WO1994018310A1 - Additif de stimulation de croissance pour milieu de culture de cellules de mammifere - Google Patents

Additif de stimulation de croissance pour milieu de culture de cellules de mammifere Download PDF

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Publication number
WO1994018310A1
WO1994018310A1 PCT/US1994/001522 US9401522W WO9418310A1 WO 1994018310 A1 WO1994018310 A1 WO 1994018310A1 US 9401522 W US9401522 W US 9401522W WO 9418310 A1 WO9418310 A1 WO 9418310A1
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cells
growth
gems
suspension
media supplement
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PCT/US1994/001522
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English (en)
Inventor
Samia S. Mankarious
Yu Ping Maguire
Stanley Tadao Enomoto
William Nash Drohan
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Baxter International Inc.
American National Red Cross
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Application filed by Baxter International Inc., American National Red Cross filed Critical Baxter International Inc.
Publication of WO1994018310A1 publication Critical patent/WO1994018310A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • C12N5/0037—Serum-free medium, which may still contain naturally-sourced components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/70—Undefined extracts
    • C12N2500/80—Undefined extracts from animals
    • C12N2500/84—Undefined extracts from animals from mammals

Definitions

  • This invention relates to a method for preparing from human plasma Fraction IV 4 a growth-enhancing media supplement for the culture of mammalian cells.
  • the invention also relates to the product of this method, that is a growth-enhancing media supplement, methods for culturing cells in this supplement, and the cell cultures resulting from these methods.
  • Cell growth in culture requires that (1) the cells stay healthy, and (2) the cells are able to go through mitosis and proliferate.
  • Mammalian cells are generally considered challenging to grow under the essentially artificial conditions of Ln vitro culture.
  • the absolute requirements for growth have not been defined for most mammalian cell types, but it is known that cells require a basal medium containing salts, glucose, amino acids, minerals, and vitamins, plus a supplement containing biological factors such as transferrin, insulin, and other growth factors. Different cell types have different requirements for survival and optimal growth.
  • the supplementing biological factors are most often derived from an animal source such as bovine serum.
  • Certain cell types will grow in media supplemented only with the albumin fraction derived from serum.
  • many cell types require other factors in serum that have not yet been identified, but are known to be necessary for the cells to thrive in an artificial medium.
  • Certain cell types require factors that are not present in adult serum but only in fetal serum. This situation has led to the extensive use of fetal bovine serum (FBS) to supplement the media used to support the growth of these cells.
  • FBS fetal bovine serum
  • the use of fetal serum has several serious drawbacks, including high cost and pending government regulations which may further decrease or eliminate the availability of fetal serum (Hodgson, J. 1993 Bio ⁇ Technology 11:49-53) .
  • MacLeod, A.J. , EP 440 509 disclosed a supplement produced from a Cohn fraction IV or Cohn fraction II and III combined, in which immunoglobulins were removed by polyethylene glycol precipitation.
  • Ng, P.K., et al., US Patent No 4,452,893 disclosed a supplement obtained from Fraction IV which is substantially free of components having a molecular weight higher than 250 kDa. These removed components were considered to be inhibitory to cell growth.
  • Congote L.F. (1987) In Vitro Cellular & Dev. Biol. 23:361, described the extraction of an erythropoietin-like factor from Cohn fraction V.
  • Cohn fraction IV contains a 50,000 kDa lipase that is heat-labile and that can elevate lipid accumulation in cultured fibroblasts.
  • Fenje, P US Patent No 3,769,415, disclosed a method for the production of a killed rabies virus vaccine containing protein from fraction V.
  • EP 415 666 (Sasai, S. , et al) disclose the use of a media supplement derived from human serum by a salting-out process to culture lymphokine-activated killer (LAK) cells.
  • EP 415 666 disclosed viral inactivation by treatment of the supplement with chemicals such as ethylene oxide or glutaraldehyde.
  • FIG. 1 depicts schematically the preparation of the growth-enhancing media supplement (GEMS) .
  • GEMS growth-enhancing media supplement
  • Figure 2 shows the molecular weights of proteins in pasteurized GEMS detected by HPLC.
  • Figure 3 shows the molecular weights of proteins, as detected by HPLC, in GEMS filtered through a virus-removing membrane.
  • Figure 4 depicts the growth of hybridoma cell line 9199 in pasteurized GEMS (PAST-G and PAST-V) and GEMS filtered for virus removal (ASAHI-G and ASAHI-V) compared with FBS (FBS- G and FBS-V) .
  • Figure 5 depicts the growth of hybridoma cell line 9105 in GEMS compared with FBS.
  • Figure 6 compares the growth of MB231 cells in 10% FBS with their growth in 0.5% FBS supplemented with GEMS.
  • Figure 7 compares the growth of peripheral blood lymphocytes (PBL) in various media supplements.
