Abstract:
A process for applying and storing adherent cells on a tissue culture plate  20  for preparing tissue culture plates for use in tissue culture experiments. In the process, adherent cells are removed from a flask using trypsin after being incubated in a CO 2  Incubator. The cell suspension is created using the adherent cells and a liquid culture medium, which is centrifuged and tested for volume using a hemacytometer. The cell suspension is placed in the wells of a tissue culture plate  20  and then incubated. A cryopreservative is deposited into each well and the tissue culture plate is sealed with an adhesive foil cover and cryogenically preserved using liquid nitrogen. The tissue culture plate  20  is then place into a plastic, waterproof, resealable bag enabling it to be stored at −80° C. for up to 3 months and shipped without damaging the morphological or functional nature of the adherent cells. The tissue culture plate forms part of a cryopreserved cell culture assembly which includes frozen adherent cells attached to the bottom wall surface of a well in the tissue culture plate.

Description:
[0001]    This application is a continuation-in-part of application Ser. No. 09/040,379, filed Mar. 18, 1998, which is herein incorporated by reference. 
     
    
     
       BACKGROUND  
         [0002]    1. Field of the Invention  
           [0003]    This invention relates to well slides and cell cultures, with the intent of improving the method of preparing cell cultures for various tissue culture experiments.  
           [0004]    2. Description of Prior Art  
           [0005]    Currently, adherent cell lines are grown on a variety of plastic containers such as flasks, Petri dishes, and multi-well tissue culture plates. Once these cell lines have reached confluency, they are treated with trypsin-containing solutions to release them from the plastic surface, counted, and either re-plated or frozen. When these cells are needed for culture, they are thawed and plated. These manipulations are time-consuming and statistically increase the chance for contamination. Numerous assays require the use of adherent cell lines at various stages of confluency and it can be difficult to have multiple plates, dishes or flasks at a particular stage of growth. At the present time, these types of cells are frozen in the non-adherent stage and require growing when thawed. Many adherent cell lines lose their particular characteristics after numerous passages.  
         SUMMARY OF THE INVENTION  
         [0006]    The present invention is directed to a cryopreserved cell culture assembly including a tissue culture plate having at least one well bounded by a bottom wall surface upon which cells can attach and grow, and a plurality of side walls forming a top rim, the rim defining a well opening. Adherent cells in a frozen state attach to the bottom wall surface. The cells contain an amount of a cryoprotectant which is effective to protect them from damage when stored frozen. In more specific aspects of the invention, the cells are animal cells, insect cells or mammalian cells, and in a still more specific aspect of the invention, the cells are receptive for infection by HIV-1, and even more specifically the receptive cells are HeLa-CD4-LTR-β-gal, HeLa-CD4-CCR5-LTR-β-gal, H9, C8166, Molt-4, Jurkat, CEMX174, HUT 78, or U87.CD4.  
           [0007]    The invention is also directed to a method of preparing a cryopreserved cell culture assembly, comprising contacting adherent cells attached to a bottom wall surface of a tissue culture plate with an amount of a cryoprotectant effective to protect said cells from damage when freezing, wherein the tissue culture plate has at least one well bounded by said bottom wall surface upon which cells can attach and grow, and a plurality of side walls forming a top rim, the rim defining a well opening. The tissue culture plate is sealed by placing a cover over said top rim and the adherent cells attached to said bottom wall surface are frozen  
           [0008]    The present invention provides plates of adherent cells which have been frozen at desired stages of confluency and which are ready for use in various assays, such as neutralization assays, and for feeder cell cultures, upon thawing. The present invention also relates to methods of preparing and assembling such plates. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0009]    [0009]FIG. 1 is a diagram flow chart of the preparation of the adherent cells and cell plates.  
         [0010]    [0010]FIG. 2 is a perspective view of a well section of a treated tissue culture plate prior to incubation.  
         [0011]    [0011]FIG. 3 is a perspective, sectional view of a well of a treated tissue culture plate after incubation.  
         [0012]    [0012]FIG. 4 shows well plate prior to submersion in liquid nitrogen.  
         [0013]    [0013]FIG. 5 shows a side view of a well treated with adherent cells after incubation.  
         [0014]    [0014]FIG. 6 shows a well plate, with an adhesive foil cover, after thawing.  
