JP2002539783A - Cell culture device and method for culturing cells - Google Patents

Abstract

Kind Code: A1 A cell culture device and a method for culturing cells. Provided are a method for culturing cells and, optionally, a magnetic field for cells desired to be cultured. A cell culture device (12) for performing a selective separation. The cell culture device (12) is preferably gas-permeable, preferably each with a frame (18), securely sealed to the frame (18) in a non-leakage seal, and between them. Two membranes (31, 32) forming a culture chamber (40) and at least one resealable opening to allow the introduction or removal of substances into or from said culture chamber (40). (23).

Description

DETAILED DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION [0001] The present invention relates generally to devices and methods for growing cells or tissue cultures in vitro. More particularly, the present invention allows for rapid and uniform gas transfer between the environment of cells contained within the cell culture vessel device and the atmosphere of the incubator in which the cell culture device is kept warm. And a cell culture device comprising at least one gas permeable membrane.

BACKGROUND OF THE INVENTION In eukaryotic cell culture systems, the culture of cells is generally under controlled conditions of pH, temperature, humidity, osmolality, ionic concentration, and gas exchange. Regarding the last, oxygen and carbon dioxide (CO ₂ ) are of particular importance for cell culture. In a general eukaryotic cell culture system, an incubator is provided in which CO ₂ is introduced so as to maintain an atmosphere of about 5% of CO ₂ in the incubator. The CO ₂ in order to maintain the pH at physiological levels, tissue culture, particularly interacts with the buffering system. Conventional cell culture vessels include tissue culture flasks, tissue culture bottles, and tissue culture plates. The inlet of CO ₂ from the incubator atmosphere to the tissue culture plate generally protrudes and loosely fits over the plate to remove particulate contaminants from entering the plate chamber (
Adaptable cover, but allows gas exchange between the incubator atmosphere and the atmosphere in the tissue culture plate. Similarly, for tissue culture flasks or bottles, a loosely fitting cap removes particulate contaminants from entering the flask or bottle chamber, but between the incubator atmosphere and the atmosphere within the flask or bottle. Allow gas exchange. More recently, the cap has been provided with a gas permeable membrane or filter, thereby allowing gas exchange by the tight fitting cap.

In addition to CO ₂ , cell culture depends on the ability to supply the cells with a sufficient amount of oxygen required for cellular respiration and metabolic functions. The supply of oxygen for cell respiration in a conventional cell culture vessel is performed in a header space of the vessel above the surface of the tissue culture medium, for example, a hollow space in the vessel. Efforts to increase the oxygen concentration in the cells to be cultured include mechanical agitation, sparging or aeration of the medium, increasing the local pressure of oxygen, and / or increasing the atmospheric pressure. Therefore, in conventional cell culture vessels, any volume or surface provided for gas exchange, which is proportional to the volume or surface of the entire vessel, may be used inefficiently and / or Limiting the rate of gas exchange or reducing the gas balance. This means that the rates of cell growth, cell density, and total cell number
Due to space, surface area, and gas exchange limitations, often low, small scale cultures (
At 15 ml or less).

[0004] The rate of gas exchange across gas permeable membranes has been described as "improved." However, gas permeable membranes have been described as not preferred for use in cell culture systems for a variety of reasons. For example, US Pat.
No. 3,228, an oxygen toxic boundary layer forms at the interface between the gas permeable membrane and the tissue culture medium; and further, cells that enter the toxic boundary layer may be irreparably damaged. Has been taught. Further, in US Pat. No. 5,707,869, the surface chemistry of gas-permeable, liquid-impermeable materials is incompatible with many cell types; It is taught that due to their nature, such substances can lead to the depletion of soluble growth factors.

Thus, cell culture devices can provide increased surface area for gas exchange as compared to conventional cell culture vessels; and achieve higher cell densities in a relatively short period of time. There is a need to provide high rates of cell growth, and even distribution of adhesion-dependent cells along the attachment surface.

SUMMARY OF THE INVENTION The present invention relates to at least one frame; two thin membranes, at least one of said membranes being gas permeable, and said membrane forming a culture chamber with respect to said frame. Being securely sealed (as a non-leakage seal); and at least one resealable opening that allows material to be introduced into or withdrawn from the culture chamber through the frame. And a cell culture device comprising:

In one preferred embodiment, the cell culture device comprises a frame in which two gas permeable membranes form and extend over and are securely sealed with a culture chamber therebetween. . The frame is sufficiently rigid to provide a housing for assembling the cell culture device of the present invention. The membrane is of a suitable thickness to provide sufficient gas permeability to accommodate cell growth in the chamber and to provide sufficient structural integrity to handle the device. In addition, the membrane may have sufficient optical clarity and transparency to observe the cell culture (eg, the color of the cell culture medium; and cell growth and morphology as can be observed by microscopy). Consists of clarity. The frame has at least one resealable opening, and preferably at least two resealable openings, allowing the substance to be introduced into or withdrawn from the cell chamber. Each opening defines a frame in which a portion of an instrument (eg, a needle or pipette or pipette tip) through which material is introduced into or withdrawn from the culture chamber is guided. It has an opening through it. In a preferred embodiment, to prevent the part of the device from piercing any of the walls formed by the membrane of the culture chamber when inserted through the resealable opening of the frame. In addition, the frame is of sufficient thickness and the opening is of a well-defined diameter.

[0008] In another preferred embodiment, the cell culture device further comprises an additional frame for accommodating the cell culture device formed by the one frame and the two gas-permeable membranes. In this embodiment, the cell culture device comprises a two-piece frame consisting of an inner frame and an outer frame. The inner frame securely seals therein at least one and preferably two gas permeable membranes, forming a culture chamber therebetween. The membrane is of a suitable thickness to provide sufficient structural integrity to handle the device. In addition, the membrane can be used to control the cell culture (eg, the color of the cell culture medium; and cell characteristics such as cell growth and morphology as can be observed under a microscope).
Consists of sufficient optical clarity and clarity to be observed. The outer frame includes at least one resealable opening that allows material to be introduced into or withdrawn from the cell chamber (eg, when aligned with the opening in the inner frame); And preferably has at least two resealable openings. The outer frame is a rigid housing formed to assemble the cell culture device of the present invention to accommodate insertion and mounting of the inner frame. The inner frame also has at least one, and preferably at least two, openings. Preferably, each opening of the inner frame is aligned with a resealable opening of the outer frame to form an aligned opening. Thus, for example, each opening of the outer frame serves as a passage in which the needle of an instrument for introducing or withdrawing material from the culture chamber into the culture chamber is guided. The needle is guided through the aligned (and resealable) openings in the outer and inner frames and is introduced into the culture chamber. In a preferred embodiment, the frame comprises a membrane wall of a culture chamber when a needle is inserted through both the resealable opening of the outer frame and the opening of the inner frame aligned therewith. The opening is of sufficient thickness and the opening is of a sufficiently limited diameter to prevent puncturing of any of the portions.

In any of the embodiments (one-piece frame structure or two-piece frame structure), the cell culture device is gas-exchanged and equilibrated and cultured in the device as compared with a conventional cell culture device. Additional clarity and clarity for observing cell oxidation, cell culture and cell properties during culture, mounting surfaces and conditions that facilitate even the distribution of attachment-dependent cells, space efficiency, versatility, and comparison Conditions that can achieve high cell densities in short periods of time and promote high rates of cell growth,
Provide unexpected combinations of features, including:

The above and other objects, features, and advantages of the present invention are described in the following detailed description of the invention, in which reference numerals are used to refer to the same or similar parts throughout the several illustrated drawings and embodiments. It will become apparent when read in connection with the accompanying drawings shown.

DETAILED DESCRIPTION OF THE INVENTION Definitions The term "gas permeable membrane", as will be described in more detail herein, is here, for the purposes of the description and claims, of cell culture. Microbial contamination that may permit the transfer of gas into and out of the chamber and (eg, the pore size is small enough to exclude the passage of microorganisms commonly encountered in cell culture contamination) And observing the cell culture (eg, of the color of the tissue culture medium containing the pH indicator; and of the characteristics of the cultured cells, such as cell growth and morphology as detectable by light microscopy). It is used to mean a liquid-impermeable biocompatible material that has optical clarity and clarity to allow. The thickness of the gas permeable membrane will depend on the desired resulting properties, including but not limited to structural integrity, degree of gas permeability, and gas transfer rate. Generally, the thickness of the gas permeable membrane will be from less than about 0.00125 inches to about 0.4 inches.
It can range up to 005 inches. In a preferred embodiment, the thickness of the membrane is in a range from about 0.0125 inches to about 0.0025 inches. The gas permeable membrane may consist of one or more membranes known in the art. The membrane generally comprises a suitable polymer, including polystyrene, polyethylene, polycarbonate, polyolefin, ethylene vinyl acetate, polypropylene, polysulfone, polytetrafluoroethylene, or a silicone copolymer. Selection of the composition of the gas permeable membrane depends on the type of cells to be cultured (eg, cells that grow attached (adhesion dependent), cells that grow in suspension and suspension (adhesion independent), and Cells that will grow or become suspended or suspended), the degree of gas permeability, the rate of gas transfer, and the optical clarity and clarity. In a preferred embodiment, the gas permeable membrane is made of polystyrene. In a more preferred embodiment, the gas permeable membrane is treated against attachment-dependent cells on one side of the membrane that will serve as a surface for attachment-dependent cells in culture. It consists of polystyrene which has been treated by ionization to improve the adhesion of the film surface. The ionization of the film renders the treated film surface more hydrophilic and uses methods known in the art including plasma discharge, corona discharge, gas plasma discharge, ion bombardment, ionizing radiation, and high intensity UV light. Can be implemented using In a preferred embodiment for promoting attachment-independent cell growth, the gas permeable membrane is not treated by ionization.