  • Figure 8 depicts the support of lytic activity in lymphokine-activated killer cells (LAK's) via culture in 2% GEMS compared with AIM V and 2% AB serum.
  • LAK's lymphokine-activated killer cells
  • Figure 9 shows the proliferation of tumor-infiltrating lymphocytes in GEMS.
  • Figure 10 depicts the growth of primary rabbit kidney cells (PRK's) in media supplemented with GEMS.
  • Figure 11 is a tabular depiction of the growth of hematopoietic progenitor CD34+ cells in media variously supplemented with lipids, GEMS (pasteurized or filtered) , or 25% FBS/HS.
  • the invention is further directed to methods for inactivating or removing viruses from the GEMS by either pasteurization or filtration through a virus-removing membrane, or both.
  • the invention is also directed to the GEMS resulting from the above processes.
  • the invention is further directed to methods for culturing mammalian cells in GEMS and the cell cultures resulting from these methods.
  • the invention method for producing GEMS begins with a starting material designated "Cohn fraction IV 4 paste" derived from human serum. While it is possible to make the Cohn fraction IV 4 paste from serum obtained from only one or a few donors, it is much preferred to make the paste from a pool of many donors, most preferably more than 3000 donors. The large pool provides the advantage of averaging out any batch to batch variations which might otherwise occur among batches obtained from only a few donors. Thus the user of GEMS is spared the time and cost of trying several batches of media supplement in order to find the one that works for a given cell.
  • Cohn fraction IV 4 refers to a paste obtained essentially as follows: Plasma from one or more human donors is collected, frozen, and then partially thawed at a controlled temperature not exceeding 6°C forming a cryo-suspension, pooled, and centrifuged at about 5000 g until all the cryo-precipitate has been separated from the cryo-suspension. The resulting supernate, the cryo-poor plasma, is collected in a temperature-controlled tank and cooled to about -5°C. The pH is adjusted to about 6.9 and the ethanol concentration adjusted to about 20% v/v by the addition of a pH 4.0 buffer/ethanol solution prechilled at about -15°C.
  • the Cohn fraction IV 4 paste is then suspended in a buffer having a pH of about 7.4 to about 8.4, most preferably 7.8, stirred to form a homogeneous suspension, and the pH is then adjusted to achieve a stable pH between 6.4 to 8.0, most preferably 7.3.
  • stable pH refers to a pH value that does not change by more than about 0.1 pH points when the suspension is stirred.
  • the suspension is then clarified, and the supernatant is collected.
  • clarification refers to any process which physically separates a precipitating solid phase from a liquid in which the solid is suspended. Clarification may be achieved via centrifugation, filtration, or decantation after a period of time sufficient to allow the solids to settle.
  • the supernatant is filtered through a sterilizing filter to remove microorganisms.
  • sterilizing filter refers to a membrane device with pore sizes averaging 0.2 ⁇ m in diameter which will allow the passage of large proteins but which will detain bacteria and any larger microorganisms.
  • the GEMS is now suitable to be added to basal medium to promote the in vitro growth of mammalian cells.
  • the GEMS may be filtered through various types of virus-removing membrane filters which have pore sizes small enough to retain viruses but large enough to allow proteins to flow through.
  • a typical virus-removing system has, in sequence, a 75nm pore membrane and one or two 35nm pore membranes.
  • pasteurize the GEMS For virus inactivation, it is preferred to pasteurize the GEMS since pasteurization, i.e. heating to 60°C for 10 hours, has been shown to kill all known viruses. Prior to pasteurization, however, it is preferred to stabilize the biological growth components via the addition of various combinations of sucrose, sorbitol, glycine, and ascorbate. This stabilization procedure ensures that unidentified biological factors present in GEMS will survive the heat treatment (pasteurization) without forming complexes with other factors, becoming degraded, or otherwise being inactivated.
  • the stabilized, pasteurized, GEMS is then subjected to a procedure to remove the stabilizers in order not to compromise the growth of the cells.
  • the various methods used for removal of stabilizers are dialysis, diafiltration, gel filtration, and size-exclusion chromotography.
  • dialysis refers to a process whereby the GEMS containing stabilizer is suspen ⁇ ed in a bag having a semipermeable membrane with pore sizes of approximately 1000 Daltons which allow the stabilizer molecules to be drawn by osmosis out of the GEMS, through the pores, and into a surrounding buffer solution having a lower osmolarity. Higher molecular weight molecules are retained within the bag.
  • the surrounding buffer solution is replaced at intervals until the stabilizers have been sufficiently removed from the GEMS in the suspended dialysis bag.
  • a preferred method for removing stabilizers involves "diafiltration” , also known as “molecular wash at constant volume” . Prior to diafiltration, the volume of the GEMS may be reduced via ultrafiltration in order to conserve the amount of buffer subsequently required for diafiltration.