     
    
     DETAILED DESCRIPTION  
       [0015]    The following reference numerals are used:  
         [0016]    [0016] 02  adherent cells;  
         [0017]    [0017] 04  flask;  
         [0018]    [0018] 06  CO 2  incubator;  
         [0019]    [0019] 08  trypsin;  
         [0020]    [0020] 10  culture medium;  
         [0021]    [0021] 12  cell suspension;  
         [0022]    [0022] 14  centrifuge tube;  
         [0023]    [0023] 16  hemacytometer;  
         [0024]    [0024] 18  microscope;  
         [0025]    [0025] 20  tissue culture plate;  
         [0026]    [0026] 22  well;  
         [0027]    [0027] 24  cryogenic preservative;  
         [0028]    [0028] 26  liquid nitrogen;  
         [0029]    [0029] 28  adhesive foil cover;  
         [0030]    [0030] 30  plastic, waterproof, resealable bag; and  
         [0031]    [0031] 32  supernatant.  
         [0032]    This invention preferably utilizes adherent cells  02 . The cells can be of any origin, including insect, plant, mammal, etc. It is preferred that these cells adhereto the surface of the tissue culture receptacle. Useful cell lines include: F4, V9, F2, epithelial, fibroblasts, T cells, such as lymphoblastoid cell lines, e.g., HeLa-CD4-LTR-β-gal (MAGI); MAGI-CCR-5. See, e.g., et al.,  J. Virol ., 71:3932-3939, 1997. In addition, T cell lines, lymphoblastoid cell lines, H9, C8166, Molt, Molt-4, CEM, Jurkat, preferably, CEMX174, HUT 78, U87.CD4. See, e.g.,  Virology,  236:208-212, 1997.  
         [0033]    In a preferred embodiment, the present invention relates to a cryopreserved cell culture assembly, comprising: a tissue culture plate having at least one well bounded by a bottom wall surface upon which cells can attach and grow, and a plurality of side walls forming a top rim, said rim defining a well opening; adherent cells attached to said bottom wall surface, wherein said cells contain an amount of a cryoprotectant which is effective to protect them from damage when stored frozen, wherein said cells are in the frozen state.  
         [0034]    By the term “tissue culture plate,” it is meant any vessel or receptacle in which cells can be grown. For example, a tissue culture plate can be a flask, a petri dish, a six-well, 12-well, 24-well, or 96-well container. It can be constructed from any suitable material, including, plastics such as polystyrene or PETG. The bottom surface of such plates, e.g., the bottom surface of the well, can be treated to facilitate growth and attachment, i.e., “tissue-culture treated.” Any substrate upon which cells adhere can be used, according to the present invention, including but not limited to, polystyrene, polyamino-treated surfaces (such as polysine, etc.), organic and inorganic membranes, polycarbonate membrane inserts, glass, and treated culture plates, e.g., poly-d-lysine and polycarbonate-treated plates. In addition to plates, tissue culture bottles, carriers, and other such culture vessels can be used.  
         [0035]    The plate preferably contains at least one-well or receptacle, where the well or receptacle has a bottom surface and a plurality of sides (circular, rectangular, etc.). The sides form at rim at their top which can be raised or unraised. Such plates are commercially available. See, e.g., U.S. Pat. No. 4,012,288 or 5,795,775. Generally, the plate has a cover or lid which can be used to seal the plate around the rims of the well. See, e.g., FIG. 6. Such cover can be attached in any suitable manner, e.g., by adhesive. It can be removable or detachable/  
         [0036]    The assembly preferably comprises adherent cells, i.e., cells which stick to the surface of the well. The surface can be treated, as mentioned above, with any agent which facilitates or enhances cell adhesion to it. The adherent cells preferably contain an amount of a cryoprotectant which is effective to protect cells from damage during the freezing process, storage process, or thawing process, e.g., to prevent ice-crystal formation. Useful cryoprotectants include, e.g., dimethylsulfoxide, glycerol, propylene glycol, ethylene glycol, trehalose, raffinose, or hydroxyethyl starch. Effective amounts of cryoprotectants, and methods of freezing, are well-known in the art and can be determined routinely.  
         [0037]    The adherent cells  02  are grown in a flask  04  and incubated in a CO 2  incubator  06  at 37° C. with 5% CO 2  levels. The flask  04  is removed from the CO 2  incubator  06  and aspirated, and 5 ml of trypsin  08  are added and allowed to stand for 10 to 15 minutes. Then 5 ml of culture medium  10  are added to the flask  04 . Using a pipette, 10 ml are removed from the cell suspension  12 . The cell suspension  12  is placed into a 15 ml centrifuge tube  14 . The cell suspension  12  is then centrifuged for 7 minutes at 1000×g. The supernatant  32  is aspirated, at which time the cell pellets are located at the bottom of the centrifuge tube  14 . The pellets are re-suspended in 10 ml of the culture medium  10 . Then, using a Pasteur pipette, one droplet is removed from the cell suspension  12  and placed on a hemacytometer  16  and covered with a slip. The hemacytometer  16  is placed on a microscope  18  to count the cells. Using the cell count, the cell suspension  12  is adjusted to result in a cell count of 4×104 per 1 ml by diluting the cell suspension  12  with a culture medium  10 .  
