Cooperation Treaty Publications WO 02/41969, WO 02/42419, and WO 02/42421 all published on May 30, 2002 to Barbera-Guillem et al. These patents teach various combinations and variations of the PCT WO 00/56870 including the use of magnetic sheets for magnetic separation, methods of adhering and removing such magnetic sheets, the use of a single rather than two semi or selectively permeable membranes, and other elements and techniques. The devices taught by these patents are substantially similar to the PCT WO 00/56870 Publication and they each share the very same limitations and shortcomings discussed herein. As such, these devices can increase the time spent by technicians on cell culture, can increase the risk of contamination of entire batches of cell cultures, can be unnecessarily wasteful of money and other resources, and can generally increase the burdens of high density cell culture.
[0047] Still another attempt to address certain of the limitations of the prior art involves the use of biological or bio-mimetic substrates to support the growth and propagation of cells. These substrates may be derived from or closely mimic the extra-cellular compounds upon which cells may grow in vivo. Such substrates include, for example, collagen, elastin, cartilage, and cellulose, all common components of the extra-cellular matrix of higher animals or plants. As an example of this type of technology, one of the teachings of U.S. Pat. No. 6,312,952 to Hicks is the use of cartilage and type I collagen arranged in layers to form a support matrix sub structure bathed in a liquid nutrient medium and upon which the cells of interest are cultured. Another teaching of the '952 patent is the use of a composite cell culture, the simultaneous culture of more than one cell type; in this case chondrocytes are provided as an accessory cell that, under the right conditions, can promote the growth and proliferation of certain cells of interest such as epithelial cells. The use of these types of substrates is reasonably well suited for certain applications, such as the culture of epithelial cells in a pseudo-epithelial arrangement to be used for transplantation or for the promotion of wound healing in vivo. However, these substrates may be poorly adapted to support the growth of other cells of interest, or may be no more useful or effective at promoting the growth of those cells than the glass, plastic, or other commonly used substrates. Furthermore, the production of the support matrix substructure can be labor intensive, time consuming, and may be expensive, depending upon the availability of the substrate of interest and the purity required for the desired application. What is more, this technology is not well suited for those applications that require high yield, high density cell culture.
[0048] There have been attempts to overcome the shortcomings of the prior art that do contemplate high density, high yield cell culture. An example of such an attempt is the use of three-dimensional arrays of microfibers as a substrate to support the culture of cells. The microfiber array is encased in a substantially sealed container, such container defining a microfiber-enclosing cavity, an entry port, and an exit port. Nutrient medium enters the container by way of the entry port, washes over and immerses the microfibers, and then leaves the container via the exit port. In this arrangement, the microfibers provide significantly more surface area than in any Of the other prior art, the constant flow of nutrient medium provides a steady supply of fresh medium and removes spent medium, and high cell yields are possible. An example of such a device is disclosed in U.S. Pat. No. 4,546,083 to Meyers et al., which teaches some of these elements as well as others.
[0049] While the microfiber arrays may increase the growth yield of the cells of interest, these fibers may be difficult and expensive to obtain or manufacture, compared to the previously described art. A plurality of variables is considered by the operator in choosing a microfiber array optimized for a particular cell of interest. These variables include, for example, available fiber surface area, fiber dimension, priming volume, flow properties, and others. A microfiber array optimized for one cell or cell line may be sub-optimal or even non-permissive for another cell or cell line, requiring additional time to optimize a new microfiber array and additional money to purchase such a microfiber array. Regardless of the particular microfiber array used in an application, the very nature of the three-dimensional array may preclude mechanical harvesting of the cells and may interfere with other harvesting methods. Additionally, dedicated machinery is used to provide a reservoir of fresh medium, to pump the medium into the entry port of the microfiber-enclosing cavity, and to collect the medium from the exit port of the microfiber-enclosing cavity. Such dedicated machinery can be expensive, difficult to maintain, and unlike the previously described incubator may not be useful for the culture of other cells or in other applications. Furthermore, any of the various pumps, valves, reservoirs, and the like used in this technology that come into contact with the nutrient medium must be sterilized before each use and must be maintained in such a way as to prevent contamination during use. This sterilization places an additional burden on the operator and maybe difficult to establish and to maintain, particularly in valves, interior compartments, and other similarly inaccessible or non-obvious locations.