  • a third alternative for the removal of stabilizers is "gel filtration” using a desalting gel.
  • the GEMS Prior to adding GEMS to the basal medium, the GEMS is subjected to a treatment with Protein A or Protein G, both of which selectively bind IgG. This treatment may be conducted by column adsorption or batch adsorption. As the GEMS is contacted with Protein A or Protein G, the trace amounts of IgG are bound and separated from the remaining GEMS components, and the IgG-free GEMS is collected.
  • Other affinity matrices known to adsorb IgG or other classes of Ig molecules may also be used, depending on which class of Ig molecules one desires to remove.
  • GEMS products of the processes described above are useful in the culture of diverse mammalian cell types.
  • GEMS alone is sufficient to supplement a basal medium, completely replacing the fetal bovine serum which would otherwise be required.
  • a human-derived supplement such as GEMS may be considered highly advantageous over fetal bovine serum for applications involving human therapy because non-human proteins are not present in GEMS.
  • GEMS can replace up to 95% of the otherwise required fetal bovine serum (FBS) , leading to a great reduction in cost and an increase in availability and convenience.
  • FBS fetal bovine serum
  • hybridomas are particularly challenging or impossible to grow in medium without FBS.
  • Several of these hybridomas have been shown to thrive and produce their desired antibody product in medium supplemented only with GEMS (Examples 3 and 4 below) .
  • the growth of hybridomas in the GEMS of the instant invention was reported in J.Tissue Culture Methods 14:39-44, February 13, 1992. This report did not disclose the method to produce the GEMS of the instant invention.
  • GEMS may be used in place of FBS in the culture of adherent cell lines such as MB157, MB231, and Vero as well as chondrocytes, epithelial cells, human diploid strains, monkey kidney cells, canine kidney cells, fibroblasts, and certain hematopoietic cells that usually require a stro al layer for growth.
  • GEMS may also be used in place of FBS to culture cell lines which do not require attachment, but rather grow in suspension.
  • non-adherent cell lines which may be cultured in GEMS include K562 cells, H69 cells, certain types of CHO cells, BHK-21 cells, HELA cells, baby hamster kidney cells (BHK) , human umbilical vein endothelial cells (HUVEC) , HEP-G2 cells, U0937 cells, human osteosarcoma cells (OS2) , and NIH 3T3 cells.
  • Peripheral blood lymphocytes are particularly challenging to grow in culture and have traditionally been thought to require FBS or whole human serum for proliferation. It has been found that 2% GEMS of the instant invention will support the proliferation of PBL's in culture to a level comparable to that afforded by 10% FBS.
  • PBL's may be activated by contact with IL-2 and/or a combination of anti-CD3 and anti-CD28 antibodies to become lymphokine-activated killer cells (LAK's) which can lyse certain target cells.
  • LAK's lymphokine-activated killer cells
  • the support of lytic activity of LAK's has likewise been thought to require FBS or whole human serum.
  • GEMS can replace FBS or whole human serum to support the proliferation and activation of LAK's.
  • Tumor infiltrating lymphocytes (TIL's) have also been considered challenging to grow in culture, usually requiring fetal serum for propagation and lytic activity.
  • TIL's Tumor infiltrating lymphocytes
  • GEMS was found to support the growth and lytic activity of TIL's derived from breast tumors or melanoma's.
  • GEMS may also support the growth and lytic activity of TIL's derived from many different types of solid tumors, allowing the genetic manipulation of TIL's in
  • Hematopoietic progenitor cells are selected from bone marrow or peripheral blood samples on the basis of antibody binding to cell-surface antigen CD34+.
  • optimal growth of CD34+ cells in culture was obtained by supplementation of the culture medium with 12.5% FBS plus 12.5% horse serum (25% FBS/HS) .
  • filtered (i.e. non- pasteurized) GEMS at 5 mg/ml or 0.2 mg/ml supports the growth of CD34+ cells to a level comparable to that of 25% FBS/HS.
  • Pasteurized GEMS also supports the growth of bone marrow-derived CD34+ cells to an extent comparable to that of 25% FBS/HS.
  • Pasteurized GEMS supports the growth of peripheral blood derived CD34+ cells at approximately one- third the rate of growth stimulated by 25% FBS/HS. A lower growth rate may be acceptable when all the advantages of GEMS are considered.
  • GEMS Cells derived from solid primary tissue, including both normal and tumor tissues, are considered particularly difficult to grow in culture because they generally require attachment to substrate as well as a diverse and undefined array of biological growth factors.
  • primary tissue notably primary rabbit kidney tissue. This finding suggests the use of GEMS to expand ex vivo cells from an individual patient's tumor for diagnostic and/or therapeutic purposes.