         [0038]    A rigid polystyrene tissue culture plate  20  with a plurality of wells  22  is then treated in the following manner. With a pipette, 1 ml of the 4×10 4  cell suspension  12  is placed in each well  22  of a polystyrene tissue culture plate  20 . The entire surface of the bottom of the well is to be uniformly covered with the cell suspension  12 ; however, when using MAGI CCR-5 (HeLa-CD4-LTR-β-gal) cells, only 30% to 50% of the well  22  bottom is to be covered with the suspension. The tissue culture plate  20  holding the cell suspension  12  is then incubated for 1 hour in the CO 2  incubator  06 , with a culture medium containing 30% fetal calf serum, at 37° C. with 5% CO 2 , allowing the cells to grow.  
         [0039]    After incubation, the tissue culture plate  20  is subjected to a freezing procedure, wherein the temperature is strictly controlled at −160° C., as follows. A 100 μl solution of a cryogenic preservative  24  at 4° C. is pipetted into each well  22  of the tissue culture plate  20 . An adhesive foil cover  28  is sealed over the top of the tissue culture plate  20 , thus protecting the adherent cells  02 , ensuring moveability of the tissue culture plate  20 , and permitting movement of the liquid nitrogen  26  around each individual well  22 . The tissue culture plate  20  is set into a container holding enough liquid nitrogen  26 , at −160° C., to reach above the level of the adherent cells  02  but below the lip of each well  22  in the tissue culture plate  20  for 1 minute. Each tissue culture plate  20  is then placed in a plastic, waterproof, resealable bag  30  and sealed. The tissue culture plate  20  is stored at −80° C. where it can be maintained for 3 months.  
         [0040]    In this regard, FIG. 1 is a flow chart of the preparation of the adherent cells  02  and the tissue culture plate  20 . FIG. 2 illustrates the bottom of the well  22  treated with the 1 ml cell suspension  12  solution prior to incubation of the tissue culture plate  20 . FIG. 3 illustrates the bottom of the well  22  treated with the 1 ml cell suspension  12  solution after incubation of tissue culture plate  20 . FIG. 4 shows a tissue culture plate  20  prior to submersion in liquid nitrogen  26 . FIG. 5 shows a side view of a well  22  treated with the adherent cells  02 . FIG. 6 shows a tissue culture plate  20 , with a foil adhesive cover, after thawing.  
         [0041]    When the treated tissue culture plate  20  going to be used, it is sufficient to place it in water at 37° C. and allow it to stand for approximately 4 to 7 minutes. The cryogenic preservative  24  is aspirated from each well  22 , followed by a wash step with 500 μl of culture medium  10  per well  22 .  
         [0042]    The plates then can be used to serve for, but are not limited to, these laboratory functions: They can be used to run various assays, such as epithelial and neutralization assays, and may be used for feeder cell cultures.  
         [0043]    Accordingly, it can be seen that the present invention would allow for plates of adherent cells to be frozen at a particular stage of confluency and be ready for use upon thawing.  
         [0044]    Although the description above contains many specificities, these should not be construed as limiting the scope of the invention but as merely providing illustrations of some of the presently preferred embodiments of this invention. Various other embodiments and ramifications are possible within its scope. For example, the invention can utilize various adherent cells  02  and various cryopreservatives  24 , and various assays and tissue culture experiments can be run using the invention after thawing. The adherent cells  02 , cryopreserved in wells  22 , using the above stated method on tissue culture plates  20 , after storage at −80° C., can be thawed and used in various assays and as feeder cells. After being cryopreserved and stored in the above stated method, the adherent cells  02  maintain both their morphological and functional characteristics, sufficient for their intended use. A number of plant, animal, and insect cells, including but not limited to, HeLa-CD4-LTR-β-gal, F4, B9, F2, epithelial, T cells, lymphoblastoid, H9, C8166, Molt, Molt-4, CEM, Jurkat, and CEM74, can be cryopreserved and stored using the above-stated method.  
