Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of culturing cells in suspension
Reexamination Certificate
2000-03-15
2002-09-24
Redding, David A. (Department: 1744)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of culturing cells in suspension
C435S394000, C435S401000, C435S402000, C435S288300, C435S288400, C435S297100, C435S297500, C435S305100, C435S305200
Reexamination Certificate
active
06455310
ABSTRACT:
FIELD OF THE INVENTION
The present invention generally relates to an apparatus and methods for growing cells or tissue culture in vitro. More particularly, the present invention relates to a cell culture apparatus containing at least one gas permeable membrane which allows rapid and uniform transfer of gases between the environment of cells contained in the cell culture container apparatus and the atmosphere of the incubator in which the cell culture apparatus is incubated.
BACKGROUND OF THE INVENTION
In eukaryotic cell culture systems, the culture of the cells is generally under conditions of controlled pH, temperature, humidity, osmolarity, ion concentrations, and exchange of gases. Regarding the latter, oxygen and carbon dioxide (CO
2
) are of particular importance to the culturing of cells. In a typical eukaryotic cell culture system, an incubator is provided in which CO
2
is infused to maintain an atmosphere of about 5% CO
2
within the incubator. The CO
2
interacts with the tissue culture medium, particularly its buffering system, in maintaining the pH near physiologic levels. Conventional cell culture containers comprise tissue culture flasks, tissue culture bottles, and tissue culture plates. Entry of CO
2
from the incubator atmosphere into a tissue culture plate generally involves a loosely fitting cover which overhangs the plate in excluding particulate contaminants from entering the plate chamber(s), but allows gas exchange between the incubator atmosphere and the atmosphere within the tissue culture plates. Similarly, for a tissue culture flasks or bottle, a loosely fitting cap excludes particulate contaminants from entering the chamber of the flask or bottle, but allows gas exchange between the incubator atmosphere and the atmosphere within the flask or bottle. More recently, a cap is provided with a gas permeable membrane or filter, thereby allowing for gas exchange with a tightly fitting cap.
In addition to CO
2
, the culturing of cells is dependent upon the ability to supply to the cells a sufficient amount of oxygen necessary for cell respiration and metabolic function. The supply of oxygen for cell respiration in conventional cell culture containers is in the header space of the container, e.g., the void space in the container that is above the surface of the tissue culture medium. Efforts to increase oxygen concentration to the cultured cells includes mechanical stirring, medium perfusion or aeration, increasing the partial pressure of oxygen, and/or increasing the atmospheric pressure. Thus, in conventional cell culture containers the volume or surface provided for gas exchange, as relative to the volume or surfaces of the whole container, is either inefficiently used and/or results in limiting the rate of gas exchange or in the equilibration of gases. This is even more noticeable in small-scale cultures (15 ml or less) in which rate of cell growth, cell densities, and total cell numbers, are frequently low due to space, surface area, and gas exchange limitations.
The rate of gas exchange across gas permeable membranes has been described as “improved”. However, gas permeable membranes have been described as undesirable for use in a cell culture system for various reasons. For example, in U.S. Pat. No. 5,523,228, it is taught that a boundary layer of oxygen toxicity forms at the interface between the gas permeable membrane and the tissue culture medium; and further, cells entering into the toxic boundary layer can be irreparably damaged. Further, in U.S. Pat. No. 5,707,869 it is taught that the chemistry of the surface of gas permeable, liquid impermeable materials is incompatible with many cell types; and additionally, due to their propensity to cause non-specific protein binding, such materials can lead to depletion of soluble growth factors.
Thus, there is a need for a cell culture apparatus that can provide an increased surface area for gas exchange as compared to conventional cell culture containers; and which also provides a high rate of cell growth in achieving a high cell density in a relatively short period of time, and with an even distribution of anchorage dependent cells along the attachment surface.
SUMMARY OF THE INVENTION
The present invention provides a cell culture apparatus comprising at least one frame; two thin membranes wherein at least one of the membranes is gas permeable, and wherein the membranes are securedly sealed to (in a leak-proof sealing with) the frame, in forming a culture chamber; and at least one resealable aperture through the frame which allows substances to be introduced into, or withdrawn from, the culture chamber.
In one preferred embodiment, the cell culture apparatus comprises a frame over which is extended and securedly sealed thereto two gas permeable membranes in forming a culture chamber therebetween. The frame is sufficiently rigid to provide a housing for assembling the cell culture apparatus of the present invention. The membranes are of suitable thickness to provide sufficient gas permeability to accommodate cell growth in the chamber, and to provide sufficient structural integrity for handling the apparatus. Further, the membranes are of a sufficient optical transparency and clarity so as to observe the cell culture (e.g., the color of the tissue culture medium; and cellular characteristics such as growth and morphology of cells, as observable by microscopy). The frame has at least one resealable aperture, and preferably at least two resealable apertures, which allows substances to be introduced into, or withdrawn from, the culture chamber. Each aperture comprises an opening through the frame which may serve as a passageway into which is guided a portion of an instrument (e.g., needle or pipette or pipette tip) for introducing a substance into or withdrawing a substance from the culture chamber. In a preferred embodiment, the frame is of sufficient thickness and the apertures are of a sufficient limiting diameter to prevent the instrument portion, when inserted through a resealable aperture of the frame, from puncturing either of the walls formed by the membranes of the culture chamber.
In another preferred embodiment, the cell culture apparatus further comprises an additional frame which accommodates the cell culture apparatus formed by the one frame and the two gas permeable membranes. In this embodiment, the cell culture apparatus comprises a two-piece frame comprising an inner frame and an outer frame. The inner frame has securedly sealed thereto at least one, and preferably two, gas permeable membranes in forming a culture chamber therebetween. The membranes are of suitable thickness to provide sufficient structural integrity for handling the apparatus. Further, the membranes are of a sufficient optical transparency and clarity so as to observe the cell culture (e.g., the color of the tissue culture medium; and cellular characteristics such as growth and morphology of cells as observable by microscopy). The outer frame has at least one resealable aperture, and preferably at least two resealable apertures, which allows substances to be introduced into, or withdrawn from, the culture chamber (e.g., when aligned with an aperture of the inner frame). The outer frame is a rigid housing shaped to accommodate the insertion and attachment of the inner frame in assembling the cell culture apparatus of the present invention. The inner frame also has at least one aperture, and preferably at least two apertures. Preferably, each aperture of the inner frame is aligned with a resealable aperture of the outer frame in forming aligned apertures. Thus, for example, each aperture of the outer frame may serve as a passageway into which is guided a needle of an instrument for introducing a substance into or withdrawing a substance from the culture chamber. The needle is guided through the aligned (and resealable) apertures of the outer frame and inner frame, and introduced into the culture chamber. In a preferred embodiment, the frame is of sufficient thickness and the apertures are of a sufficient limiting diameter t
Benesch Friedlander Coplan & Aronoff LLP
BioCrystal Ltd.
Miller Raymond A.
Redding David A.
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