The term “membrane” is used herein for the purposes of the description and claims to support the growth of cells that are cultured in the absence of a gas permeable membrane; or other source of gas exchange. Membrane ("non-permeable membrane") that can exchange gas enough to perform
) Is used to mean "Membrane" means a gas permeable membrane in combination with either another gas permeable membrane or a non-permeable membrane. "Impermeable membrane"
It is possible to exclude microbial contamination (eg, the pore size is small enough to exclude the passage of microorganisms commonly encountered in cell culture contamination).
And a liquid-permeable, biocompatible material that is optically transparent and clear to allow observation during the cell culture process. The choice of thickness and / or composition of the impermeable membrane will depend on the desired resulting properties, including but not limited to structural integrity. Generally, the thickness of the impermeable membrane is about 0.0
It can range from less than 0125 inches to about 0.009 inches. The non-permeable membrane may consist of one or more membranes known in the art. The impermeable membrane can be treated by ionization to improve adhesion to the adhesion-dependent cells on one side that will serve as a surface for attachment of the adhesion-dependent cells during culture.

The term “magnetic sheet” is used herein, for the purposes of the description and the claims, to refer to magnetic particles (or magnetic composites with one or more biological cells) disposed adjacent thereto. Particle) is used to mean at least one substantially flat sheet having a magnetic field of sufficient strength to attract and securely hold the particles in place. The magnetic sheet is substantially rigid (as will become more apparent from the examples below), wherein the releasably fixed frame can then be removed by pulling the frame from the magnetic sheet. (Including varying degrees of stiffness as is known to those skilled in the art); or pulling the magnetic sheet from a frame allows the magnetic sheet to be separated from a frame removably secured thereto. A flexible (highly flexible) magnetic sheet with sufficient flexibility to allow The magnetic sheet may be a linear and parallel alternating north and south pole magnetized material. Magnetic sheets include, but are not limited to, sheets of fine magnetic powder, such as barium ferrite, incorporated in a thermoplastic binder; sheets of plastic or vinyl material infused with ferromagnetic material; A sheet of synthetic resin material embedded therein; magnetic particles embedded in a polymer sheet, typically 0.7 mm or 0.030 inches thick; vinyl material containing a magnetic substance dispersed therethrough; or Including other suitable materials having properties compatible with their intended purpose.
As will be apparent to those skilled in the art, the thickness of the magnetic sheet is not limited, but may be desired, such as the composition of the sheet material, whether the magnetic sheet comprises one or more sheets. Will vary depending on factors including magnetic field strength and pole spacing. In that regard, and for purposes of explanation, but not limitation, the magnetic sheet may range from about 0.2 mm to about 5 mm. An illustrative example of a magnetic sheet that can be purchased commercially and that is useful for making magnetic separation devices according to the present invention is found in Ohio, Marietta, Ohio.
io, Marietta) Magnetic Specialty Company (Magneti)
c Specialty, Inc. ) From the trademark "Promag (PROMAG)" (
Strontium ferrite-based material); and Electrodyne (Ele) of Batavia, Ohio.
ctrodyne Co. , Inc. ) A bonding material consisting of neodymium, iron and boron can be obtained. Commercially available examples of magnetic sheets have a magnetic field strength as measured by a Gauss meter, including but not limited to about 150 to about 600 Gauss.

The term “tissue culture medium” is used herein for the purposes of the description and claims to refer to sufficient nutrients (eg, vitamins, amino acids, essential nutrients, salts, and the like) and living cells ( Or a solution used to provide properties (eg, osmolality, buffering) to maintain and support viable cells in tissues. Commercially available tissue culture media are known to those skilled in the art.

Example 1 This example describes various embodiments of a cell culture device according to the present invention. However, in all embodiments of the cell culture device according to the invention described here in this and the following examples, an essential feature is that the culture chamber formed therein has at least one gas permeable Is composed of a conductive film. Now, referring to FIGS. 1 and 2, in one embodiment, the cell culture device 12 includes a frame 18. Frame 18 is made of a suitable plastic, thermoplastic, synthetic or natural material that can be fabricated into a framework structure, thereby achieving the structural integrity required for its intended purpose. It will consist of a basic biocompatible composition that can be included. It should be apparent to those skilled in the art that wide selection latitude can be exercised in selecting suitable materials for forming and / or manufacturing the frame 18. The dimensions of the cell culture device 12, and the frame 18, include, but are not limited to, the desired liquid volume of the culture chamber formed therein, including the dimensions of the culture chamber.
It will depend on one or more factors. In a preferred embodiment, the cell culture device 12 has a generally rectangular shape to allow it to be fitted and held in a substantially proper position by a standard mechanical stage sample holder for the microscope. No. In a more preferred embodiment, cell culture device 12 (and frame 18) has a length ranging from about 10 cm to about 13.5 cm, a width ranging from about 7 cm to about 9 cm, and about 0.2 cm to about 9 cm. 2
. A height in the range of up to 0 cm; or sufficient to be fitted and held substantially in place by the standard mechanical stage sample holder of the microscope (eg, containing a 96 well microtiter plate) Has dimensions. In a further preferred embodiment, cell culture device 12 has a length of about 12.7 cm, a width of about 8.5 cm, and a height of about 0.5 cm; using a conventional cell culture vessel, Provided is a cell culture device that allows culturing cells in an incubator space that is less than required for culturing cells at a growing rate or corresponding cell density.

Referring to FIGS. 1-7, frame 18 accommodates alignment, contact and fixation (by non-leakage sealing) of membranes 31 and 32 thereto when assembling cell culture device 12 of the present invention. This is a housing having a shape to be formed.
At least one of the two membranes 31 and 32 is a gas permeable membrane; and in a preferred embodiment, both membranes 31 and 32 are gas permeable membranes. Membrane 31 and 32 will be secured to frame 18 with non-leakage sealing using means that may include mechanical, chemical or other suitable means. For example, a clamping mechanism, which is a mechanical means such as a non-permanent locking means, can be used to secure the membranes 31 and 32 to the frame 18 forming a non-leakage seal.
In other examples, chemical means such as the use of an adhesive (including a bonding agent) may be used to secure the membranes 31 and 32 to the frame 18 in a non-leakage seal. The adhesive may be a double-sided adhesive tape, a polymer adhesive, an acrylic pressure-sensitive adhesive, a hot melt adhesive, a rubber cement, or any other form of adhesive or bonding agent useful for the purposes associated with the present invention. May also be in the form of Other suitable means include one or more of heat bonding, sonic welding, a press-fit sealing to form a non-leakage seal, and a molding process, wherein the membrane is an integral part of the frame. And more. For example, in the use of an adhesive, the adhesive may be such that in the process of assembling the cell culture device according to the present invention, a portion of the membrane extending on the frame contacts the adhesive on the surface of the frame. In addition, a non-leakable seal is added between the frame and the membrane that extends over the frame (see, eg, FIG. 3) and is secured to the frame in the formation of the culture chamber, resulting in a non-leakable seal. Is applied in such a way as to cause a force along the horizontal axis of the relevant part of the membrane fixed to the frame.

As will be apparent to those skilled in the art from the description herein, one or more membranes (or portions thereof) are cultured in a culture chamber of a cell culture device according to the present invention. It can be removed from the frame 18 to facilitate further manipulation of the cells. In one embodiment, where the attachment-dependent cells are cultured and both membranes 31 and 32 are fixed to the frame 18, the membrane to which the attachment-dependent cells are attached dissociates it from the frame. Or, it is detached from the frame by peeling. Alternatively, a membrane that does not serve as an attachment surface for the attachment-dependent cells is cut or stripped from the frame, leaving the membrane to which the attachment-dependent cells adhere to remains fixed to the frame. It is. In each case, the attachment-dependent cells attached to the membrane are then directly colored using standard dyes or colorants and methods for coloring known to those skilled in the art. For example, the attached cells can be analyzed by fluorescence microscopy, phase contrast microscopy, Nomarsky contrast microscopy, electronic scanning microscopy, and associated imaging (eg,
(Photo or digital imaging).
Alternatively, the attached cells will be gently scraped from the membrane in situations where it is desired to harvest cells from the membrane without enzymatic treatment (eg, trypsinization). Such a situation requires that the trypsin-sensitive cell surface molecule be intact; for example, further analysis directly in flow cytometry analysis or in a functional bioassay (eg, cytotoxicity assay) is required. If so, further use or analysis of the harvested cells may be included. As illustrated in FIG. 2, the frame 18 may further have a plurality of cut cuts 88 that may facilitate further manipulation of cells cultured in the cell device according to the invention. These cuts are cut along the cut cuts of the frame having a membrane fixed thereon and having attachment-dependent cells adhered thereon, and the membrane cut along the plane of the cut cuts, resulting in As such, by allowing more than one piece of frame to which a strip of membrane is attached, it allows the formation of multiple strips of the slide glass type for microscopic observation.

As shown in FIGS. 2 to 6, the frame 18 has at least one opening 2
3, and preferably at least two openings. Here, as already described, the opening 23 of the frame 18 serves as a passage through which instruments for introducing or drawing out or discharging substances from the culture chamber are guided. Will be. Therefore, the diameter of each opening 23 is
The relevant part of the instrument required for its intended purpose to introduce or withdraw material from the culture chamber or to remove material from it (for reference purposes only; )). As will be apparent to those skilled in the art, the diameter of each opening 23 will depend on the type of instrument used for the intended purpose, and the size of the tip of the instrument. For example, if the device is a syringe and needle assembly, the diameter of each opening 23 will be sufficient to allow a standard needle to pass therethrough (eg, about 1 mm to about 2 mm in diameter). The tip of the needle is guided through the opening 23 of the frame 18 and is introduced into the culture chamber 40. In a preferred embodiment, the frame is of sufficient thickness, and one or more openings 23, when inserted through the openings 23, form the tip of the device by the membranes 31 and 32. Culture chamber 40
Of a diameter that is sufficiently limited to prevent contact with and puncture any of the walls of the wall. The at least one opening 23 is resealable by any suitable means known in the art; for example, a cup, plug, or other suitable means. Referring to FIG. 6, and in one preferred embodiment, the opening 23 is substantially filled and sealed with a material comprising a gasket 24 that is sufficiently flexible to self-seale, and the "resealable opening" Section "23" is formed, thereby allowing re-sealing after needle entry and needle withdrawal. Such materials are known to those of skill in the art, and are not limited to one or more rubbers, silicones, silicone rubbers or other elastomeric materials compatible with the intended purpose. May be included. In another embodiment, the opening 23 allows for entry by a needle and resealing after needle withdrawal to prevent leakage from the opening 23 to the outside of the culture chamber 40 of the cell culture device 12. A resealable opening 23 is formed, partially filled and sealed by a gasket.