         [0045]    The invention method utilizes the tissue culture plate  20  wherein the adherent cells  02  are receptive to infection by a plurality of viruses including, but not limited to, HIV, SIV, BIV, and SHIV. The tissue culture plate  20  utilizes the adherent cells  02  listed above as a means for making this invention function. The tissue culture plate  20  also utilizes a foil adhesive cover  28  to enhance storage capabilities and to protect the adherent cells  02  in the wells  22  from contamination. The adhesive foil  28  also protects the wells  22  while allowing even flow and uniform cooling of the cells  02  with liquid nitrogen  26 . The method also utilizes a plastic, waterproof, resealable bag  30  for storage and labeling purposes. In order for the invention to obtain consistent volumes of cells  02  per will  22 , the invention requires the use of one of two methods of cell counting prior to treatment of the tissue culture plates  20 . The first method of cell counting requires the pipetting of 1 ml of cell suspension  12  into a collection vial, placing the vial into a cell counter and reading the digital output, and then diluting the cell suspension  12  until its count reads 4×10 4  per ml. The data resulting from performing these tests eliminate the necessity for volume and sterility testing to be done later and eliminate varying results while performing other assays and in determining the consumption of feeder cells. The adherent cells  02  are incubated in the wells  22  of the tissue culture plate  20  in a culture medium  10  including DMEM supplemented with 5% heat-inactivated fetal calf serum and 50 units/ml of penicillin/streptomycin. The adherent cells  02  are cryopreserved on a rigid, polystyrene tissue culture plate  20  by treating the adherent cells  02  on a tissue culture plate  20  with a cryopreservant  24 . The cryopreservant  24  may be a 10% glycerol or DMSO solution or both, with 30% fetal calf serum. The adhesive foil  28  is placed over the wells  22  on the tissue culture plate  20 , and the plate  20  is set into liquid nitrogen  26  at −160° C. and allowed to stand for 1 minute. The tissue culture plate  20  can then be stored in a waterproof, plastic, resealable bag  30  at −80° C. There are various methods of incubation. One method of incubation is to place the tissue culture plate  20  in water at 37° C. for 4 to 7 minutes. The outlined steps eliminate the variability in thawing.  
         [0046]    The cryopreserved adherent cells  02  can be stored for at least 3 months. The adherent cells  02 , cryopreserved in the tissue culture plate  20  utilizing the above method can be shipped to and utilized by laboratories that do not have the facilities to produce the cultured cells. The invention also provides for more accurate and consistent results arising from the set number of cells  02  per well  22 .  
       EXAMPLES  
     Example 1  
       [0047]    The following example further illustrates one preferred embodiment of the method of the present invention, but is neither intended nor should be considered as limitative in scope. Using HeLa-CD4-LTR-β-gal cells  02  in a flask  04 , they are incubated in an incubator  06  at 37° C. at 5% CO 2  levels. The flask  04  bearing HeLa-CD4-LTR-β-gal cells  02  is removed from the incubator  06  and aspirated. Next, 5 ml of the culture medium  10  is added to the flask  04 , wherein the culture medium is comprised as follows:  
         [0048]    RPMI 1640 10% heat-inactivated fetal serum;  
         [0049]    10% fetal calf serum;  
         [0050]    10 mM gentamicin;  
         [0051]    200 mM of L-glutamine; and  
         [0052]    non-essential amino acids and vitamins.  
         [0053]    Using a pipette, 10 ml of the cell suspension  12  is removed from the flask  04  and placed into a 15 ml. centrifuge tube. The tube is centrifuged for 7 minutes at 1000×g. The cells form pellets at the bottom of the supernatant  32 , whereupon the supernatant  32  is aspirated out of the tube and the cell pellets are re-suspended in 10 ml. of the culture medium  10 . One droplet from the suspension  12  is removed by a Pasteur pipette, placed on a hemacytometer  16 , and covered with a slip. The hemacytometer  16  is placed under a microscope  18  and the cells are counted. Using the cell count, the number of cells  02  per milliter is adjusted by diluting the cell suspension  12  with the culture medium  10  until the cell count reaches 4×10 4  per ml. Then 1 ml of the cell suspension  12  is pipetted in each well  22  of a rigid styrene plastic well plate  20 . The suspension  12  is only permitted to cover up to 30% to 50% of the bottom of the wells. The amount of culture medium per well varies with the size of the tissue culture plate wells and the plurality of wells, e.g., 200λ for a 96-well plate, 500λ for a 48-well plate, 1 ml for a 24-well plate, 3 ml for a 12-well plate, and 5 ml for a 6-well plate. The current embodiment utilizes a 24-well plate. At this point, the plate  20  is incubated for 1 hour, with a culture medium containing 30% fetal calf serum, in the CO 2  incubator  06  at 37° C. with 5% CO 2 , allowing cells  02  to grow in the well  22 . The medium  10  is then aspirated from each well  22  and the plate  20  is prepared for cryogenic preservation as follows: 100 μl of 10% DMSO solution, with 30% fetal calf serum at 4° C. is pipetted into each well  22 . An adhesive foil cover  28  is sealed over the top of the tissue culture plate  20 , thus protecting the adherent cells  02 , ensuring moveability of the tissue culture plate  20 , and permitting movement of the liquid nitrogen  26  around each individual well  22 . The tissue culture plate  20  is immersed in a container holding 200 ml of liquid nitrogen  26  at −160° C. The operator is careful not to allow any liquid nitrogen  26  to get into any individual well  22 . The tissue culture plate  20  is allowed to stand in the liquid nitrogen  26  for 1 minute. The plate  20  is then placed in a plastic, waterproof, resealable bag  30  and stored at −80° C. To use, the waterproof, resealable bag  30  is allowed to warm to room temperature by placing it into container of water at 37° C. for approximately 4 to 7 minutes. The tissue culture plate  20  is then removed from the bag  30 . The adhesive foil  28  is removed to expose the wells  22  for inoculation. The adherent cells  02  are then infected with HIV and used for infectivity testing for HIV.  