  • the GEMS of the instant invention serves as a replacement for animal serum in general, and fetal serum in particular, when cells are grown in culture for a variety of medical purposes.
  • GEMS replaces FBS for the culture of certain hybridomas that are difficult or impossible to grow in serum-free media formulations. GEMS may thus make monoclonal antibody production from hybridomas more economical as well as more acceptable to regulatory agencies because of the absence of non-human proteins in the monoclonal antibody product.
  • GEMS replaces FBS for the culture of established cell lines that were formerly thought to require FBS.
  • GEMS is particularly valuable for the culture of transformed cell lines expressing recombinant protein products. It is expected that recombinant protein products will be more easily purified from GEMS supplemented media as compared with FBS-supplemented media due to the absence of non-human proteins in GEMS.
  • GEMS supports the in vitro growth and activity of cells of the immune system, including PBL'S, LAK'S, TIL'S, hematopoietic stem cells and hematopoietic progenitors.
  • PBL'S PBL'S
  • LAK'S LAK'S
  • TIL'S hematopoietic stem cells
  • hematopoietic progenitors hematopoietic progenitors.
  • GEMS can be used to generate large numbers of immune- system cells for the purposes of adoptive immunotherapy whereby autologous or allogeneic cells are generated in culture for replacement to the patient when needed.
  • Lymphocytes generated in culture may also be genetically altered to express on their surfaces antigen-recognition proteins such that LAK's or TIL's will be targeted to pathological cells in the patient for therapy or diagnosis.
  • GEMS can be used in conjunction with individual extracellular matrix components such as collagen and fetuin or with complete extracellular matrices such as those derived from bovine cornea or human embryonic cells.
  • GEMS can potentially be used in primary culture to amplify the number of cells isolated from a human solid tumor, thereby allowing the development of therapy and diagnostic tests based on an individual's unique tumor cell type. Given a sufficient quantity of the patient's tumor cells, antibodies may be raised specifically against those tumor cells. The specific antibodies may be conjugated with a diagnostic label or a toxin to form a "silver bullet" directed against the individual patient's tumor tissues and metastatic cells.
  • Cohn Fraction IV 4 paste was obtained essentially as described in Cohn, E.J., et al., (1946) J. Am. Chem. Soc. 68:465. Briefly, frozen plasma from several donors was collected from plasma centers across the U.S. The frozen plasma was partially thawed at a controlled temperature not exceeding 6°C forming a cryo-suspension, pooled, and centrifuged at about 5000g until all the cryo-precipitate had been separated from the cryo-suspension. The resulting supernate, the cryo-poor plasma, was collected in a temperature-controlled tank and cooled to about -5°C.
  • the pH was adjusted to about 6.9 and the ethanol concentration adjusted to about 20% v/v by the addition of a pH 4.0 buffer/ethanol solution prechilled at about -15°C. All precipitation steps with ethanol and subsequent centrifugation were done at about -5°C.
  • the resulting suspension was mixed to achieve complete precipitation and then centrifuged at about 5000 g.
  • the centrifugate (supernate, Fraction I+II+III) was collected.
  • the Fraction I+II+III centrifugate (supernate) was adjusted to a pH of about 5.2 while maintaining the same alcohol cencentration with a prechilled pH 4.0 buffer/ethanol solution.
  • the resulting suspension was mixed to achieve complete precipitation and centrifuged at about 5000 g.
  • the centrifugate (supernate, Fraction IV-l) was adjusted to about 140 meq/L by the simultaneous addition of a pH 5.95 buffer and IN sodium bicarbonate solution. Ethanol was then added to a final concentration of about 40% v/v. The suspension was mixed to achieve complete precipitation and centrifuged at about 5000 g. The resulting precipitate, Fraction IV 4 paste, was collected.
  • This supernatant comprised the growth-enhancing media supplement (GEMS) .
  • GEMS growth-enhancing media supplement
  • the stabilized supplement was placed in a water bath containing an ice/water mix. A timer was set to begin heating at 10PM and to stop at 9AM, (11 hours) . The tubes were kept submerged to the liquid fill level. The incubator was tested to assure that it would reach 60°C during the first hour of heating.
  • the pasteurized GEMS was stored at 5°C when diafiltration or desalting was planned within 24 hours. For longer periods prior to desalting, the supplement was stored at -25°C.
  • the pasteurized GEMS was rendered essentially free of the stablizers by concentrating the GEMS to approximately half of its volume using an ultrafiltration system with PTGC membranes (polysulfone membranes with nominal molecular weight cut-off of 10,000) , followed by molecular washing and diafiltration using the same UF system with ImM HEPES, 0.9% NaCl pH 7.2.
  • the buffer was used in an amount approximately ten times the volume of the media concentrate. After diafiltration, the GEMS was adjusted to a protein concentration of approximately 50 mg/ml protein with lOmM HEPES, 0.9% NaCl, pH 7.2 buffer.