[0050] A related but distinct attempt to surmount the limitations of the prior art are those devices sometimes referred to by those skilled in the art as chemostats. In one arrangement, such devices pump nutrient medium from a reservoir into a well or wells containing the cells to be cultured. As a given well fills, the nutrient medium flows into the next well and so on until the nutrient medium fills the final well and flows into a second reservoir configured to capture spent medium. The chemostat may also be configured with a single well. Such chemostats may be suited for the study of a particular culture over an extended period of time. Chemostats are, however, not well adapted to high yield cell culture and are also subject to many of the same limitations as the microfiber array technology. To the extent that a given well of the chemostat is substantially similar to the cell culture flask or to the well of the multiple well plate, the chemostat is also subject to many of the same limitations of available surface area, effective gas diffusion, and the like. Furthermore, where the chemostat is configured with a plurality of wells, each well of the chemostat receives nutrient medium from the same reservoir. This technology is therefore not well suited for the simultaneous culture of cells or cell lines with different nutritional needs. This technology is also not suited for the simultaneous culture of different cells or cell lines with the same or similar nutritional requirements, since cells from a given well may spill into a subsequent well and formed an undesirable mixed culture. An example of a chemostat that discloses, for example, some of these elements is found in U.S. Pat. No. 6,271,027 B1 to Sarem et al.
[0051] The need remains for a cell culture apparatus that both provides sufficient surface area in a manageable volume without the need for cumbersome manipulations and allows for the efficient exchange of gas, nutrients, and waste without excessive risk of contamination or the use of excessive headspace. While many of the prior art devices were aimed to improve the art of such devices, none has achieved the optimized and effective capabilities and widespread compatibility of the instant invention. The present invention meets the herein described and other needs without adding any complexity, inefficiencies, or significant costs to implementation in existing applications and environments. The various embodiments of the present invention disclosed are readily adapted for preferable ease of manufacture, low fabrication and setup costs, effectiveness of operation, and for wide compatibility with extant cell culture technologies.
SUMMARY OF INVENTION
[0052] In its most general configuration, the present invention advances the state of the art with a variety of new capabilities and overcomes many of the shortcomings of prior devices in new and novel ways. In one of the many preferable configurations, a cell culture device includes substantially planar anterior and posterior shells or walls or faces that are arranged in a substantially confronting relationship, and which are joined by respective opposing dextral and sinistral laterally opposed longitudinal edges, and opposing superior and inferior peripheral lateral edges. The shells or walls or faces and the edges together define a media reservoir or chamber or cistern. Optionally, at least one of the anterior and posterior walls or faces or shells and the edges are preferably formed with at least one circumfluent periphery that defines at least one optional respirator aperture. The optional at least one respirator is formed from a gas permeable film or membrane that seals the optional at least one respirator aperture about the periphery. The device also further incorporates at least one fluid transfer port that is formed in least one of the shells and edges and that is in fluid communication with the media reservoir or chamber or cistern. At least one gas valvule is also formed in at least one of the shells and edges of the cell culture device and is also in fluid communication with the media reservoir. The valvule is adapted to equalize positive and vacuum pressure within the cistern to ambient atmospheric pressure as fluid is communicated through the at least one port. The at least one gas valvule is preferably hydrophobic and can be adapted to prevent liquid flow there through either by selection of an appropriately capable material, or by incorporating an additional valving device, or by including a combination thereof.
[0053] The cell culture device also is further adapted so that the surface area of the optional respirator membrane or film is approximately between 1% and 10% of the surface area of the media reservoir, and more preferably approximately between 1.5% and 5%. The optional membrane or film can also be formed from a sheet material to have a thickness approximately between 0.09 and 0.14 millimeters.
[0054] Preferably, the media reservoir is adapted to receive approximately between 20 milliliters and 140 milliliters of a fluid mixture, and more preferably at least about 25 milliliters. In various embodiments and depending upon the desired uses and applications, the preferred cell culture device is adapted with the joint that is formed between the respective lateral, superior, and inferior peripheral edges being a releasably or permanently hermetically sealed joint.
[0055] In various modifications and configurations, the cell culture device may have the media reservoir being formed with a lateral dimension between the laterally opposing longitudinal edges of approximately between 6.5 centimeters and 9 centimeters, a longitudinal dimension between opposing superior and inferior lateral edges of approximately between 11 and 13 centimeters, and a dimension between interior surfaces of the anterior and posterior shells or walls or faces of approximately between 2 millimeters and 6 millimeters.