       Example 2  
       [0054]    The following example further illustrates one preferred embodiment of the method of the present invention, but is neither intended nor should be considered as limitative in scope. Using MDCK cells  02  in a flask  04 , they are incubated in an incubator  06  at 37° C. at 5% CO 2  levels. The flask  04  bearing MDCK cells  02  is removed from the incubator  06  and aspirated. Next, 5 ml of the culture medium  10  is added to the flask  04 , wherein the culture medium is comprised as follows:  
         [0055]    RPMI 1640 10% heat-inactivated fetal serum;  
         [0056]    5% fetal calf serum;  
         [0057]    10 mM gentamicin;  
         [0058]    200 mM of L-glutamine; and  
         [0059]    non-essential amino acids and vitamins.  
         [0060]    Using a pipette, 10 ml of the cell suspension  12  is removed from the flask  04  and placed into a 15 ml centrifuge tube. The tube is centrifuged for 7 minutes at 1000×g. The cells form pellets at the bottom of the supernatant  32  whereupon the supernatant  32  is aspirated out of the tube and the cell pellets are re-suspended in 10 ml of the culture medium  10 . One droplet from the suspension  12  is removed by a Pasteur pipette, placed on a hemacytometer  16 , and covered with a slip. The hemacytometer  16  is placed under a microscope  18  and the cells are counted. Using the cell count, the number of cells  02  per milliliter is adjusted by diluting the cell suspension  12  with the culture medium  10  until the cell count reaches 4×10 4  per ml. Then 3 ml of the cell suspension  12  is pipetted in each well  22  of a rigid styrene plastic well plate  20 . The suspension  12  is uniformly applied to 100% of the bottom of the wells  22 . The current embodiment utilizes a 12-well plate.  
         [0061]    At this point, the plate  20  is incubated for 1 hour, with a culture medium containing 30% fetal calf serum, in the CO 2  incubator  06  at 37° C. at 5% CO 2 , allowing cells  02  to grow in the well  22 . The medium  10  is then aspirated from each well  22  and the plate  20  is prepared for cryogenic preservation as follows: 100 μl of 10% glycerol solution, with 30% fetal calf serum at 4° C., is pipetted into each well  22 . An adhesive foil cover  28  is sealed over the top of the tissue culture plate  20 , thus protecting the adherent cells  02 , ensuring moveability of the tissue culture plate  20 , and permitting movement of the liquid nitrogen  26  around each individual well  22 . The tissue culture plate  20  is immersed in a container holding 200 ml of liquid nitrogen  26  at −160° C. The operator is careful not to allow any liquid nitrogen  26  to get into any individual well  22 . The tissue culture plate  20  is allowed to stand in the liquid nitrogen  26  for 1 minute. The plate  20  is then placed in a plastic, waterproof, resealable bag  30  and stored at −80° C. To use, the waterproof, resealable bag  30  is allowed to warm to room temperature by placing it into container of water at 37° C. for approximately 4 to 7 minutes. The tissue culture plate  20  is then removed from the bag  30 . The adhesive foil  28  is removed to expose the wells  22  for inoculation. The adherent cells  02  are then used in viral plaque assays.  
         [0062]    Thus the scope of the invention should be determined by the appended claims and their legal equivalents, rather than by the examples given.  
         [0063]    Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The preceding preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.  
         [0064]    The entire disclosure of all applications, patents and publications, cited above and in the figures are hereby incorporated by reference.  
         [0065]    From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.