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WO1986001531A1 - Procede pour degager de micro-porteurs des cellules dependant d'ancrage - Google Patents

Procede pour degager de micro-porteurs des cellules dependant d'ancrage Download PDF

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Publication number
WO1986001531A1
WO1986001531A1 PCT/US1985/001615 US8501615W WO8601531A1 WO 1986001531 A1 WO1986001531 A1 WO 1986001531A1 US 8501615 W US8501615 W US 8501615W WO 8601531 A1 WO8601531 A1 WO 8601531A1
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microcarriers
cells
cell
growth
culture
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PCT/US1985/001615
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English (en)
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Donald J. Giard
Wei-Shou Hu
Daniel I. C. Wang
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Massachusetts Institute Of Technology
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • 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
    • C12N5/0068—General culture methods using substrates
    • C12N5/0075—General culture methods using substrates using microcarriers
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20211—Vesiculovirus, e.g. vesicular stomatitis Indiana virus
    • C12N2760/20251—Methods of production or purification of viral material

Definitions

  • This invention is in the field of cell biology and pertains to a method of detaching anchoragedependent cells from positively charged microcarriers or other positively-charged substrates.
  • mammalian cells used for the production of biologicals are anchorage-dependent, that is, they grow only when they can attach themselves to a surface. Indeed, most normal mammalian cells are anchorage-dependent.
  • anchorage-dependent cells are grown in small flasks or in roller bottles - cylindrical vessels that are oriented with their long axis horizontal and are continuously rotated. However, these vessels are not generally suitable for large-scale processes.
  • Alternative methods for cell culturing have been proposed, including plastic bags, stacked plates, spiral films, glass bead propagators, artificial capillaries, and microcarriers.
  • microcarriers to enlarge the surface area in a single bioreactor for attachment and growth of anchorage-dependent cells. See van Wezel et al., (1967) "Growth of Cell Strains and Primary Cells on Microcarriers in Homogenous Culture” Nature 216, 64-65.
  • An example of microcarriers is a positively-charged bead measuring about 50 to several hundred microns in diameter and typically composed of diethylaminoethyl (DEAE)-substituted dextran.
  • DEE diethylaminoethyl
  • S/V growth surface to vessel volume
  • a much higher ratio of growth surface to vessel volume can be obtained with microcarriers in comparison to both traditional and newly developed alternative techniques.
  • the increase in S/V attainable allows the construction of a single-unit homogeneous or quasihomogeneous batch or semi-batch propagator for high volumetric productivity.
  • a single stirred tank vessel with simple feedback control for pH and pO 2 presents a homogeneous environment for a large number of cells and eliminates the need for expensive and space consuming, controlled environment incubators.
  • the total number of operations required per unit of cells produced is drastically reduced.
  • microcarriers offer economies of capital, space and manpower in the production of anchorage-dependent cells, relative to previous production methods.
  • Microcarriers also offer the advantage of environmental continuity because the cells are grown in a single controlled environment. They provide the potential for growing anchorage-dependent mammalian cells under one set of environmental conditions which can be regulated to provide constant, optimal cell growth. To exploit microcarrier technology, it is necessary to serially propagate cells entirely on microcarriers, from initial inoculum through scaleup to final harvest of the cells or cell products. Use of microcarriers, however, generally has been limited to the final stage of the scale-up procedure. The principal obstacle to their use has been the difficulty in detaching cells from the microcarriers in a viable condition, which is a prerequisite if the culture is to serve as an inoculum for a subsequent larger-scale batch.
  • Crespi and Thilly Biotechnology and Bioengineering 23, 983-993 (1981) have shown that when the anchorage-dependent cell line LLC-MK 2 and the non-anchorage-dependent CHO-K1 cell line are grown in media with a reduced calcium concentration, continuous subculture is possible by the simple expedient of periodically adding fresh microcarriers suspended in fresh medium.
  • the lower calcium concentration apparently affects the cell-microcarrier bond, permitting a bead-to-bead transfer that does not occur at normal calcium concentrations.
  • This technique is limited, however, to those cell lines both capable of growing in a reduced calcium medium and susceptible to the reducedcalcium effect on adherence. Many other cell lines, including human and chick fibroblasts, require higher levels of calcium for optimal growth, and so the technique of Crespi and Thilly may not have widespread utility.
  • This invention constitutes a method of detaching cells from positively-charged cell culture microcarriers such as DEAE-substituted polydextran beads or from other positively-charged substrates.