  • Encephalomyocarditis viruses and Sindbis viruses were added to test samples of GEMS prior to pasteurization step 10. After completion of pasteurization, no viral activity was detectable by an assay based on changes in morphology of target cells (Reed and Muench (1938) "A simple method of estimating fifty percent endpoints.” Am. J. Hyg. 27:493-497) .
  • HPLC analysis of the pasteurized GEMS demonstrated prominent protein peaks corresponding to molecular weights of about 171 kDA, 362 kDa, and 1,044 kDa (Fig. 2) .
  • the supplement was prepared as in Example 1 through step 6.
  • the resulting supernatant from step 6 was passed through virus-removing membrane filters, PLANOVA , Asahi Chemical Industry Co. , Tokyo, Japan.
  • the supernatant was first passed through a PLANOVA filter having a mean pore size of 72 +/-4 nm (PLANOVA 75) and then through 1 or 2 PLANOVA's having a mean pore size of 35 +/-2 nm (PLANOVA 35) .
  • HPLC analysis of the PLANOVA-filtered GEMS demonstrated prominent protein peaks corresponding to molecular weights of about 151 kDa, 382 kDa, and 538 kDa (Fig. 3) .
  • Basal medium contained RPMI/Ham's F12 and sodium bicarbonate Penicillin/Streptomycin and glutamine.
  • Frozen cell lines were thawed, assessed for viability by trypan blue exclusion, and resuspended at 10 cells/ml in BM.
  • the cells were plated at 10 c/ml in 20 ml of BM + 20% FCS in 75 cm tissue culture flasks.
  • FCS in BM The concentration of FCS in BM was stepped down from 20% to 4% slowly. At each stage 20%, 10%, 5%, 4%, three splits were completed i.e. viability ) 85% and cell density of 5 x 10 c/ml per split.
  • FCS + BM and 2% FCS + 2% GEM + BM When the culture was stable at 4% FCS + BM and 2% FCS + 2% GEM + BM, experimentation was begun. For each experiment or monthly test of the stability of GEMS a 4% FCS (2 mg/ml total protein) control was run. Two T75 flasks containing a volume of 20 ml at a cell density of 5 x 10 c/ml for each cell line were used as starting cultures.
  • Each stationary T75 flask contained 18 ml of the required media condition, to which 2 ml cell suspension was added to yield a final volume of 20 ml.
  • the flasks were incubated at 37°C for 56-96 hours in a 6% C0 2 -in-air atmosphere. Cell densities were calculated using a hemacytometer and magnification 10 on the microscope.
  • 18 ml of each cell suspension was transferred to 50 ml centrifuge tubes and centrifuged at 1000 revolutions per minute for 15 minutes. Thirteen ml of cell supernatant was aspirated off, and cells were resuspended in 5 ml. The next flask was seeded at 2 x 10 c/ml.
  • split #3 4 ml of supernatant were removed for antibody testing and cells were resuspended in 5 ml of media and again seeded at 2 x 10 c/ml.
  • the fourth split was like split #3.
  • the procedure for split #3 was repeated except 4 flasks were seeded in 4% FCS and 2% FCS + 2% GEM to increase the cell density in preparation for the next experiment.
  • the concentration of Human Immunoglobulin M was determined using a double antibody sandwich type ELISA.
  • the human IgM ELISA was used to quantitate the amount of human IgM in samples from the anti-CLPS monoclonal antibody supernatants, produced from cell line 78-1-2.
  • the concentration of the mouse immunoglobulin G was also determined by ELISA. This test was used to quantitate the amount of murine monoclonal antibody that is produced from Cell lines 12A8, PCV310, F8/1-5-6 and 88-60.
  • the concentration of the Factor VIII product from the cell lines CHO D-28 and C-127 was determined using a one stage activated partial thromboplastin time assay (APTT) .
  • Micronized silica was used as an activator and human Factor VIII deficient plasma was used as a substrate.
  • the APTT assay is thus a screening test for deficiencies in the intrinsic system of coagulation of which Factor VIII is a part.
  • the test sample was added to Factor VIII deficient plasma, and the degree of correction of the clotting time was measured.
  • the test clotting time was then compared to a standard curve constructed using various dilutions of a lyophilized reference of known potency. The potency of the sample was estimated from the standard curve.
  • the doubling times for this cell line were 18.5hr with 10% FCS; 29.5hr with 2% FCS; 25.6hr with GEMS at 2 mg/ml and 40.9hr with GEMS at 1 mg/ml.
  • the CHO cell line when grown in GEMS at 2 mg/ml did support the cells and the FVIII product expression was found to be greater than that with FCS at 2%.
  • GEMS also supported the expression of biologically active recombinant human Factor VIII from transfected C-127 cells, a murine mammary epithelial cell line which adhered to the flasks and grew well in GEMS without pretreatment of the flask's surface with any exogenous extracellular matrix or FBS.