[0056] In any of the preferred arrangements and configurations, the media reservoir is preferably defined by an internal surface area of the shells or faces or walls and edges that bounds an internal volume whereby the ratio between the volume and the surface area is approximately between 100 microliters per square centimeter and 1000 microliters per square centimeter, or more and depending upon the desired application. Moreover, and again depending upon the proposed applications and uses, the cell and tissue culture device can be adapted to have the media reservoir being defined by a plurality of internal surfaces of the shells and walls and faces and edges wherein substantially all of the surfaces are adapted to support growth of cells. In the alternative, only selected portions of the internal surfaces can be so adapted whereby certain other portions are adapted to inhibit such cell and or tissue growth.
[0057] The anterior and posterior shells can be, in various arrangements, be formed from a substantially transparent thermoplastic material. In alternative configurations, the thermoplastic material can be modified with a pigment that is selected for its capability to filter photonic energy outside the range of between approximately 500 and 600 nanometers, and even more preferably between about 550 and 570 nanometers, so that the energy absorbed by the cells and tissues being cultured can be closely controlled, among other possible uses and purposes.
[0058] In still more alternative configurations, the cell culture device may have the at least one fluid transfer port and or the gas valvule are adapted to communicate fluid (liquid and or gas) with the media reservoir through a siphon lock lumen formed with at least one fluid path that bends through at least one angle of approximately between 45 and 135
degrees of arc so as to equalize hydrostatic pressure against the port to minimize the possibility of leaks during use, handling, and related operations. In any of the preceding embodiments, the at least one fluid transfer port can also preferably incorporate a resealable elastomeric septum adapted to releasably receive a means to communicate a fluid through the port that can include needles of all types and various types of needleless connectors and lumens and various types of what are known to those skilled in the art as pipetter tips.
[0059] The contemplated at least one gas valvule incorporates a filtration element is also preferably adapted to pass only and or primarily gaseous atoms and molecules and to prevent the passage of particles having an average diametrical dimension of approximately between 0.1 and 0.3
microns. The filtration element or elements may also be formed from a material that is or that incorporates a hydrophobic material capable of minimizing and or eliminating the possibility that liquid will pass through the gas valvule. In alternative modifications, the filtration element can be formed from an assembly of at least 2 layers with a first layer being adapted to prevent the passage of particles having an average diametral dimension of at least between about 80 to 120 microns, and a second layer being adapted to prevent the passage of particles having an average diametral dimension of at least between about 0.1 to 0.3 microns. In yet other alternative arrangements, the filtration element is constructed or formed from a hybrid filter medium having a filtration property wherein the size of the particles that are filtered and or passed changes across a cross-section of the filter medium such that at a first exterior surface the medium is adapted to prevent the passage of particles having an average diametral dimension of at least between about 80 to 120 microns, and whereby at a second opposite exterior surface the medium is adapted to prevent the passage of particles having an average diametral dimension of at least between about 0.1 to 0.3 microns.
[0060] In still more optional variations of any of the preceding embodiments, modifications, and alternative configurations, the cell culture device may be modified wherein the anterior and posterior shells, faces, or walls and the laterally opposed peripheral longitudinal edges are adapted to form a body, which can be formed in any number of ways including extrusion methods, to have a superior body edge and an inferior body edge, wherein the superior and inferior peripheral lateral edges are further formed on respective superior and inferior end manifolds adapted to be respectively engaged with the superior and inferior body edges to further define the cistern. The manifolds and the body can be further formed with various lumens and channels that can be configured to infuse and aspirate fluids, including gases and liquids to and from the cistern, chamber, or reservoir of the cell and tissue culture device.
[0061] Other configurations of the instant cell and tissue culture device further contemplate an insulative and protective container that is formed with at least one interior cavity sized and adapted to receive one or more cell culture devices. The container can be further adapted to incorporate a means for controlling the temperature of the device, which means can include a power source, a thermo electric heat semiconductor pump, various control electronics, and heat conducting plates that can, in operation, control the temperature of the cell and tissue culture device for purposes of cooling and or warming the contents thereof.
[0062] These variations, modifications, and alterations of the various preferred embodiments may be used either alone or in combination with one another as can be better understood by those with skill in the art with reference to the following detailed description of the preferred embodiments and the accompanying figures and drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0063] Without limiting the scope of the present invention as claimed herein and reference is now made to the drawings and figures, wherein like reference numerals, and like numerals with primes, across the several drawings, figures, and views refer to identical, corresponding, or equivalent elements, components, features, and parts. In the various figures and drawings, as needed for purposes of better describing the aspects of the instant invention, various reference symbols and letters are used to identify significant features, dimensions, objects, and arrangements of elements described herein and in connection with the several figures and illustrations.