  • Microcarriers laden with attached cells are exposed to a proteolytic agent (s), such as the enzyme trypsin, at a relatively high pH, that is, a pH of about 7.8 to about 10.0, a pH of about 8.2 being preferred.
  • a proteolytic agent such as the enzyme trypsin
  • the cell-laden microcarriers are subjected to mild shear by, for example, passage through a glass bead column. Typically more than 90% of the cells are released in a viable state by this treatment.
  • the released cells can be inoculated into a new culture vessel with additional microcarriers and fresh medium and propagated further. Cell growth and cell-product formation is unimpaired by the detachment process.
  • Figure 1 illustrates the morphological changes in FS-4 cells brought about by trypsinization at high pH of the cells attached to microcarriers.
  • Figure 2 illustrates the effect of pH on cell detachment from microcarriers by trypsinization.
  • Figure 3 illustrates the kinetics of FS-4 cell growth in microcarrier culture.
  • the arrows indicate the time of high pH trypsinization and inoculation into a new culture.
  • FIG. 4 illustrates the effect of pH on the proteolytic activity of trypsin.
  • Figure 5 illustrates the serial propagation of FS-4 cells on microcarriers of a selected diameter.
  • the arrow indicates time of high pH trypsinization and inoculation into a fresh culture.
  • Figure 6 illustrates the serial propagation of Vero cells on microcarriers.
  • Figure 7 illustrates vesicular stomatitis virus production by Vero cells serially propagated on microcarriers using the high pH trypsinization method of cell detachment.
  • cells are transferred from cell-covered microcarriers to new bare microcarriers.
  • Cells grown on conventional surfaces such as Petri dishes or roller bottles, can be detached by treatment with a proteolytic agent, most often trypsin, along with mild mechanical perturbation. These cells are then used as the inoculum of another culture.
  • This method of detachment is ill-suited for detaching most types of anchorage-dependent cells from microcarriers.
  • cells grown in microcarrier culture generally cannot be used to inoculate another culture. Instead, cells grown in roller bottles are required for the inoculation of a microcarrier culture.
  • This invention provides a method of detaching cells from positively-charged substrates, such as microcarriers.
  • the method permits direct inoculation of large microcarrier cultures from a seed microcarrier culture and consequently, eliminates the laborious task of preparing large number of roller bottle cultures for inoculation. It permits serial cultivation of anchorage-dependent cells, from seed-culture to larger-scale productive cultures, entirely on microcarriers (or on other positively-charged substrates) .
  • cells can be detached from positively-charged microcarriers in viable condition by treating the cell-laden microcarriers with a proteolytic agent at a pH above the pH of normal culture conditions, that is, at a pH above about 7.8 to about 10.0, and then generating a shear force between the microcarrier and the cells to remove the cells from the microcarrier.
  • a proteolytic agent at a pH above the pH of normal culture conditions, that is, at a pH above about 7.8 to about 10.0
  • Initial or seed microcarrier cultures of anchorage-dependent cells can be established by standard techniques. See e.g., Levine, D. W., Wang, D.I.C., and Thilly, W. G. (1979) Biotechnol. Bioeng. 21, 821-845.
  • Cells can be detached from microcarriers in seed culture by the method of this invention when they reach any desired growth stage. Cells are usually detached when they reach confluence.
  • cellladen microcarriers are separated from the growth medium. Any suitable method of separation may be used.
  • the cell-laden microcarriers may be allowed to settle in the culture vessel and the medium supernatant removed by decanting or by suction.
  • all or a portion of the medium containing suspended cell-laden microcarriers can be transferred from the growth vessel to a separate vessel for separation.
  • the cell-laden microcarriers Before proteolytic treatment, the cell-laden microcarriers should be washed to remove any residual medium. When the cells have been grown in serum-supplemented medium, cell-laden microcarriers must be washed thoroughly because serum will inhibit the activity of the proteolytic agents and interferes with the detachment process.
  • the cells may be washed with an aqueous isotonic buffer, such as phosphate buffered saline (PBS) or HEPES.
  • PBS phosphate buffered saline
  • HEPES phosphate buffered saline
  • the cell-laden microcarriers are then treated with a proteolytic agent at pH 7.8-10.0.
  • the settled microcarriers may be resuspended in an aqueous isotonic buffer containing the proteolytic agent, adjusted to the selected pH within the operative range.