Now, turning to the membrane forming the wall of the culture chamber in the cell culture device according to the invention, the use of a gas-permeable membrane as an attachment surface in the culture chamber of the cell culture device according to the invention, It is observed that during culture, attachment-dependent cells tend to grow relatively uniformly over the entire attachment surface (including the edges of the chamber). The absence of significant diversity in cell attachment and growth across the attachment surface is due to the gas (e.g., e.g., It is believed to be due to the relative absence of diversity in (oxygen and carbon dioxide) exchange. Therefore, in the cell culture device according to the present invention, the gas permeable membrane is higher,
It provides an attachment surface that aids in the density of attachment-dependent cells, and therefore space efficiency.
This is a consequence of conventional cell culture vessels reported to show heterogeneous gas exchange on individual attachment surfaces; and, therefore, cells for growing depending on their spatial relationship to the gradient of gas exchange. Is advantageous for the diversity in their abilities.

As previously described herein, the membrane will be securely sealed to the frame by a method that provides a non-leakage seal. In a preferred embodiment, and referring now to FIG. 3, a suitable adhesive 56 is provided between the membrane 31 and the surface of the frame 18 and between the membrane 32 and the surface of the frame 18 (opposite the frame 18). Are interdispersed and cured between. Adhesives are known to those skilled in the art as polymeric adhesives, pressure-sensitive acrylic adhesives, hot melt adhesives, rubber cements, solvent-based bonding agents, or adhesives useful for the purposes accompanying this invention. It is known, including any other forms. From the description contained herein, as will be apparent to those skilled in the art, the distance between the membrane 31 and the membrane 32 depends on the size (eg, height) of the frame 18. Although there are no general relative restrictions on either the shape or size of the culture chamber 40, in a preferred embodiment for culturing to achieve a high cell density, the average distance between the membranes 31 and 32 is , About 1
mm to about 20 mm. In a more preferred embodiment, the average distance between membrane 31 and membrane 32 is about 4 mm.

In a preferred embodiment, and with reference to FIG. 3, when the cell culture device 12 is incubated in a standard CO ₂ incubator, the membrane 31
A gas permeable membrane located on the side of the frame 18 and securely sealed so that the cell culture device is closest to the incubator shelf on which it is placed. In this preferred embodiment, the membrane 31 will physically or wholly physically contact the incubator shelf. In such a position, and when the attachment-dependent cells are being cultured in a cell culture device, the membrane 31 serves as a surface to which said attachment-dependent cells will attach,
It is optimally useful for uniform gas exchange. In a further preferred embodiment, membranes 31 and 32 are gas permeable membranes. The advantage of having both membranes 31 and 32, consisting of gas permeable membranes, as will be disclosed herein in more detail, is that they are relatively uniform in the culture medium and useful for cells to be cultured. Gas exchange and equilibrium. In addition, both gas-permeable membranes simultaneously serve as surfaces for the attachment of attachment-dependent cells cultured in the cell culture device according to the invention. That is, as shown in FIG. 6, the adhesion-dependent cells are introduced into the cell culture device, and the introduced cells are sufficient to allow the cells to settle due to gravity and adhesion to the gas-permeable membrane 31. Time (for example, depending on the cell type,
(Minimum 60 minutes to 4 hours). The cell culture device will then be rotated 180 ° so that it is guided to allow additional attachment-dependent cells to settle by gravity and attachment to the gas permeable membrane 32. .

In another alternative, the adherence-dependent cells are first introduced to allow them to settle by gravity and adherence to the gas-permeable membrane 32; and then the cell culture device is added To allow the adherent dependent cells to settle due to gravity and adherence to the gas permeable membrane 31.
You may make it rotate by 80 degrees. In yet another alternative, the adhesion-dependent cells are introduced into a cell culture device, and the introduced cells are subjected to gravity and some cells adhere to the inner surface of the first gas-permeable membrane. Sufficient time to allow colonization by (eg, a minimum of 60 minutes to 4 minutes, depending on the cell type).
And the cell culture device is settled by the unattached attachment-dependent cells by gravity and attachment to the inner surface of the gas-permeable membrane opposite the first gas-permeable membrane. Rotate 180 ° to allow cells to grow on both membranes forming the walls of the cell culture chamber. Thus, by rotating the cell culture device according to the present invention to allow both membranes 31 and 32 to simultaneously serve as a surface for attachment-dependent cell attachment, space efficiency, It provides versatility and conditions that can achieve high cell densities within a relatively short period of time to facilitate high rates of cell growth. In an additional embodiment, in which only one membrane is used as the surface for attachment, the attachment-dependent cells are introduced into a cell culture device, and the introduced cells are subjected to gravity and gas permeable membranes. The cells are incubated for a sufficient time (depending on the cell type) to allow them to settle by attachment of the cells to 31. The cell culture device is suspended from the membrane 31 in culture, so that the translocated cells are grown therefrom.
Rotated by 80 °.

Both membranes 31 and 32 can be viewed during culture to allow observation, such as of the color of the tissue culture medium, and cell characteristics (eg, cell growth and morphology as by microscopy). Consists of sufficient optical clarity and clarity. More specifically, whether the cells are arranged and / or from the culture of a cell culture device into the incubator to generate a gas, the diffusion of CO ₂ into the tissue culture medium with a pH indicator, pH indicator culture (Generally changing from an initial dark pink to a reddish orange to an orangeish color). Membranes 31 and 32 consist of sufficient optical clarity and clarity to observe the color change of the pH indicator of the culture medium during culture. Additionally,
Both membranes 31 and 32 can be used to determine the cell shape, cell number, cell culture, in which the cultured cells are generally observed by light microscopy when the cell culture device is placed on a microscope stage for analysis. And sufficient optical clarity and clarity so that they can be analyzed visually for additional cellular properties.

In an alternative embodiment of the cell culture device according to the present invention, there are multiple culture chambers. In a further preferred alternative embodiment of the cell culture device having multiple culture chambers, the number of culture chambers ranges between two and ten. Now, with reference to FIGS. 2, 4 and 5, what is provided is:
FIG. 4 is a top view of a frame 18 having one culture chamber, two culture chambers, and four culture chambers formed therein, respectively. In a cell culture device having a large number of culture chambers, one membrane 31 and one membrane 32
It extends over the opposing surface of the frame 18 and is securely sealed, and when forming multiple culture chambers, along any portion of the frame 18 it provides a division of the frame for the formation of multiple culture chambers. Will. Each culture chamber is formed by its own membrane 31 and membrane 32; however, it is also practiced to require the frame 18 to have multiple membranes 31 and multiple membranes 32 sealed therein. Each cell culture chamber has at least one opening 23
And, in a more preferred embodiment, having two openings 23 is also shown in FIGS. As previously described herein, desirably, each opening 23 is resealable. Referring again to FIG. 4, if there are two openings 23 per culture chamber, a first opening 23 located at one end of the culture chamber and an opposite end of the culture chamber. It is desirable to have the second opening 23. Such an arrangement may facilitate a process that provides a vent to the chamber and removes air bubbles from the chamber. For illustrative purposes, but not limitation, and where:
The cell culture device 12 is about 12.7 cm long, about 8.5 cm wide, and about 0.5 cm long.
Having a height of 4 cm, each of the two chambers illustrated in FIG. 4 holds about 2.5 to about 7.5 ml of tissue culture medium; and each of the four chambers illustrated in FIG. About 1.25 to about 3.75 ml of tissue culture medium.

In an additional embodiment, the bioreactor is a plurality of cell cultures according to the present invention linked to operate properly by providing a liquid passage connecting the culture chambers of the plurality of cell culture devices. Equipment. To facilitate culturing cells in such a bioreactor environment, each of the plurality of cell culture devices directs the flow of tissue culture media through the passageway and between the culture chambers of the interconnected cell culture devices. It may be interconnected to other cell culture devices by means of a connecting passage which allows. One example of the interconnection of a first cell culture device to a second cell culture device to form a bioreactor is through the use of one piece of sterile tubing inserted between two syringe needles. One needle has a resealable opening 23
Into the culture chamber of the first cell culture device; and a second needle is inserted into the resealable opening 23, through the frame, and into the second cell. It reaches the inside of the culture chamber of the culture device. The one-piece tubing then has one open end operatively connected to the first needle, and the opposite open end of the tubing is operatively connected to the first needle. Would be connected to two needles. The tubing disposed between the two needles and connected thereto for proper operation is provided between the culture chamber of the first cell culture device and the culture chamber of the second cell culture device. Provide liquid distribution contact. Using similar methodology and multiple tubing, multiple cell culture devices are operatively connected to form a bioreactor.

Example 2 In this example, another embodiment of the cell culture device according to the present invention is described. In this embodiment, and with reference to FIGS. 8 and 9, the cell culture device 12 further includes an outer frame 16 for forming a two-piece frame consisting of the outer frame 16 and the frame 18. Various embodiments of the frame 18 and membranes 31 and 32 have been previously described in detail in Example 1 and FIG.
From FIG. 7 to FIG. The outer frame 16 may be fabricated into a framework structure, thereby achieving a suitable plastic, thermoplastic, synthetic, or natural, achieving the required structural integrity for its intended purpose. Would consist of a basic biocompatible composition that could have the following materials: The dimensions of the cell culture device 12 and the outer frames 16 and 18 may be one or more, including, but not limited to, the desired liquid volume of the culture chamber formed therein, and the dimensions of the culture chamber. It will depend on more factors. In a preferred embodiment, the cell culture device 12 has a generally rectangular shape to allow it to be fitted and held in a substantially proper position by a standard mechanical stage sample holder for the microscope. No. In a further preferred embodiment, the cell culture device 12 (and the outer frame 16) is about 10 cm
A length ranging from about 7 cm to about 13.5 cm, a width ranging from about 7 cm to about 9 cm,
And a height ranging from about 0.5 cm to about 2.0 cm; or a standard mechanical stage sample holder for the microscope (e.g., containing a 96-well microtiter plate) and substantially suitable It has dimensions sufficient to be held in position. In a more preferred embodiment, cell culture device 12 (and outer frame 16) has a length of about 12.7 cm, a width of about 8.5 cm, and a height of about 1 cm; A cell culture device is provided that allows for culturing cells at a corresponding growth rate or corresponding cell density with less incubator space than required for culturing cells. In a more preferred embodiment, the frame 18 has a length in the range of about 4 cm to about 11.5 cm, a width in the range of about 4 cm to about 7.5 cm, and about 0.2 cm to about 0.8 cm. With a height in the range up to. In a most preferred embodiment,
Frame 18 has a length of about 11 cm, a width of about 6 cm, and a height of about 0.3 cm.

