Sealed culture chamber

Details Sealed culture chamber Sealed culture chamber

2000-12-08

2003-02-18

Redding, David A. (Department: 1744)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Method of culturing cells in suspension

C435S371000, C435S286600, C435S297500, C435S303100, C435S305400, C435S285200

Reexamination Certificate

active

06521451

ABSTRACT:

BACKGROUND OF THE INVENTION
Cell culture techniques have long been established for particular cell and tissue types and uses, and continuation of cell viability depends on many factors. One common problem is contamination of cultures in the incubator by airborne contaminants such as mold spores and opportunistic bacteria. The problem lies in the incubator conditions; incubators are generally humidified to close to 100% in order to inhibit evaporation of water from the culture media within the culture dishes. The standard solution to this problem is decontamination of the incubator, a time-consuming task and difficult or impossible to do completely. There is no guarantee that a recently cleaned incubator will remain mold-free for long, especially if already-infected cultures are returned to it. The warm, humid environment found in mammalian and bacterial cell incubators is ideal for the proliferation of mold and bacteria.
The use of completely sealed culture dishes to prevent evaporation of the water in culture media is not possible in the culture of cells, because a transport of gasses to and from the culture chamber is necessary to ensure cell viability. Cells consume oxygen and produce carbon dioxide, and these gasses must be maintained in the culture space within certain levels to prevent pH changes in the medium and provide adequate oxygen for cellular metabolism. To maintain physiological pH around 7.3, most culture media use a buffering system in which there is an equilibrium between dissolved CO
2
(bicarbonate anion) and a well-regulated 5% (v/v) CO
2
atmosphere in the incubator. When brought into room atmosphere (less than about 0.1% CO
2
), bicarbonate leaves the medium as gaseous CO
2
and the pH drifts up to over 8.5. Some media designed for ambient CO
2
levels (such as Hibernate, Life Technologies, Inc., Gaithersburg Md.) use additional buffers, such as MOPS or HEPES, to maintain proper pH. However, HEPES and other synthetic organic buffers maybe highly phototoxic, even under standard fluorescent ceiling lights (Spierenburg, G. T. et al. (1984),
Cancer Research
44(5):2253-2254; Lepe-Zuniga, J. L., et al. (1987),
Journal of immunological Methods
103(1): 145-145).
A need exists for a cell culture system that reduces the risk of contamination, but prevents evaporation of water from cultures and supplies cells with the gasses necessary for viability.
SUMMARY OF THE INVENTION
A system for culturing cells has been developed that eliminates the problem of infection from the incubator and prevents water loss from evaporation of medium, yet permits the exchange of necessary gasses. The container covers and culture dishes of the invention have permitted culture of cells for prolonged periods, longer than the standard culture time of about 2 weeks, up to 12 months or longer.
In one embodiment, the invention includes a gas permeable cover for a container, which comprises a solid support sized to fit the opening of the container to be covered. The support has at least one hole which is covered by a permeable section, preferably a membrane, which is sufficiently permeable to oxygen and carbon dioxide that cultures of live cells can be sustained. The permeable section is substantially impermeable to water and water vapor. “Substantially impermeable to water” as used herein means that water and water vapor passes across the permeable section at a rate less than about 5 mol/m
2
·day at 25° C. and 1 atmosphere. Finally, the cover has a seal portion which forms a water- and gas-tight seal between the cover and the container when the cover is placed upon or engages the container as configured for use.
Preferably, the gas permeable section of the cover passes water and water vapor at a rate of less than about 3 mol/m
2
·day, more preferably less than about 2 mol/m
2
·day, most preferably less than about 1 mol/m
2
·day at 25° C. and 1 atmosphere. The permeable section preferably passes oxygen gas at a rate of greater than about 0.25 mol/m
2
·day, more preferably at a rate of greater than about 0.5 mol/m
2
·day, and most preferably at a rate of greater than about 0.8 mol/m
2
·day at 25° C. and 1 atmosphere. It preferably also passes carbon dioxide gas at a rate of greater than about 0.5 mol/m
2
·day, more preferably greater than about 1.0 mol/m
2
·day, and most preferably greater than about 1.5 mol/m
2
·day at 25° C. and 1 atmosphere. These parameters provide the advantage that the cell culture does not dry out over time and thus does not have to be maintained in a humidified incubator. Because humidified incubators often harbor contaminating microorganisms, the ability to culture cells in a reduced humidity incubator reduces the growth of such microorganisms, and thus also reduces risk of contamination of the culture by opportunistic microorganisms.
In another embodiment, the invention includes a culture dish for the culture of viable cells comprising a culture portion having a bottom part and elevated walls surrounding the bottom part, the culture portion being capable of containing the cells, and a lid portion having a permeable section, the permeable section being sufficiently permeable to oxygen and carbon dioxide that viable cells can be sustained, and substantially impermeable to water and water vapor. The lid portion has a seal region which can form a seal that is both water-tight and gas-tight between the culture portion and the walls of the culture portion when the two are configured for use. Preferably, the water- and gas-tight seal is a friction seal, created by e.g., the weight of the lid portion against the walls of the culture dish or a gasket or other material sufficiently elastic/flexible to conform to the walls.
In preferred embodiments, the permeable section is fixedly attached onto the lid portion, such as by glue, thermal, or ultrasonic bonding. Alternatively, the lid portion, or the entire vessel, can be made of substantially entirely the same material as the permeable section. The permeable section is preferably comprised of an optically clear and flat membrane, preferably a fluorinated ethylene-propylene film. This allows for imaging or surveillance of the culture without removing the cover, reducing the risk of contamination, evaporation, and pH changes. Preferably, the permeable section is impermeable to viruses, mold, bacteria, and fungi.
In other embodiments, the dish further comprises at least one electrode, more preferably an array of electrodes. In still other embodiments, the dish further comprises at least one infusion and/or outflow pipe or port for changing medium or infusing drugs, for example.
The invention also features a method of culturing cells comprising providing cells and culture medium in a culture vessel, where the culture vessel comprises a culture container portion capable of containing the cells and a cover portion. The cover portion has a solid support sized to fit the opening of the container and has at least one hole which is covered by a permeable section that is sufficiently permeable to oxygen and carbon dioxide so that live cells can be sustained. It is also substantially impermeable to water and water vapor. The cover also has a seal portion which forms a water- and gas-tight seal between the cover and the container when the cover is placed on the container as configured for use. The method includes incubating the cells under conditions physiologically compatible with cell viability. Cells can be maintained for long periods with this method by periodically changing a sufficient amount of the culture medium such that the cells have sufficient nutrients and toxic levels of waste are removed, because evaporation of the medium is greatly reduced. The relative impermeability also allows the cells to be incubated in an incubator that is not humidified, but rather kept at ambient relative humidity.


REFERENCES:
patent: 3941662 (1976-03-01), Munder et al.
patent: 3948732 (1976-04-01), Hadad et al.
patent: 5817509 (1998-10-01), Stevens et al.
patent: 5858770 (1999-01-01), Perlman
patent: 5863792 (1999-01-01), Tyndorf et al.
Barbera

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