  • Factor VIII was expressed from these C-127 cells at levels equal to or greater than that seen with 10% FBS supp1ement.
  • the use of a higher concentration of GEMS may increase the amount of Factor VIII produced. Addition of Von
  • Willebrand's Factor is expected to increase the stability of the FVIII product.
  • GEMS was found to be stable for at least 5 months when stored as a 50 mg/ml concentrated solution at 5°C or as a mixture with basal media at a final concentration of 2 mg/ml. It could be kept at -25°C, 21- 25°C, or 35°C with only a small loss in potency.
  • EXAMPLE 4 Growth of hybridoma cell lines 9199 (Mouse/human) and 9105 (Rat/rat) using GEMS.
  • Cell line 9199 (Mouse/Human) hybridoma secreting human immunoglobulin and cell line 9105 (Rat/Rat) hybridoma secreting rat immunoglobulin were evaluated for growth support and secretion in medium supplemented with GEMS. These cell lines were chosen because both were found to be particularly challenging to grow in serum-free medium.
  • Samples of conditioned supernatant were collected at regular intervals and assayed for secreted immunoglobulin concentrations.
  • the concentration of secreted antibody was slightly greater in medium supplemented with both GEMS products compared to five percent FBS and serum free medium (MF28) .
  • the concentration of secreted antibody was approximately equivalent to five percent FBS in medium supplemented with both GEMS products.
  • GEMS When GEMS is to be used to support the growth of hybridomas secreting human IgG, it may be desirable to remove the IgG component from the GEMS in order to simplify purification of the product from the culture media. Both pasteurized and Asahi filtered GEMS products are suitable for IgG removal.
  • HBS 0.02 M HEPES, 0.9% NaCl, pH 7.4
  • the gel was washed with approximately 200 ml of HBS per gram of lyophilized Protein A gel at 23° C.
  • the BioRad column was poured and packed at approximately 1.5 ml/min.
  • the column was loaded with either of the above listed forms of GEMS at 1 ml/min.
  • the flow-through material was recovered as GEMS having human IgG essentially removed.
  • Ig immunoglobulins
  • the Ig bound to the column was eluted with 50 ml of IM acetic acid, 0.1M glycine-HCl, pH 3.0.
  • Assays for IgG, IgA, and IgM content were performed for the flow-through materials as well as the eluted Ig.
  • Assays for human serum albumin (HSA) were performed in order to use HSA content as an internal control based on the assumption that HSA does not bind to Protein A.
  • IgM content was tested by the well-known ELISA assay.
  • IgG, IgA, and HSA were tested by radial immunodiffusion assay (RID) .
  • RID radial immunodiffusion assay
  • a test sample antigen was applied to a well cut into a gel matrix incorporated with the antiserum corresponding to the antigen to be tested. As the antigen diffused through the gel, it reacted with the antiarum to form a precipitin ring. The resulting diameter of the precipitin ring was measured and compared to the diameter of the precipitin rings of the antigen standards of known concentrations to quantitate the test sample antigen.
  • CHOK 1 cells (ATCC CCL 61) were harvested from serum containing medium and washed once with RPMI:Ham's F12 serum free medium. Aliquo_s of cells were dispensed into duplicate tissue culture wells (lX10E6/well) containing RPMI:Ham's F12 supplemented with GEMS at a 2 mg/ml concentration. The sets of wells were pretreated (O/N at 37 deg. C, 7% C02) with 10% FBS, collagen type VI (0.025, 0.05, 0.1, 0.5 mg/ml) or fetuin (0.07, 0.13, 0.25, 0.50 mg/ml) . The cultures were incubated at 37°C, 7% C0 2 and observed daily for plating efficiency, cell growth and morphology. Cultures were harvested and compared to 10% FBS control wells at 72 hours.
  • RPMI 1640 Ham's F12 supplemented with GEMS (lot 2) (with 10% FBS pretreatment) .
  • the test was compared to 10% FBS supplemented RPMI 1640: Ham's F12 medium (positive control) .
  • Cells were seeded at 1X10 into duplicate sets of T25 flasks. Duplicate flasks were harvested on days 1, 2, 3, 6 and 7, cell yields were recorded, growth curves were prepared, and population doubling times calculated.
  • GEMS resulted in higher cell density than basal medium alone when the substrate was pretreated with attachment factors.
  • Addition of Fetuin at 0.5 mg/ml to GEMS resulted in slightly improved morphology up to 48 hours.
  • Addition of Collagen to GEMS resulted in a slight tendency towards flattening out of cells by 72 hours.
  • FBS or FBS pretreatment cells attach but do not flatten out.
  • Cell morphology was approximately the same in base medium alone as base medium with GEMS.