[0064] FIG. 1 is an elevated isometric view, not to scale, of the cell culture flask according to the principles of the instant invention;
[0065] FIG. 2 is an elevated, rotated, exploded, and reduced scale isometric view of the device of FIG. 1;
[0066] FIG. 3 is an elevated, rotated, and exploded isometric view, in reduced scale, of the device of FIG. 2;
[0067] FIG. 4 is a detail view, rotated and in enlarged scale, of a portion of the device of FIG. 2, with certain structure removed for purposes of illustration;
[0068] FIG. 5 is a detail view, rotated and in enlarged scale, of a portion of the device of FIG. 3, with certain structure removed for clarity;
[0069] FIG. 6 is a detail view, rotated and in enlarged scale, of a portion of the device of FIG. 2, with certain structure removed for purposes of illustration;
[0070] FIG. 7 is a detail view, rotated and in enlarged scale, of a portion of the device of FIG. 3, with certain structure removed for clarity;
[0071] FIG. 8 is a detail view, rotated and in reduced scale, of the superior portion of the anterior side of the cell culture flask of FIG. 1;
[0072] FIG. 9 is a detail view, rotated and in reduced scale, of the device of FIG. 8 in operation;
[0073] FIG. 10 is a detail view, rotated and in reduced scale, of the superior portion of the anterior side of the cell culture flask of FIG. 1
reflecting an alternative configuration;
[0074] FIGS. 11 and 12 are detail views of the device of FIG. 10 in operation;
[0075] FIGS. 13 and 14 are perspective views, rotated and in reduced scale, of the cell culture flask of FIG. 1 and reflecting modified embodiments;
[0076] FIGS. 1 and 17, are plan detail views, rotated and in enlarged scale, of selected elements of FIG. 13 and with various structure removed for purposes of illustration;
[0077] FIG. 18 is a plan detail view, rotated and in enlarged scale, of selected elements of FIG. 14 and with various structure removed for purposes of explanation;
[0078] FIG. 19 is a plan view, rotated and in reduced scale, of the device of FIG. 1;
[0079] FIG. 20 is a section view, rotated and in enlarged scale and taken along section line 20-20, of the cell culture flask of FIG. 19;
[0080] FIG. 21 is a detail section view, rotated and in enlarged scale and taken along section line 21-21, of the device of FIG. 19;
[0081] FIG. 22 is a detailed section view, in enlarged scale and taken about detail view lines 22-22, of the device if FIG. 21;
[0082] FIGS. 23 and 24 are perspective views, rotated and in reduced scale, of the device of FIG. 1 and shown in operation;
[0083] FIG. 25 is a detail partial-section and perspective view, rotated and in enlarged scale, of various components of the cell culture flask of FIGS. 23 and 24 during use;
[0084] FIG. 26 is a partial detail section view, in enlarged scale and taken about detail view line 26-26, of the cell culture flask of FIG. 21
in operation;
[0085] FIG. 27 is a partial detail section view of the flask of FIG. 26
during continued use;
[0086] FIG. 28 is a partial detail section view of the flask of FIGS. 26
and 27 during continued operation;
[0087] FIG. 29 is an elevated perspective view, in reduced scale, of the device of FIG. 1 with hidden lines depicted for purposes of illustration;
[0088] FIGS. 30 and 31 are perspective and rotated views, in reduced scale, of the flask of FIG. 1 shown in use;
[0089] FIGS. 32 and 33 are diagrammatic and schematic representations, in modified scale, of alternatives and variations of certain elements and components of the cell culture and tissue flask or vessel of FIGS. 1 and 29;
[0090] FIGS. 34, 35, and 36 are diagrammatic and schematic cross-sectional representations, in modified scale, of various additional variations, features, and elements of the cell culture and tissue flask or vessel of FIGS. 1 and 29;
[0091] FIG. 37 is a partial detail view, in enlarged scale and rotated and taken about detail view line 37-37, of an optionally modified configuration of the various features and elements of the cell culture and tissue flask or vessel of FIG. 19;
[0092] FIG. 38 is a partial detail section view, in enlarged scale and rotated and taken about detail view line 38-38, of the flask or device of FIG. 37;
[0093] FIG. 39 is a partial detail view of the flask or device of FIG. 38
shown in operation;
[0094] FIG. 40 is a plan view, in reduced scale and rotated, of an alternative arrangement of the cell and tissue culture device according to the instant invention;
[0095] FIG. 41 is a plan view, in reduced scale and rotated, of another variation of the vessel or flask according to the principles of the instant invention;