Referring now to FIGS. 9-12, the outer frame 16 is provided with a frame 18 (with membranes 31 and 32 therein) for assembly of the cell culture device 12 of the present invention.
Is a rigid housing formed to accommodate alignment, contact and locking of the (non-leakage, fixed). The fixing of the frame 18 to the outer frame 16 is such that at least the opening 23 of the frame 18 is aligned with the openings 21 and 23, at least one of the aligned openings being resealable. It is required that a non-leakage seal be formed at a position where it abuts and is aligned with the opening 21 of the outer frame 16 in order to form the seal. In one embodiment, frame 18 (where membranes 31 and 32 are sealed and secured) is fitted into a recess in outer frame 16 (eg, FIG. 9).
And then the frame 18 and the outer frame 16 are permanently integrated by sonic welding, bonding, gluing, or other suitable means known in the art for this intended purpose. Fixed to The integral securing of the outer frame 16 and the frame 18 includes contacting and securing the outer frame 16 to the frame 18; the outer frame 16 may be secured to a frame (eg, a membrane 31 or Contacting and fixing any of them); and combinations thereof.

In another embodiment, frame 18 (where membranes 31 and 32 are securely sealed) is fitted into a recess in outer frame 16 (see, eg, FIG. 9) and frame 18 Is then detachably fixed to the outer frame 16 in a non-permanent manner. Means for releasably securing frame 18 to outer frame 16 may include mechanical means, chemical means, or other suitable means. For example, snap fitting, pressure fitting, non-permanent locking means, clamping mechanism, or tongue and groove fitting arrangement,
Mechanical means such as can be used to form a non-leakage seal and removably secure frame 18 to outer frame 16. In another example, chemical means, such as the use of a non-permanent adhesive or non-permanent bonding agent, form a non-leakable seal to removably secure frame 18 to outer frame 16. Can be used. Non-permanent adhesive or non-permanent bonding agent, double-sided adhesive tape,
Polymer adhesive, acrylic pressure-sensitive adhesive, hot melt adhesive, rubber cement,
Alternatively, it may be in the form of any other form of adhesive or bonding agent useful for the purposes associated with the present invention. Removably securing the outer frame 16 and the frame 18 integrally includes contacting and securing the outer frame 16 to the frame 18; , Contacting and fixing to the membrane 31 or the membrane 32); and combinations thereof. In another embodiment in which the frame 18 is detachably fixed to the outer frame 16, the outer frame 1
6 is one membrane (membrane 3) in the recess into which the frame 18 will eventually fit.
Either 1 or 32) is secured by non-leakage sealing. The outer frame 16, having the membrane so attached, is contacted and fitted to the frame 18 with the second membrane attached thereto by non-leakage sealing, so as to form a culture chamber therefrom, And secured, where the second membrane is on the side of the frame 18 opposite to which the outer frame 16 is placed, contacted and secured. Frame 18 is recessed in the outer frame in a manner to be releasably secured.
In the process of assembling the cell culture device according to the present invention, the pressure causes a downward force along the horizontal axis of the outer frame, and consequently the outer frame and the frame 18 Is applied to cause non-leakage sealing and formation of a culture chamber. External frame 16
Removably securing the frame 18 with the frame 18 includes contacting and securing the outer frame 16 to the frame 18; and contacting and securing the membrane securely sealed to the outer frame 16 to the frame 18. And combinations thereof.

As will be apparent to those skilled in the art from the description herein, frame 18
Removably securing to the outer frame 16 will facilitate further manipulation of the cells to be cultured in the culture chamber of the cell culture device according to the present invention. In one embodiment in which the adhesion-dependent cells are cultured and both membranes 31 and 32 are secured to frame 18, frame 18 may be removed from outer frame 16. The membrane to which the adhesion-dependent cells are attached,
It is detached from the inner frame by cutting or stripping it from the inner frame. Alternatively, a membrane that does not serve as an attachment surface for the attachment-dependent cells may be cut out of the frame 18 while leaving the membrane to which the attachment-dependent cells adhere to remains fixed to the frame 18 or Stripped off. In each case, the attachment-dependent cells attached to the membrane are then directly colored using standard dyes or colorants and methods for coloring known to those skilled in the art; or else If removed (as described in detail above in Example 1). Similarly, in an assembly where one membrane is fixed to the outer frame, the other membrane is fixed to the frame 18, and the outer frame and the frame 18 are detachably fixed to form a cell culture device. After the cells to be cultured in the culture device are cultured, the frame 18 may be removed from the outer frame. With either membrane (or both) to which the cultured cells are attached, the attached cells are further manipulated thereon (eg,
It can be stained, observed, imaged, harvested by gentle stripping, used in functional assays, etc.) and used as a culture medium (whether still attached to or separated from each frame). In further embodiments, the outer frame, frame 18, or a combination thereof, may further have a cut cut across each frame. The cut cuts may include the removal of the outer frame of the cells cultured in the cell device according to the present invention from the frame 18; facilitating and manipulating the access of the membrane to which the attachment-dependent cells have been attached; And / or facilitating removal of cultured cells (eg, either adherence-dependent or adherence-independent); and attaching the frame having a membrane immobilized thereon and having adherence-dependent cells adhered thereon. Slides for microscopy by dividing along the cut cuts and cutting the membrane along the plane of the cut cuts, resulting in more than one piece of frame having a strip of membrane attached thereto Further operations may be facilitated, including one or more of the formation of multiple strips of the glass type.

As shown in FIGS. 8 and 10, the outer frame 16 has at least one opening 21 and preferably at least two openings. At least one opening 21 of outer frame 16 is aligned with at least one opening 23 of frame 18 to form an aligned opening, and at least one opening of the aligned opening is Resealable. Thus, for example, the opening 21 in the outer frame would serve as a passage through which a needle for introducing or withdrawing material from or withdrawing material from the culture chamber is guided. Therefore, the diameter of each of the openings 21 and 23 is sufficient to allow a standard needle to pass therethrough (eg, about 1 mm in diameter).
From about 2 mm). The needle has an aligned opening (of the outer frame 16) 2
1 and 23 (of the frame 18) and the culture chamber 40
Introduced within. In a preferred embodiment, the frame is of sufficient thickness, and one or more openings 21 and 23, when inserted through aligned openings 21 and 23, Membranes 31 and 3
2 has a diameter that is sufficiently restricted to prevent it from contacting and piercing any of the walls of the culture chamber 40 formed by the culture chamber 2. Referring to FIG. 10, in one preferred embodiment, the opening 21 is substantially filled and sealed with a material comprising a gasket 24 that is sufficiently flexible for self-sealing, and the "resealable opening" 21 thereby permitting re-seal after needle entry and needle withdrawal. Such materials are known to those of skill in the art, and are not limited to one or more rubbers, silicones, silicone rubbers or other elastomeric materials compatible with the intended purpose. May be included. In another embodiment, the opening 21 is
Forming a resealable opening 21 that allows re-seal after entry and withdrawal of the needle so as to prevent leakage from the opening 21 to the outside of the culture chamber 40 of the cell culture device 12, Partially filled and sealed by a gasket. In a further embodiment, the aligned openings 21 and 2
At 3, a resealable opening 21 and a resealable opening 23 are formed to prevent leakage from the aligned openings 21 and 23 of the cell culture device 12 to allow for entry by a needle and Opening 21 is filled and sealed with gasket 24 and opening 23 is partially or substantially filled, in a manner that allows for resealing after needle withdrawal.

In a more preferred embodiment, and with reference to FIGS. 8 and 9, the inclined portion 14 of the outer frame 16 is inclined about 35 ° with respect to the plane of the at least one gas permeable membrane. . This angulation or tilting of the portion 14 uses the cell culture device 12 and, when the adherence surfaces and conditions are to be observed, the adherence dependence over the entire surface of the membrane to which the cells are attached in the culture chamber 40. It is advantageous to promote a uniform distribution of the cells. Therefore, for full microscopic observation of the cells in the cell culture device growing on the edge of the cell chamber 40, the standard microscope objective is tilted about 35 ° with respect to the mechanical stage at the tip. As such, portion 14 allows the user to position a standard microscope objective in a manner that allows the user to fully view such cells. In that regard, and as described in more detail herein in Example 1, the use of a gas permeable membrane as an attachment surface in a culture chamber of a cell culture device according to the present invention generally results in cell attachment. It produces an unexpected lack of significant variability and growth over the entire attachment surface. In a preferred embodiment for culturing to achieve a high density of cells, although there is no general restriction on the height of the culture chamber, the average distance between membranes 31 and 32 is (ie,
The membranes are spaced a distance apart), ranging from about 1 mm to about 5 mm. In a more preferred embodiment, the average distance between membranes 31 and 32 is about 3
mm.