  • GEMS at 1:25 dilution supported CHOKI 1 cell growth equivalent to 10% FBS containing medium when the substrate was pretreated with FBS.
  • the log phase(0-48 hours) population doubling times were: GEMS - ⁇ .7 hours 10 % FBS -11.5 hours
  • the population doubling time was also equivalent to that for 10% FBS (1 2 hours and 11.6 hours, respectively) and the cell yields were equivalent (1.6 X 10 6 and 1.7 X 10 6 respectively)
  • EXAMPLE 7 Use of GEMS for growth of cell lines MB231, H69, and K562.
  • MB231 is a human breast cell line which requires attachment
  • H69 is a human small cell lung line
  • K56 2 is a human promyelocyte cell line. The latter two cell lines can grow in suspension.
  • Initial test conditions were: 2.5% and 1% fetal bovine serum (FBS) with and without 5% GEMS, 2.5% GEMS, 1.25% GEMS, 0.625% GEMS, and 0.312% GEMS. After the first 3 days of culture, cells were transferred to 0% and 1% FBS with and without GEMS at the above concentrations and then cultured for an additional 12 days.
  • FBS fetal bovine serum
  • MB231 cells were cultured in 96-well microtiter plates using varying concentrations of FBS (10%, 2%, 0.5%) , GEMS (0%, 2%, 4%), with or without laminin and fibronectin at lOO ⁇ g/ml.
  • MTT is a soluble tetrazonium salt which is reduced to insoluble, colored formazans by the metabolic activity of living cells.
  • the crystal violet assay was used to determine cell number based on its staining of protein.
  • EXAMPLE 8 GEMS used to support the growth and lytic activity of human lymphocytes.
  • Human lymphocytes were obtained from peripheral blood mononuclear cells and tumor specimens. For collection of peripheral blood lymphocytes, 50 ml of whole heparinized blood was separated on Ficoll-Hypaque and the mononuclear cells were harvested from the interface. The cells were washed 3X and resuspended for counting.
  • AIM V serum-free medium, GIBCO
  • the supplement consisted of hydrocortisone (0.5 ⁇ g/ml) , insulin (10 ⁇ g/ml) , EGF (5 ng/ml) , T 3 (10 " M) , selenium (2 ng/ml) , linoleic acid BSA (5 ⁇ g/ml) .
  • the supplemented RPMI without GEMS also received transferrin (20 ⁇ g/ml) .
  • Lymphocytes at 2-5 X 10 cells/ml were either:
  • Lytic assay A standard Cr release assay was used to determine the lytic activity of activated lymphocytes on
  • K562 cells K562 cells. K562 cells were first loaded with Cr 51 and subsequently added to the cells to be tested for lytic activity at an effector to target ratio of 2.5:1, 10:1, and 25:1. The plates were incubated at 37°C for 4 hours after which 100 ⁇ l of supernatant was removed from each well and counted in a scintillation counter. % cytotoxicity was calculated using the formula
  • Tumor infiltrating lymphocytes were isolated from solid specimens obtained from surgery or malignant fluid specimens. Solid specimens were minced, and, if necessary, digested with enzymes. Malignant fluid specimens were washed and resuspended in medium containing either IL-2 or medium containing a combination of anti-CD3, anti-CD28, and IL-2. Cells were incubated at 37°C in a fully humidified incubator.
  • Figure 7 exemplifies the results of a typical PBL proliferation experiment conducted over 25 days. Until day 15, 2% GEMS supported growth to an extent essentially equivalent to that afforded by HDL, HB104, and 2% AB serum.
  • Figure 8 shows that 2% GEMS supported the lytic activity of lymphokine-actvated killer (LAK) cells. Results using 2% GEMS were typically comparable to results obtained using AIM V or 2% AB serum.
  • TIL's tumor-infiltrating lymophocytes
  • Primary cells were obtained from rabbit kidney and grown in glass tubes containing RPMI plus either 10% FBS as a control, or lower concentrations of FBS supplemented with 2% GEMS. After several days in culture, cell counts were compared as shown in Figure 10. GEMS supplemented 1% or 2% FBS to support growth of PRK cells to a level comparable to that obtained with 10% FBS.
  • EXAMPLE 10 The use of GEMS to sustain the proliferation of CD34+ hematopoietic progenitor cells in serum-free medium.
  • CD34+ cells were purified using Dynal immunomagnetic beads, from bone marrow or leukapheresis products from patients who had been mobilized with G-CSF during the recovery from chemotherapy induced leukopenia. The selected CD34+ cells were then seeded into suspension cultures at 10 /ml.
  • the control media called HLTM (for human long term culture medium) , was a McCoy 5A base medium supplemented with amino acids, vitamins, monothioglycerol and hydrocortisone and contained 12.5% fetal bovine serum (FBS) and 12.5% horse serum (HS) from pretested batches. This media has been previously shown to give optimal performance in the proliferation and differentiation of CD34+ cells.