[0096] FIG. 42 is a partial section view, in enlarged scale and rotated and taken about section line 42-42 of either of the cell and tissue culture vessels or flasks of FIGS. 40 and 41;
[0097] FIG. 43 is a partial section view of the flask or vessel of FIG. 42
shown in operation;
[0098] FIG. 44 is a partial section detail view, in enlarged scale and rotated and taken about section line 44-44, of the cell culture flask of FIG. 19;
[0099] FIGS. 45 and 46 are partial section detail views of alternative arrangements of features of the flask of FIG. 44;
[0100] FIGS. 47, 48, 49, and 50 are partial section detail views, rotated and in enlarged scale, of various other arrangements of the features of the flask of instant invention of FIG. 1 and the other figures herein;
[0101] FIG. 51A is a side view, in modified scale and rotated, illustrating optional features and elements of the flask or vessel of FIGS. 1, 19, and the other figures herein;
[0102] FIG. 51B is a partial section view, in enlarged scale and rotated and taken about section line 51B-51B, of the flask or vessel of FIG. 5A;
[0103] FIG. 52A is a side view, in modified scale and rotated, that depicts additional possible features and elements of the flask or vessel of FIGS. 1, 19, and the other figures herein;
[0104] FIG. 52B is a partial section view, in enlarged scale and rotated and taken about section line 52B-52B, of the flask or vessel of FIG. 52A;
[0105] FIG. 53 is a plan view, not to scale, of an alternative configuration of the cell culture flask of FIG. 1;
[0106] FIG. 54 is a detail section view, in enlarged scale and rotated and taken about section line 54-54, of the cell culture flask of FIG. 53;
[0107] FIG. 55 is a plan view, not to scale, of an alternative configuration of the cell culture flask of FIG. 1;
[0108] FIG. 56 is a cross section view, rotated and in enlarged scale and taken about section line 56-56, of the flask of FIG. 55;
[0109] FIG. 57 is a section view, rotated and in enlarged scale and taken about section line 57-57, of the cell culture flask of FIG. 55;
[0110] FIG. 58 is a section view of an alternative arrangement of features of the flask of FIG. 57;
[0111] FIG. 59 is a section view, rotated and in enlarged scale and taken about section line 59-59, of the flask of FIG. 55;
[0112] FIGS. 60, 61, and 62 are section views having optional and alternative configurations of the features and elements of the flask of FIG. 59;
[0113] FIG. 63 is a plan view, not to scale, of optional features and elements of the cell culture flask of FIGS. 1, 19, and the other figures herein;
[0114] FIG. 64 is a plan view, not to scale, of optional features and elements of the cell culture flask of FIGS. 1, 19, and the other figures herein;
[0115] FIG. 65 is an elevated perspective diagrammatic view, not to scale, of an alternative embodiment of a cell culture flask according to the principles of the instant invention;
[0116] FIG. 66 is a plan view, in modified scale, of another alternative configuration of the cell culture flask of FIG. 65 according to the principles of the instant invention;
[0117] FIG. 67 is a partially exploded plan view, in similar scale, of the flask of FIG. 66;
[0118] FIG. 68 is a partially exploded view, in modified scale and with various elements rotated for illustration purposes, of the flask of FIGS. 66 and 67;
[0119] FIG. 69 is another partially exploded view, in similar scale and with various elements rotated, of the cell culture flask of FIGS. 65, 66, 67, and 68;
[0120] FIG. 70 is a partial section view, rotated and in enlarged scale and taken about section line 70-70, of the flask of FIG. 66;
[0121] FIG. 71 is an exploded detail section view of the flask of FIG. 70;
[0122] FIG. 72 is an elevated perspective view, in enlarged scale and rotated, of certain optionally modified elements of the flask of FIG. 68;
[0123] FIG. 73 is an elevated perspective view, in reduced scale, of various optionally configured components of the elements of FIG. 72;
[0124] FIG. 74 is an elevated perspective and assembly view, in reduced scale and rotated, of some of the components of FIGS. 72 & 73;
[0125] FIG. 75 is an exploded and elevated perspective view, rotated and in enlarged scale, of certain components and elements of the cell culture flask of FIGS. 65, 66, and 67;
[0126] FIG. 76 is a partially assembled and partially exploded view, in similar scale, of various components of the vessel or flask of FIGS. 65, 66, 67, and 75;
[0127] FIG. 77 is an elevated perspective assembled view, in modified scale, of the flask of FIGS. 65, 66, 67, and 75;
[0128] FIG. 78 is an elevated perspective view, rotated and in modified scale, of various optional features and elements compatible for use with the cell culture flask or vessel according to the principles of the instant invention and as reflected in any of the various figures including, for purposes of example without limitation, FIGS. 1, 19, 65, 66, and the other figures herein;