In a preferred embodiment, and with reference to FIG. 10, the membrane 31 does not physically contact the shelf of the incubator, but when the cell culture device 12 is incubated in a standard CO ₂ incubator. , A portion of the outer frame 16 physically contacts the shelf of the incubator (ie, there is a space between the membrane 31 and the shelf of the incubator). This arrangement also allows one or more cell culture devices to be stacked while substantially maintaining space to allow gas exchange over the width and length of each respective membrane 31. In a more preferred embodiment, both membranes 31 and 32 are gas permeable membranes having advantages as described in more detail above in Example 1. In an alternative embodiment of the two-frame configuration of the cell culture device according to the invention, and as described in more detail above in Example 1, the frame 18 comprises
It may consist of multiple culture chambers. In additional embodiments, where the cell culture device may include one or more culture chambers, the outer frame may include one or more frames 18 (e.g., a series of grooves) (with each frame including at least one culture chamber). Or by a recess); and the cell culture device is configured with an outer frame in which one or more frames 18 are housed and secured therein. Thus, the cell culture device will serve as a bioreactor with multiple culture chambers. The plurality of culture chambers may be provided by using sterile tubing inserted between two syringe needles, as described in more detail in Example 1 above, to allow for the flow of tissue culture media between each other. As such, they may be interconnected. For example, 1
One needle is inserted into the aligned openings 21 and 23 and leads into the culture chamber of the first cell culture device consisting of the frame 18; and the second needle is inserted into the aligned openings 21 and 23 and into the culture chamber of the second cell culture device consisting of the frame 18. The tubing located between the two needles and connected to them to work properly is the first frame 1
Liquid communication is provided between the culture chambers 8 and the culture chambers of the second frame 18. Using similar methodology and multiple tubing, multiple frames 18 are operatively connected to form a bioreactor.

Example 3 In this example, whether the cells are individual cells (cells grown independently of forming tissue-like structures; an illustrative example is a cell line) ,
Or cells that form a tissue (generally a mesh or network of cells that have their intercellular material to form a structured or organized tissue), or a combination thereof. Various embodiments have been described that use a cell culture device according to the present invention for culturing cells. Individual cells that can be cultured in a cell culture device include, but are not limited to, animal cells, insect cells, mammalian cells, human cells, transgenic cells, genetically engineered cells, transformed cells, cell lines It will be apparent to one of skill in the art that the cell has one or more cell types, including, adhesion-dependent cells, and adhesion-independent cells. It will also be apparent to one skilled in the art that tissues formed by cells in culture can also be cultured in a cell culture device according to the present invention. The cell culture devices of the present invention generally provide a specific type of cells to be cultured, as long as the culture medium provides sufficient nutrients and properties (eg, osmotic pressure) to maintain and support cell growth. It will be further apparent to those skilled in the art that the invention is not limited to tissue culture media capable of maintaining cell growth.

In one embodiment, the adhesion-dependent cells are cultured in a cell culture device according to the present invention. For the attachment-dependent cells to grow, at least one membrane of the culture chamber will be attached to the cells and have sufficient hydrophilicity to promote the adhesion of the cells desired to be cultured. And therefore has an inner surface that can serve as a surface to which cells can attach and grow. The at least one membrane may have a unique chemical property that promotes such adhesion, or the inner surface may promote such adhesion (eg, electrically, ionically (Chemically or chemically). Wherein such treatment may include one or more of chemical etching of the inner surface, ionizing the inner surface, or coating the inner surface with a suitable coating reactant; It will be apparent to those skilled in the art. Suitable coating reactants are known to those skilled in the art, including gelatin, collagen, fibronectin, sticky proteins (sticky proteins), sticky peptides (see, eg, PCT International Application No. PCT / US95 / 00817).

In the method of culturing cells using the cell culture device according to the present invention, the cells are cultured so as to form a suspension in an appropriate amount of tissue culture medium to support cell growth. Suspending the cells to be deposited (eg, attachment-dependent or attachment-independent cells); and in an instrument (eg, a syringe and needle combination) for injecting the suspension into the cell culture device. Introducing said suspension into said cell culture chamber of said cell culture device using sterile (sterile) techniques and through a resealable opening; Inserting a needle tip) (or, for a cell culture device, further comprising an outer frame, through a set of aligned openings; for example, a resealable opening 21 and a correspondingly aligned opening. Discharging the suspension from the device (eg, from a syringe chamber and through the tip of a needle) and into the cell culture chamber; and removing the suspension from the cell culture device. Withdrawing a portion (eg, the tip of a needle); a cell culture incubator (an incubator that provides sufficient conditions for cell growth as is known in the art; including, for example, appropriate temperatures and CO ₂ concentrations) Keeping the cell culture device containing the suspension of culture medium and cells in an environment close to controlled atmosphere conditions).

It is noted here that the step of introducing cells and culture medium into the cell culture device according to the invention can be performed in a sterile environment or in a non-sterile environment where it is provided that aseptic techniques are used. You. This is because the cell culture device itself can provide a sterile, sealed environment (when sterilized using conventional means known in the art). For the aseptic approach, and in an exemplary embodiment, the gasket 24 is first wiped with alcohol, and then the sterile needle 61 of the syringe 60 (containing the suspension of cells to be cultured) is opened. (Eg, as shown in FIG. 6) or a series of aligned openings (eg, FIG. 1)
(As shown at 0), with the resulting introduction of the needle 61 into the culture chamber 40. The cell suspension will then be expelled from the syringe 40, through the needle 61 and into the culture chamber 40. Needle 61
It is then withdrawn from the cell culture device 12 and upon withdrawal, the perforations resulting from the needle 61 are spontaneously sealed by the gasket 24 to prevent leakage from the cell culture chamber 40.

There are a number of ways to introduce one or more substances into the culture chamber of a cell culture device. Materials that will be introduced into the culture chamber of the cell culture device include tissue culture media alone, tissue culture media containing cells, cells suspended in physiological buffers, and drugs or cytokins or growth factors or enzymes (eg, ,
It will be apparent to those skilled in the art that it will include one or more of (trypsin solution) or other biological reactants for treating cells cultured therein. Depending on factors such as the amount of material introduced into the cell culture chamber, and the size of the opening, it will often be necessary to provide a drainage of the culture chamber. Therefore, in the method for culturing cells in the cell culture, the step of introducing the cell suspension may further include a draining step. Evacuation is a process that allows air or gas to be withdrawn from the culture chamber when the substance is introduced into the culture chamber. Evacuation would be required to relieve pressure on the membrane of the cell culture chamber due to the injection of air during the process of introducing the substance into the culture chamber. In one embodiment, the same resealable opening 23 is used both to introduce material in the culture chamber and to allow air or gas to be withdrawn from the culture chamber. In one embodiment, a portion of the material is introduced into the culture chamber, and a syringe and needle containing the remaining material to be introduced are withdrawn from the cell culture chamber. The cell culture device is then tilted or rotated until air or gas bubbles (collectively “bubbles”) are located near the same opening 23. A second syringe and needle, the syringe being empty, is inserted into the same opening 23 with the needle tip inside the bubble; and the syringe plunger is then ejected from the culture chamber. Pull back to remove bubbles from the culture chamber in the process. The second syringe and needle are then removed from the opening 23 and the first syringe and needle containing the remaining substance to be introduced introduces the remainder of the substance into the culture chamber. Is reinserted into and through the same opening. As will be apparent to those skilled in the art, and depending on the amount of material to be introduced or whether bubbles are desired or on the size of the culture chamber, the first syringe and needle and the second syringe and needle There may be multiple exchanges between

In one alternative embodiment, one or more to introduce a substance into the culture chamber and to allow air or gas or a solution (eg, culture medium) to be displaced from the culture chamber. Opening is used. In embodiments using one or more openings, a needle connected to a syringe containing the substance to be introduced into the culture chamber is inserted into the first opening 23. The first
A second needle, which is a needle only (not connected to a syringe), is inserted into the second opening 23 which provides access to the same culture chamber as the opening 23 of the second needle. . The cell culture device uses a syringe and a needle inserted through the first opening, and when the substance is slowly introduced into the culture chamber, in the process of discharging the culture chamber, the cell opening is inserted into the second opening. It is rotated or tilted in such a way that gas or air is exhausted through the inserted second needle. After the substance has been introduced into the culture chamber, the syringe and needle are
May be removed from the second opening and the second needle may be removed from the second opening. The process of draining may include connecting the first opening to the second opening, such as at the opposite end of the cell culture chamber and cell culture device, as illustrated in FIGS. 2, 4 and 8. Can be facilitated by being spaced from the opening of the

[0039] 1 present in the culture medium of cells to be cultured in the cell culture device according to the present invention.
In one embodiment including a method for collecting one or more substances, having a controlled delivery device (eg, a drip control as in an iv bag for intravenous application) and A needle connected to a bag or similar container containing fresh tissue culture medium to be introduced into the culture chamber is inserted into the first opening. A second needle is inserted into the second opening, providing access to the same culture chamber as the first opening. The second needle may be connected to one end of a one-piece tubing, and the opposite end of the tubing is introduced into or connected to a collection container. Therefore, as fresh tissue culture medium is drip into the culture chamber of the cell culture device, it is withdrawn through the second opening and collected from the culture chamber of the cell culture device, A culture medium from cultured cells containing cell products released during culture (eg, a method for harvesting monoclonal antibodies from hybridoma cells or a method for harvesting recombinant protein from transfected cells) At).

In a preferred embodiment, and for applications where a high growth rate of cells is preferred, in a method of culturing cells in a cell culture device according to the present invention, a cell culture chamber is desired to be grown. It is completely filled with cell culture media or other suitable growth media for culturing cells or tissues. It will be apparent to those skilled in the art that the culture chamber means will be completely filled without leaving substantially any headspace in the culture chamber. It may be possible to steepen the initial rise in the pH of the tissue culture medium within the first 1-2 hours in conventional cell culture vessels, mainly due to the distribution of dissolved CO ₂ between the culture medium and the gas phase. Attention. The pH change, cells sufficiently earlier than is possible to generate significant amounts of CO _2, resulting primarily within the first hour of incubation.
The resulting change in pH, by the cells, to provide a CO ₂ through the cell respiration,
Negatively affects the rate of cell growth until it is sufficient to restore optimal CO ₂ levels. However, unlike the conventional cell culture vessels described above, the cell culture device according to the invention can be filled in such a way that no substantial headspace is created. In addition, the cell culture device of the present invention provides an unexpected capacity for gas balance that is believed to result from the expanded gas exchange exhibited by the use and arrangement of one or more gas permeable membranes. provide. These features of the cell culture device according to the present invention cooperate to prevent sharp changes in the initial pH as observed in conventional cell culture vessels. By preventing such abrupt changes in the pH of the culture medium, the initial culture conditions achieve a pH equilibrium that further aids in optimal cell growth. Therefore, contrary to the cell culture device according to the present invention, a disadvantage of the conventional cell culture vessel is that the conventional cell culture vessel creates an upper space between the top of the vessel and the surface of the tissue culture medium. It is necessary. In addition, in the method of culturing cells in a cell culture device according to the present invention, the culture chamber is filled in a manner that there is no substantial head space, and the cell culture device is capable of detecting foam formation. Alternatively, it is tilted or gently vibrated to mix the cells with the culture medium contained therein without causing cell trauma.