  • the growth factors IL-3 (30OU/ml) , G-CSF(30OU/ml) , GM-CSF(300U/ml) and SCF(20ng/ml) were added to all of the cultures.
  • the base medias to which GEMS was added also included Iscove's modified Dulbecco's medium (IMDM) without serum.
  • IMDM Iscove's modified Dulbecco's medium
  • the cultures were then harvested and the proliferation index was determined by dividing the final number of cells by the initial number.
  • a sample of the cultured cells was stained with antibodies to CD15 and CDllb and analyzed by flow cytometry to determine the differentiation state of the cells. This phenotyping protocol defines a population of CD15+CDllb- cells that represent myelocytes and promyelocytes.
  • Propidium iodide was also added to these samples to identify the non-viable cells present.
  • Cell samples from the cultures were also plated in methyl cellulose cultures containing recombinant growth factors and incubated for an additional 14 days to estimate the number of cells that form colonies containing >50 cells and the proportion of granulocyte-macrophage colony forming cells, macrophage colony forming cells, erythroid colony forming cells (BFU-E) and mixed colony forming cells. These data are presented as indices of the final numbers of colonies divided by the initial numbers of colonies. In addition the numbers of cluster forming cells which produce less than 50 cells was estimated and presented in a similar manner.
  • samples of 2500 CD34+ cells/well were incubated in 96 well microtiter plates containing control media or different concentrations of GEMS for 7 days and pulsed overnight with H-Thymidine. The cells were harvested onto filters and counted in a scintillation counter to determine cpm.
  • IMDM base medium supplemented with GEMS.
  • CD34+ cells Four preparations of selected CD34+ cells were evaluated: two preparations from bone marrow and two from leukapheresis products. Filtered GEMS at 5 mg/ml or 0.2 mg/ml in a McCoy's base medium had equivalent proliferation of cell numbers (PI day 11) when compared to the control HLTM medium containing 25% FBS/HS. Viabilities and the percent of GM colony forming cells were slightly lower in the GEMS supplemented media but CD15+ CDllb- cells were in similar proportions.
  • the cultures containing pasteurized GEMS produced only one third the number of cells as the control when McCoys 5A base was used with peripheral blood CD34+ cells but performed in a similar manner to filtered GEMS or the control when IMDM base was used with bone marrow derived CD34+ cells.

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Abstract

Méthodes de production d'un additif de stimulation de croissance en milieu à partir de la fraction de Cohn IV4 dérivée du sérum humain. Ces méthodes portent également sur la culture de diverses séries de cellules de mammifères dans cet additif, lequel se substitue avantageusement dans de telles cultures au sérum de f÷tus bovin.
PCT/US1994/001522 1993-02-12 1994-02-14 Additif de stimulation de croissance pour milieu de culture de cellules de mammifere WO1994018310A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2284254A1 (fr) 2009-07-28 2011-02-16 Grifols, S.A. Milieux pour la culture de cellules de mammiferes comprenant les surnagenants obtenus a partir des etapes de fractionnement Cohn et leurs utilisations.

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0440509A2 (fr) * 1990-02-02 1991-08-07 Common Services Agency Nouveaux ingrédients pour milieu de croissance cellulaire et son procédé de préparation

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0440509A2 (fr) * 1990-02-02 1991-08-07 Common Services Agency Nouveaux ingrédients pour milieu de croissance cellulaire et son procédé de préparation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ADVANCES IN BIOCHEMICAL ENGINEERING, Volume 37, issued March 1988, A.J. MACLEOD, "The Use of Plasma Protein Fractions as Medium Supplements for Animal Cell Culture", pages 41-56. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2284254A1 (fr) 2009-07-28 2011-02-16 Grifols, S.A. Milieux pour la culture de cellules de mammiferes comprenant les surnagenants obtenus a partir des etapes de fractionnement Cohn et leurs utilisations.
ES2360782A1 (es) * 2009-07-28 2011-06-09 Grifols, S.A. Suplemento de medios para cultivo celular y metodo para su preparación a partir de derivados plasmáticos de origen humano.
US8252590B2 (en) 2009-07-28 2012-08-28 Grifols, S.A. Mammalian cell culture media which comprise supernatant from cohn fractionation stages and use thereof
EP2826854A1 (fr) 2009-07-28 2015-01-21 Grifols, S.A. Milieu de culture de cellules mammifères comprenant un surnageant à partir d'étapes de fractionnement de Cohn et utilisation associée
EP3059305A1 (fr) * 2009-07-28 2016-08-24 Grifols, S.A. Milieu de culture de cellules mammiferes comprenant un surnageant a partir d'etapes de fractionnement de cohn et utilisation associee

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