[0129] FIG. 79 is a side view, rotated and in enlarged scale, of another alternative configuration of the flask of FIG. 78;
[0130] FIG. 80 is a partial section view, rotated and in enlarged scale and taken approximately about section line 80-80, of optionally modified features of the flask of FIG. 78;
[0131] FIG. 81 is a partial section view, in modified scale, reflecting a diagrammatic illustration of optional configurations of the features and components of the flask of FIG. 80;
[0132] FIGS. 82, 83, and 84 are additional optional arrangements of features and elements of the flask of FIG. 80;
[0133] FIGS. 85 and 86 are elevated perspective views, rotated and in modified scale, of optional additional features and components of the cell culture flask of FIGS. 1, 19, 65, 66, and other figures herein;
[0134] FIG. 87 is a side section view, in enlarged scale and rotated and with certain structure removed for illustration purposes, of certain components of the flask of FIGS. 85 and 86;
[0135] FIG. 88 is an elevated perspective view, not to scale, of an alternative configuration of the flask of FIGS. 85, 86, and 87;
[0136] FIG. 89 is a schematic and diagrammatic perspective view, in enlarged scale, of various components of the flask of FIGS. 85, 86, and 88, with certain structure removed for purposes of further illustration;
[0137] FIG. 90 is a section view, rotated and in enlarged scale, of alternative arrangements of the flask of FIGS. 85, 86, 87, and 88;
[0138] FIG. 91 is a section view, in similar scale, of another optionally modified configuration of the flask of FIG. 90; and
[0139] FIG. 92 is a section view in enlarged scale, of the flask of FIG. 91, with various structure repositioned for purposes of further illustration.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0140] The state of the art of cell culture devices is significantly advanced on several avenues by the present invention. Industrial production of high-density and certain low-density cell cultures is needed for myriad applications including, for example, medical, research, military, veterinarian, agricultural, and related endeavors. There are many difficulties in producing large scale and economically efficient high-throughput, high-volume, and high-density cell cultures for such applications and endeavors, which difficulties are especially prevalent in the high-precision production of biologically synthesized molecules needed for the preparation of antibodies, vaccines, biological reagents and response modifiers, and the like. Each of these pursuits are often plagued with microbiological and cross-cell line contamination issues, ineffective or inefficient culture devices having only limited surface area available for adherent cell growth, and problems attendant to media and gas replenishment, to name a few of the more troublesome concerns.
[0141] The instant invention addresses all of these issues in new, novel, and heretofore unknown ways that not only overcome the shortcomings of the prior art attempts, but which also address such shortcomings without significant changes to conventional procedures and practices and with reductions in cost or operational constraints. Moreover, the instant invention accomplishes such advances and improvements without abandoning the long-used conservative strategies, regulations, and protocols well-established in the scientific, research, medical, and industrial communities that have the need for such improved cell culture devices. These benefits are accomplished in ways that enable sterile and compartmentalized high-volume or high-throughput cell and tissue culture with a precision and with a confidence of success that has never before been possible.
[0142] For purposes of illustrating the present invention, the terms bioreactor, cell or tissue culture device, apparatus, cell factory, container, culture tube, cluster dish, dish, flask, ELISA plate, multi-well plate (including single, double, quadruple, 4 by 6, and 12 by 8 type multi-well or 96 well plates), micro-incubator, micro-carrier, microplate, microslide and chamber slide, microtiter plate, roller bottle, spinner flask, vessel, high-density cell culture, and plurals and combinations of such descriptive phrases, all are intended to refer generally to any item capable of being used for purposes of culturing, handling, manipulating, storing, analyzing, and otherwise establishing, supporting, harvesting, and using cells and by-products thereof in vitro or otherwise for a variety of purposes as set forth and as contemplated herein.