In an additional embodiment of a method of culturing cells using a cell culture device according to the present invention, the method comprises the steps of: providing a suitable amount of tissue culture medium to support cell growth;
Forming a suspension to suspend the adherence-dependent cells to be cultured; injecting the suspension into the cell culture device through one or more resealable openings Introducing said suspension into a suitable instrument to perform said method using sterile (sterile) techniques and through one or more resealable openings, and said cells of said cell culture device. Inserting the tip of the instrument into a culture chamber; passing the tip of the instrument and releasing the suspension into the cell culture chamber; and the tip of the instrument from the cell culture device. Withdrawing;
The cell culture device containing the suspension of culture medium and cells is allowed to settle for a time sufficient to allow the cells to settle by gravity and contact and adhesion to the membrane provided as the attachment surface ( For example, 30 minutes to 3 hours depending on the cell type). The method then comprises rotating the cell culture device 180 ° and positioning the cell culture device in the incubator so that the cells are kept warm in a translocated manner in the culture chamber. That is, the cells are attached to a membrane that serves as the upper wall of the culture chamber for an incubator shelf on which the cell culture device is mounted. Therefore, in this embodiment, the cells are suspended in the culture chamber and are grown suspended along the surface of the upper membrane. This causes cell debris to drop from the suspended culture to the lower membrane by gravity,
There, such separated cell debris will be easily removed from the culture chamber by a suction and / or washing step.

In an exemplary embodiment of culturing cells in a cell culture device according to the present invention, a cell growth rate (measured by doubling time) is calculated. In this example, in each of two tubes, 1 × 10 ⁶ cells of the same mammalian cell line were filled with carboxyfluorescein diacetate succinimidyl ester stain (10 μM final concentration “CFSE”). 20 ml of the same tissue culture medium contained was mixed. CFSE is a stain that becomes internally bound by the treated cells. The cells are allowed to incubate at 37 ° C. for 2 hours. The CFSE-treated cells from one tube are introduced into the culture chamber of the cell culture device according to the invention, where the cells grow,
Cells are cultured along a wall consisting of a gas permeable membrane having a total surface area of 50 cm ² . The CFSE-treated cells from the second tube are introduced into a conventional tissue culture flask having a total surface area of 75 cm ² on which the cells grow. The cell culture device and properly drained tissue culture flask are then 5% C
Incubate together at 37 ° C. in the same culture incubator with O ₂ . As shown in Table 1, the cell culture devices and tissue culture flasks were periodically and temporarily removed from the incubator, placed under a fluorescence microscope, and the amount of fluorescence intensity of individual cultured cells. Is weighed. Since the amount of dye per cell decreases in direct proportion to the division of the cells in culture, the doubling time of the cells was measured when the fluorescence intensity of the cells to be cultured was at the beginning of the culture ("0 hours") It is expressed by the time at which a value indicating 50% of the value of the fluorescence intensity is reached. As shown in Table 1, and as calculated from the fluorescence intensity ("FI"), the doubling time of the cells cultured in the cell culture device ("Device A") according to the invention is 12 hours. In contrast, the cell doubling time in a conventional tissue culture flask ("Device B") was 20 hours. In addition, the results in Table 1 show that at least during the first 36 hours of culture, the growth rate of cells cultured in the cell culture device ("Device A") according to the present invention, It shows that the growth rate of cells cultured in culture flasks ("Device B") is unexpectedly and significantly greater. (Table 1) Culture time FI of cells in device A FI of cells in device B 0 40 40 6 32 38 12 20 30 18 18 15 22 24 11 17 30 5 15 36 0 11 42 0 8 480 0 4 54 0 260 0 Example 4 The cell culture device may be used for magnetic separation applications. In this example, various embodiments are described in which the cell culture device further comprises a magnetic sheet detachably secured thereto. In one embodiment,
1, FIG. 7, FIG. 9, FIG. 11, and FIG.
Is selected from the group consisting of: the magnetic sheet is in close proximity to or in contact with substantially all of the surface of the membrane 31 (all or a portion of the membrane forming the wall of the culture chamber 40). Cell culture device 12
It extends upwardly and is detachably fixed to it. The term "close proximity" will become more apparent from the following description, as the magnetic sheet is held in position along the membrane 31 in the process of magnetic separation by the magnetic attraction of magnetic particles. Means sufficiently close (without physical contact) to the membrane 31 that the magnetic field is strong enough to act. As will be apparent to those skilled in the art, such extreme proximity will depend on factors including, but not limited to, the size of the magnetic particles used in the separation process, and the thickness of the membrane 31. There will be. In the magnetic sheet, the magnetic particles disposed in the culture chamber 40 are attracted by the magnetic field strength of the detachably fixed magnetic sheet, and the magnetic particles are placed in a proper position (the inner wall of the culture chamber 40 including the inner surface of the membrane 31). Along) will be releasably secured to the cell culture device. For example, the magnetic sheet 51 detachably secured to the cell culture device 12 may include one or more fasteners, one or more fasteners, as will be more apparent to those skilled in the art from the following description. Clamps, non-permanent adhesives, sealed vacuum fasteners, and ferromagnetic materials to which the magnetic sheet can be coupled by magnetic attraction, combinations thereof, or other suitable materials having properties consistent with its intended purpose. And will be achieved by means selected from the group consisting of:

In a preferred embodiment, the magnetic sheet 51 has a shape of substantially the same dimensions as the membrane placed in contact with it, and the magnetic sheet 51 is formed by using a non-permanent adhesive to Fixed detachably. The magnetic sheet is detachably secured along and to a portion of the cell culture device selected from the group consisting of a frame bottom, a membrane bottom, and combinations thereof. The non-permanent adhesive is "removable" with sufficiently low tack to allow the magnetic sheet to be removed from contact with a cell culture device that is releasably secured.
Adhesive. That is, the non-permanent adhesive prevents the magnetic sheet from being detached from the cell culture device without substantially damaging any surface of the cell culture device or the magnetic sheet when they are separated from each other. An adhesive of suitable peel strength that is acceptable. In a more preferred embodiment, the adhesive additionally has an initial and adequate cohesive strength that controls and suppresses the substantial transfer of cohesive residue, especially to surfaces other than the laminated surface. (The latter may include one or more frames, a membrane in close proximity to the magnetic sheet, or a magnetic sheet). In that regard, and in a preferred embodiment, the non-permanent adhesive is substantially free of the adhesive, or any residue thereof, from interfering with the optical clarity of the membrane of the cell culture device to which it is detachably secured. It is applied to the magnetic sheet so as to avoid a temporary transition. The non-permanent adhesive is a double-sided adhesive tape, a polymer adhesive, a pressure-sensitive acrylic adhesive,
It may be in the form of a hot melt adhesive, rubber cement, or any other form of adhesive for said purpose with the present invention. It is known in the art that double-sided pressure-sensitive adhesive tapes have an adhesive on both sides of the film or carrier (carrier), and the film or carrier functions as a support on which the adhesive is applied. In one embodiment, the non-permanent adhesive allows the magnetic sheet to be removed from the cell culture device; and, optionally, if desired, subsequent to the removal, the magnetic sheet can be attached to the cell culture device. "Replaceable" adhesive that allows it to be repositioned against and re-attached in a form that is releasably secured by the application of light pressure to the cell culture device and / or magnetic sheet Consists of The repositionable adhesive can be repeatedly adhered to and removed from the substrate without substantial loss of tackiness (for a discussion of such adhesives, see, for example, US Pat. , 663,241). An illustrative example of a high performance pressure sensitive adhesive useful in the present invention is "MACtac, Inc." of Stow, Ohio, from "MACtac, Inc."
MACbond IB-3628 "; and commercially available from Adhesives Research, Inc. under the trade name AR-7840. In an alternative embodiment, frame 18 and / or outer frame 16 comprises molded plastic with ferromagnetic material embedded therein. The ferromagnetic material may have particles, one or more filaments, one or more wires, and the like. For purposes of illustration, frame 18 (or outer frame 16 as illustrated in FIG. 12) is made of molded plastic with ferromagnetic material embedded therein, and The magnetic sheet 51 can be detachably fixed to the cell culture device by magnetic attraction to the embedded particles.

In a method of magnetic separation using a detachably fixed cell culture device, and here using the method described in the previous example, it is introduced into the culture chamber: (a A) a liquid comprising a mixed population of cells having therein a population of cells of interest (eg, a cell type desired to be sequestered from a liquid comprising a mixed population of cell types); and (b) coated with a ligand A magnetic separation reactant having magnetic particles, wherein the ligand has binding specificity and affinity for the cells desired to be separated and for achieving magnetic separation.
After mixing the contents for a time sufficient for the contact and binding interaction between the magnetic separation reactant and the cell of interest to occur, the magnetic separation reactant forms a composite through the ligand coating. Contact and bind target cells present in the fluid. It will be apparent to those skilled in the art that the fluid comprising the mixed population of cells, and the magnetic separation reactants, are first mixed and then the mixture is introduced into the cell culture device to perform the magnetic separation. In either embodiment, the formed composition is attracted (by magnetic attraction) and along the plane of the cell culture device to which the magnetic sheet is detachably secured, the membrane 31 Contact the wall of the culture chamber 40 including the inner surface of the culture chamber. The magnetic sheet 51 has a magnetic field strength sufficient to hold the composite (and any magnetic separation reactants not bound to cells) in place along the membrane 31. As noted earlier herein, it should be noted that physical contact between the membrane 31 and the magnetic sheet 51 is not required for the magnetic sheet 51 to hold the composite in place along the membrane 31. Is done. Further, in embodiments where the magnetic sheet 51 and membrane 31 are not in physical contact before the fluid, cells and magnetic separation reactant are added to the cell culture device 12, such a magnetic material may be added to the culture chamber 40 when added to the culture chamber 40. The components may be of sufficient weight to depress membrane 31 into contact with magnetic sheet 51.

After allowing the composition to remain in place along the membrane 31 for a sufficient period of time, the fluid is removed from the cell culture device. In the negative selection method, the removed fluid is used because a specific cell population is depleted.
In the process of positive selection, the fluid removed is retained in the mixed cell population because the cell type desired to be separated is held in place by magnetic attraction and as a composite in the culture chamber. Contains substantially all (unnecessary) residues of In this positive selection process, the culture chamber 40 is used to remove the unbound cells from the target cells via magnetic attraction as part of a compound with the magnetic separation reactant. It will be washed with a solution (eg, tissue culture medium or physiological solution). In one embodiment where it is desired to culture positively selected cells, an appropriate amount and type of tissue culture medium for the cell type is introduced there into a culture chamber; Removed from the culture device (e.g., by a pull or pull operation), thereby removing the magnetic force holding the compound in place in the culture chamber, and thereby releasing the compound into the culture medium; The cell culture device is then placed in a cell culture incubator and kept warm. The cell culture device 12 and the magnetic sheet 51
Note that both are generally rectangular. However, in one embodiment, the cell culture device facilitates removal of the magnetic sheet 51 from the cell culture device 12 so as to facilitate gripping of the protruding edge of the magnetic sheet 51. It has one of the four corners of the frame separation.

In order to explain the method of magnetic separation using the cell culture apparatus (which is further provided with a magnetic sheet detachably attached thereto), a 1.5 × 10 ⁶ T47-D (
A mixture of relatively slowly growing human breast cancer cell lines) and a cell mixture of 1.5 × 10 ⁶ Jurkat cells (human T cell lines) were used. Commercially available magnetic reactants, including magnetic beads with an anti-epithelial cell marker antibody (BER-EP4) applied thereto, provide an approximate ratio of 6 beads per cell in a small tube in an approximate total volume of 500 μl. 3 with the cell mixture and stirring
Mix for 0 minutes. The mixture with cells and magnetic reactants is then:
A magnetic sheet having a magnetic field strength of 525 gauss is introduced into the cell culture device containing 5 ml of the tissue culture medium in a mixed manner and then detached to the cell culture device along the membrane forming the bottom wall of the culture chamber. Fixed possible. The cell culture device with the magnetic sheet detachably secured thereto was placed on a rotating platform for 10 minutes, and then the tissue culture medium was removed from the cell culture device. The removed tissue culture medium, and its cell contents, were placed in a separation vessel and were the result of a negative selection process. Although a washing step could optionally be performed within the positive selection process, no washing step was performed in this example. Rather, the magnetic sheet is removed from the cell culture device, 20 ml of tissue culture medium is added to and mixed with the positively selected cells in the cell culture device, and then the cell culture device is exposed to 5% CO _2. 3 in a week in an incubator
It was kept at 7 ° C. After one week of culture, adherence-dependent cells (collected by trypsinization) and adherence-independent cells in the culture are collected and labeled with anti-CD labeled with phycoerythrin (to detect Jurkat cells).
Stained with anti-epithelial cell marker antibody labeled with antibody 45 and with fluorescein isothiocyanate (to detect T47-D cells) and analyzed and quantified by flow cytometry. The amount of each cell type detected in the culture was then expressed as a percentage of the total cell count of the culture. As shown in Table 2, in the positive selection process, three independent experiments using the cell culture device further provided with a magnetic sheet releasably secured thereto resulted in a positive selection efficiency greater than 99%. Showed sex. Optionally, in culture of positively selected cells, the magnetic beads may be removed first prior to culture, and in this example, the positively selected cells are cultured Note that it was still bound to the beads at the beginning of the reaction. (Table 2)-Positive selection Experiment number Cell count Jurkat cells T47-D cells 13.9 × 10 ⁶ 0.25% 99.55% 2 5.4 × 10 ⁶ 0.40% 99.30% 3 5.1 × 10 ⁶ 0.20% 99.65% Cells isolated by negative selection Was also cultured for one week, harvested, and stained and analyzed for flow cytometry using the methods described herein. The amount of each cell type detected in the culture was then expressed as a percentage of the total cell count of the culture. As shown in Table 3, in a negative selection process, three independent experiments with the cell culture device further comprising a magnetic sheet releasably secured thereto were about 95% or higher than 95% The effectiveness of the negative selection was shown. (Table 3)-Negative selection Experiment number Cell count Jurkat cells T47-D cells 11.8 × 10 ⁶ 95.0% 5.0% 2 7.0 × 10 ⁶ 98.5% 1.5% 3 2.3 × 10 ⁶ 94.9% 2.6% Example 5 According to the invention Cell culture devices may also be used in applications where recombinant DNA molecules have been introduced into cells to be cultured. In this embodiment, an effective amount of the vector is used so that the vector comes into contact with the cells to be cultured contained in the culture chamber of the cell culture device (for introduction into the cells to be cultured). The use of a cell culture device in which a vector has been introduced into cells by being introduced into the cell is described. A method for introducing a substance into a cell culture device is described in Example 3 herein.
May be used to introduce a vector into a cell culture device, as described in. As will be apparent to those skilled in the art, the vector may include a plasmid vector, a viral vector, an expression vector, or a combination thereof. As will be apparent to one of skill in the art, the vector includes a recombinant DNA molecule comprising the vector linked to affect (eg, to a promoter) the desired DNA molecule to be expressed by the cell containing the vector. You may go out. As is known to those skilled in the art of molecular biology, animal cells (and more particularly mammalian cells) or plant cells use expression vectors to facilitate expression of the desired gene into gene production in the cell. It is a common cell into which such a recombinant DNA molecule is desired to be introduced.

For example, cultured cells housed in a cell culture device may be incubated with a mixture containing a certain amount of recombinant DNA molecules desired to be introduced into the cells, and a transfection reaction. Good. In an illustrative example, a commercially available plasmid encoding a green fluorescent protein ("pTracer") is introduced by transfection into cells cultured in a cell culture device. Was. In this embodiment, the cell culture device comprises:
Seed with 1 × 10 ⁶ T47-D cells and allowed to grow to approximately 50-60% fusion to the attachment surface. In a small tube, 500 μg of plasmid DNA was mixed with 900 μl of serum-free tissue culture medium. In another tube, 120 μl of the commercially available lipid-containing, transfection reaction was mixed with 600 μl of tissue culture medium. After a 30 minute incubation at room temperature, the contents of the two tubes are mixed and the mixture is
It was kept warm for 0 minutes. The mixture was further mixed with 18 ml of tissue culture medium.
After the tissue culture medium has been removed from the cell culture device and the cultured cells have been washed once with the culture medium, a mixture of the tissue culture medium, the transfection reaction product and the vector is introduced through the opening, and the cultured cells are introduced. Is released into the culture chamber such that it contacts the adherent support. The cell culture device then allows the contact between the cultured cells and the mixture (comprising the tissue culture medium, the transfection reaction and the vector) to determine the uptake and introduction of the vector by the cultured cells. Incubated at 37 ° C. overnight in 5% CO ₂ to facilitate. The tissue culture medium was then removed from the cell culture device and replaced with a tissue culture medium lacking phenol red as a pH indicator. The cell culture device was then mounted on the stage of a UV microscope and the cultured cells were analyzed and weighed for the fluorescence produced by the described green fluorescent protein. The number of cells positive for the green fluorescence and the total number of cells in culture were weighed to determine the percentage of cells successfully transfected ("transfection efficiency"). The resulting transfection efficiency was about 20%. As will be apparent to one of skill in the art, the transfection efficiency is not limited, but is optimized for the type of cells in which it is desired to introduce the vector, the conditions for transfection. (Eg, the amount of DNA per cell number, the culture medium used for transfection, the time of the transfection process), and factors that include the type and amount of transfection reaction used, and Will do. It will also be apparent to one of skill in the art that using similar steps, vectors may also be introduced into attachment-dependent cells cultured in the cell culture device according to the present invention.

The foregoing description of certain embodiments of the invention has been described in detail for purposes of explanation. In view of the description and illustration, other skilled in the art will readily adapt the present invention to adapt the present invention for various applications without departing from the basic concept. And / or adaptations, and such modifications and / or adaptations are intended to be within the meaning and scope of the appended claims.

[Brief description of the drawings]

FIG. 1 is a side view of one embodiment of a cell culture device according to the present invention.

FIG. 2 is a top view of one embodiment of the cell culture device according to the present invention.

FIG. 3 is a partial cross section obtained through a 4-4 cut of FIG. 2;

FIG. 4 is a top view of another embodiment of the cell culture device according to the present invention.

FIG. 5 is a top view of one further embodiment of the cell culture device of the present invention.

FIG. 6 is a partial cross-sectional view of a cell culture device showing one embodiment in which a sample is introduced or withdrawn from a culture chamber.

FIG. 7 is a partial cross-sectional view of the cell culture device showing one embodiment in which a magnetic sheet is detachably fixed to a contact portion with respect to the cell culture device.

FIG. 8 is a top view of another embodiment of the cell culture device according to the present invention.

9 is a side view taken along the line 2-2 in FIG. 8;

FIG. 10 is a partial cross-sectional view of a cell culture device showing one embodiment in which a sample is introduced or withdrawn from a culture chamber.

FIG. 11 is a partial cross-sectional view of the cell culture device showing one embodiment in which a metal frame is fixed to an outer frame and a magnetic sheet is held at a predetermined position by magnetic attraction to the metal frame.