[0143] With reference now to the various figures and specifically FIGS. 1, 2, and 3 the instant invention is directed to a cell and tissue culture flask, device, or vessel 100 that incorporates, among many other features, a posterior face, wall, or shell 110 and an anterior face, wall, or shell 140. Although any of a variety of shapes and sizes of cell culture devices or vessels 100 is contemplated by the instant invention, the illustrative configurations of FIGS. 1, 2, and 3 depict the walls or shells 110, 140 being generally planar and rectangular in shape with circumfluent penpheral edges, which in multipart embodiments can be adapted to registered with one another in a confronting relationship, and which in all embodiments propose that the walls or shells 110, 140
permanently and or releaseably mate with one another. As discussed in more detail herein, the walls or shells 110, 140 can be preferably also adapted for remating upon release and separation either by way of new and novel mating interfaces adapted for use alone and or in connection with a specialized release tool, which tool is not shown herein but which can be understood in principle by those skilled in the, relevant arts.
[0144] The posterior shell or wall 110 further includes an interior surface 115 generally circumscribed by a superior peripheral lateral edge 120 that is longitudinally opposed to an inferior peripheral lateral edge 125 and respective and laterally opposed dextral and sinistral peripheral longitudinal edges 130, 135. The anterior wall or shell 140 is preferably also formed with peripheral edges adapted to sealingly mate with the peripheral edges 120, 125, 130, 135 of the posterior shell or wall 110. More specifically, the anterior wall or shell 140 includes an interior surface 145 generally encircled by a superior peripheral lateral edge 150
longitudinally opposite an inferior peripheral lateral edge 155, and respective laterally opposed dextral and sinistral peripheral longitudinal edges 160, 165.
[0145] When the shells or walls 110, 140 are assembled together (FIG. 1), the interior surfaces 115 and 145 define what maybe referred to herein as an interior chamber, reservoir, or cistern 170 for containing nutrient media (not shown) and the cell culture (not shown) during use and operation of the preferred cell and tissue culture flask 100. As stated, the instant cell culture vessel or device 100 is directed to a variety of preferred shaped and configurations that may be equally suitable for purposes of improved cell culture capability. Additionally, the instant configurations reflected in the figures, including specifically FIGS. 1, 2, and 3, are for purposes of illustration but not limitation depicted with the interior chamber, reservoir, or cistern 170 that in one variation preferably has a volumetric capacity to hold between about 10
and 140 milliliters of media, cell and tissue culture, and constituents thereof. Even more preferably, the present invention is directed to one or more embodiments having a preferred volumetric capacity range corresponding to one of a number of different sizes of cell culture devices with one such size being that of the cell culture flask 100
depicted in the FIGS. 1, 2, 3. The device or vessel 100 preferably is sized whereby the reservoir or cistern 170 has a volumetric capacity of approximately between 20 milliliters and 35 milliliters, and more preferably between about 20 and 30 milliliters, and even more preferably a volumetric capacity that can receive at least about 25 milliliters of media, cell and tissue culture, and constituents thereof.
[0146] To establish the desired volumetric capacities the cell culture devices or vessels including device 100 that are contemplated herein generally are adapted to have dimensional sizes and shape profiles that are configured for compatibility with a large variety of existing and widely used scientific, clinical, and industrial peripheral equipment. Such equipment is readily available in use with existing prior art cell culture items having selected sizes, shapes, and configurations as further set forth herein. For example, the assembled cell culture device 100 is preferably adapted to have an exterior dimension between the respective posterior and anterior, sinistral and dextral opposing peripheral longitudinal edges 130, 135, 160, 165 of between about 6 and 9
centimeters, and more preferably about 7 and 8.5 centimeters, and even more preferably approximately 8.4 centimeters across.
[0147] In the exemplary and demonstrative configuration of FIGS. 1, 2, and 3 the assembled cell culture device 100 also preferably has an external longitudinal dimension between the respective posterior and anterior, superior and inferior peripheral edges 120, 125, 150, 155 of about between 10 and 14 centimeters, and more preferably approximately between 11 and 13 centimeters, and even more preferably about 12.6 centimeters. Additionally, the preferred cell culture device 100 is arranged whereby the exterior thickness of the assembled respective anterior and posterior shells or walls 110, 140 of the device 100 is approximately between 3 and 20 millimeters and more preferably in the range of about 4 t