FIG. 12 is a partial cross-sectional view of one embodiment of a cell culture device showing one embodiment in which the outer frame includes metal particles embedded therein and the magnetic sheet is held in place by magnetic attraction to the metal particles.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 5/10 C12N 15/00 A 15/09 5/00 B (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY) , KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, D E, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK , LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZWF term (reference) 4B024 AA20 CA01 DA02 EA04 GA11 GA18 HA13 4B029 AA02 AA11 AA12 AA21 AA23 BB11 CC01 CC02 CC11 DF05 DG064B AB01 BA01 BA25 BC03 BC07 BC21 BD14 CA60

Claims (40)

[Claims] 1. A frame comprising: a housing; and at least one opening having an opening through the frame, the at least one opening being resealable, comprising: a frame; and two membranes. At least one of the membranes is a gas permeable membrane that forms a culture chamber between the two membranes and the frame and is securely sealed to the frame as a non-leakage seal. And a cell culture device wherein the membrane has sufficient optical clarity and clarity to allow observation of the cell culture. 2. The cell culture device according to claim 1, wherein the frame has two or more resealable openings. 3. The first of the two membranes comprises a gas permeable membrane,
The cell culture device according to claim 1, wherein a second one of the two membranes includes a non-permeable membrane. 4. The method of claim 1, wherein each of the two membranes comprises a gas permeable membrane.
The cell culture device according to item 1. 5. The cell culture device according to claim 1, wherein the at least one gas-permeable membrane has a surface that has been treated to improve cell attachment. 6. The cell culture device according to claim 1, wherein the cells are selected from the group consisting of individual cells, cells forming a tissue, and a combination thereof. 7. The cell culture device according to claim 1, wherein the cell culture device has a substantially rectangular shape. 8. The cell culture device has a length in a range from about 10 centimeters (cm) to about 13.5 cm, a width in a range from about 7 cm to about 9 cm, and about 0.2 cm to 2 cm. 2. The cell culture device according to claim 1, having a height in the range of: 9. The cell culture device according to claim 8, wherein the two membranes are separated by a distance in a range from about 1 millimeter (mm) to about 5 mm. 10. The cell culture device according to claim 1, wherein the frame further has a cut cut used to divide the frame along the cut. 11. The cell culture device of claim 1, wherein each opening comprises a self-sealing gasket material forming a resealable opening. 12. The cell culture device of claim 1, wherein the adhesive is interdispersed and cured between each membrane and the frame to provide a non-leakage seal. 13. The method according to claim 13, further comprising a plurality of culture chambers formed between two membranes and the frame, wherein each of the plurality of culture chambers has at least one culture chamber.
The cell culture device of claim 1, accessible by two resealable openings. 14. The frame of claim 1, further comprising an outer frame having a housing in which the frame of claim 1 is fitted and fixed, wherein the outer frame forms an aligned opening. Having at least one opening aligned with the opening of the frame and being in close contact with the non-leakage seal, and at least one opening of the aligned opening has a resealable opening. The cell culture device according to claim 1. 15. The cell culture device according to claim 14, wherein the frame according to claim 1 is fitted and fixed in a concave portion of the external frame. 16. The cell culture device according to claim 14, wherein the outer frame has a slope portion inclined at an angle of 35 ° with respect to a plane of the at least one gas-permeable membrane. 17. The cell culture device further includes a magnetic sheet detachably fixed thereto, wherein the magnetic sheet extends over the cell culture device and attaches the magnetic sheet to the cell culture device. 2. The method according to claim 1, wherein the membrane is formed in a position selected from the group consisting of a group consisting of: The cell culture device according to any one of the preceding claims. 18. The magnetic sheet according to claim 1, wherein one or more fasteners, one or more fasteners,
By means selected from the group consisting of one or more clamps, a non-permanent adhesive, a sealed vacuum fastener, a ferromagnetic material for bonding said magnetic sheet via magnetic attraction, and combinations thereof. 18. The cell culture device according to claim 17, which is detachably fixed. 19. The cell culture according to claim 17, wherein the magnetic sheet is detachably fixed to a part of the cell culture device selected from the group consisting of the frame, the membrane, and a combination thereof. apparatus. 20. The cell culture device further comprises a magnetic sheet detachably fixed thereto, wherein the magnetic sheet extends over the cell culture device and attaches the magnetic sheet to the cell culture device. 15. The membrane forming the culture chamber of the above, is disposed in a position selected from the group consisting of a group consisting of a group consisting of: The cell culture device according to item 1. 21. The magnetic sheet according to claim 1, wherein the magnetic sheet comprises one or more fasteners,
By means selected from the group consisting of one or more clamps, a non-permanent adhesive, a sealed vacuum fastener, a ferromagnetic material for bonding said magnetic sheet via magnetic attraction, and combinations thereof. 21. The cell culture device according to claim 20, which is detachably fixed. 22. The cell culture device according to claim 20, wherein the magnetic sheet is detachably fixed to a part of the cell culture device selected from the group consisting of a frame, the membrane, and a combination thereof. . 23. A cell culture comprising a plurality of the cell culture devices according to claim 1, wherein a fluid passage is provided between the plurality of cell culture devices, and the fluid passage is connected by the fluid passages. Bioreactor that enables fluid flow transfer of the culture chamber of the device. 24. A method of culturing cells in the cell culture device of claim 1, comprising: (a) suspending cells in an appropriate amount of tissue culture medium to support cell growth. Forming a suspension to suspend the cells to be cultured; (b) introducing the suspension into an instrument for injecting the suspension into the cell culture device; (c) C.) Inserting a portion of said device through said resealable opening and into said cell culture chamber of said cell culture device; and, d) said suspension from said device and into said cell culture chamber. (E) withdrawing the part of the instrument from the cell culture device; and (f) storing the suspension of culture medium and cells in a cell culture incubator. To keep warm Law. 25. The method of claim 24, further comprising, after step (d), discharging to remove air released into the culture chamber during step (d). 26. The method according to claim 24, wherein the cells to be cultured are adhesion-dependent cells. 27. The method according to claim 24, wherein the cells to be cultured are adhesion-independent cells. 28. After the attachment-dependent cells adhere to the membrane of the cell culture device in step (f), the method comprises culturing the cells in a transposed manner in the culture chamber. 27. The method of claim 26, further comprising rotating the cell culture device 180 degrees. 29. After the attachment-dependent cells adhere to the membrane of the cell culture device in step (f), the method comprises: (g) rotating the cell culture device by 180 °; and (h) 27. The method of claim 26, further comprising repeating steps (c) through (f), wherein each of the two membranes provides an attachment surface on which cells are cultured.
The method described in. 30. A method for culturing cells in the cell culture device of claim 14, comprising: (a) suspending cells in a suitable amount of tissue culture medium to support cell growth. Forming a suspension to suspend the cells to be cultured; (b) introducing the suspension into an instrument for injecting the suspension into the cell culture device; (c) C.) Inserting the portion of the device through the aligned opening and into the cell culture chamber of the cell culture device; d) the suspension from the device and into the cell culture chamber. (E) withdrawing the part of the instrument from the cell culture device; and (f) storing the suspension of culture medium and cells in a cell culture incubator. To keep warm How. 31. The method of claim 30, further comprising, after step (d), evacuating to remove air released into the culture chamber during step (d). 32. The method of claim 30, wherein the cells to be cultured are adhesion-dependent cells. 33. The method according to claim 30, wherein the cells to be cultured are adhesion-independent cells. 34. After the attachment-dependent cells adhere to the membrane of the cell culture device in step (f), the method comprises culturing the cells in a transposed manner in the culture chamber. 33. The method of claim 32, further comprising rotating the cell culture device 180 degrees. 35. After the attachment-dependent cells adhere to the membrane of the cell culture device in step (f), the method comprises: (g) rotating the cell culture device by 180 °; and (h) 33. A further step comprising repeating steps (c) to (f), wherein each of said two membranes provides an attachment surface on which cells are cultured.
The method described in. 36. A magnetic separation method using the cell culture device according to claim 17, comprising: (a) a cell type population desired to be isolated by magnetic separation; A liquid containing a mixed population of cells, present with a magnetic separation reactant (reagent) having binding specificity for the cell type desired to be separated, is combined with the magnetic separation reactant and the Mixing so as to form a mixture consisting of a compound with a cell of a sexual type; and (b) mixing the mixture in the culture chamber of the cell culture device with a culture chamber formed by a membrane. Introducing the magnetic sheet along a releasably fixed plane such that the mixture comes into contact with the wall; and (c) allowing the composite to respond to the magnetic field strength of the magnetic sheet. Incubating the mixture in the cell culture device for a time sufficient to contact and retain a location along the wall of the culture chamber due to the suction of the composition. 37. The method further comprising removing the mixture remaining unconstrained in the culture chamber from the cell culture device, and wherein the removed mixture comprises:
37. The method of claim 36, comprising cells separated by a process of negative selection. 38. The cell of claim 37, wherein after the removal of the mixture, the cells having the compound confined in the culture chamber and remaining in the cell culture device include cells separated by a positive selection process. the method of. 39. The method of introducing a tissue culture medium into the culture chamber, removing the magnetic sheet from the cell culture device, and culturing cells separated by the positive selection process. 39. The method of claim 38, further comprising keeping the culture device warm. 40. A method for introducing a recombinant DNA molecule into cells cultured in the cell culture device according to claim 1, wherein the method comprises: (a) transfection reaction product (reagent); Mixing the recombinant DNA molecule with the vector so as to form a mixture containing the recombinant DNA molecule; and (b) mixing the mixture containing the recombinant DNA molecule into a culture medium containing the recombinant DNA molecule. Mixing with a culture medium, (c) introducing the culture medium containing the recombinant DNA molecule into the culture chamber so as to contact cells cultured in the cell culture device, and (d) the culture. Transferring the culture medium containing the recombinant DNA molecule in contact with the cells to be cultured into cells to be cultured in the culture chamber. To facilitate the introduction of recombinant DNA molecules, the method having to be kept warm.