  • the optimum pH within the prescribed range is dependent upon a host of factors including the particular proteolytic agent, the type of microcarrier, the type of cell and the conditions of culture and can be determined for any combination of these factors by routine experimentation.
  • proteolytic treatment of about 10-15 minutes duration is sufficient to obtain a high degree of detachment.
  • a chelating agent such as ethylenediamine tetraacetic acid (EDTA) may be added to the solution of the proteolytic agent.
  • the chelating agent acts as a scavenger of divalent cations such as calcium and magnesium. Divalent cations are thought to play a role in cell attachment to substrates, and their removal may aid in detachment.
  • Suitable proteolytic agents are the proteases trypsin, pronase, collagenase and proteinase K. Mixtures of two or more of these enzymes may be used.
  • the concentration of the proteolytic agent (s) should be that which yields a high degree of detachment with l ⁇ inimal loss of cell viability.
  • the preferred proteolytic agent is trypsin.
  • trypsin preparations are available commercially.
  • a frequently-used trypsin preparation is Bacto-Difco 1:250.
  • a concentration of about 0.10.2% (0.1-0.2 g trypsin preparation per 100 ml buffer solution) can be used for the detachment process.
  • FS-4 cells grown on DEAE-dextran microcarriers are detached readily by a 0.2% solution of this trypsin preparation at pH between 8.2 and 9.0; the detached cells reattach to and grow on fresh microcarriers without significant loss of viability.
  • pH 8.2 can be used routinely and provide satisfactory results.
  • Another suitable trypsin preparation is twice recrystallized trypsin supplied by Worthington Diagnostics. A 1:500 dilution of a 4.0% stock solution of this trypsin preparation yields excellent detachment.
  • Temperature is a factor which may affect proteolysis at elevated pH.
  • the temperature at which cells are trypsinized can affect viability during subculturing.
  • McKeehan et al. reported that cells trypsinized at low temperature (4°C) showed a significant improvement in subsequent clonal growth.
  • McKeehan, W. L. et al. "The Use of Low-Temperature Subculturing and Culture Surface Coated with Basic Polymers to Reduce the Requirement for Serum Macromolecules" In: The Growth Requirements of Vertebrate Cells In Vitro, pp. 118-150, Cambridge University Press, 1981.
  • FS-4 cells are detached and successfully cultured after high pH trypsinization at room temperature.
  • the combination of high pH, low temperature and, possibly, a reduced trypsin concentration may improve cell viability.
  • the most effective combination of parameters can be determined for any particular type of anchorage-dependent cell by routine experimentation.
  • Additional factors which influence cell detachment by this process include the composition of the growth medium, the concentration of serum supplement, the degree of confluency of the cells at the time of detachment and the length of time that the cells have been in a confluent stage. As these conditions vary, concentration of proteolytic agent, temperature, the concentration of any chelating agent and the pH can be adjusted to provide optimum detachment and cell viability.
  • a shear force can be generated in a number of ways. For example, sufficient shear force may be provided by simply pipetting, repeatedly if necessary, a reconstituted suspension of the treated microcarriers. For large cultures, a convenient way of providing effective shear is to pass the treated microcarriers through a column packed with glass beads. Glass beads of about 3 mm in diameter are suitable; column dimensions may vary depending upon the amount of microcarriers. A snear force is generated during passage of the microcarriers through the column which is sufficient for detachment of cells. The column effluent containing microcarriers and detached cells is collected and can be used directly as an inoculant for a fresh culture.
  • the method of this invention is believed to be useful for detaching anchorage-dependent cells from any positively-charged substrate suitable for their attachment.
  • substrates may be fibers, plates or beads.
  • Typical microcarriers are comprised of porous beads having a positively charged surface.
  • the beads are made of hydroxyl-containing polymers such as dextran, dextrin, starch, cellulose, polyglucose and substituted derivatives of these polymers.
  • the hydroxyl groups provide sites for attachment of a charge-supplying group which most often are tertiary amines.
  • Some examples of commercially available positively charged microcarriers are ion exchange resins sold under the trade names DEA ⁇ -Sephadex A50, and DEAE-Sephadex A25 by Pharmacia.
  • These microcarriers comprise dextran polymers substituted with diethlyaminoethyl (DEAE) groups, generally designated DEAE-substituted dextran beads.
  • DEAE diethlyaminoethyl
  • human diploid fibroblasts (FS-4 strain) were serially propagated on DEAE-dextran microcarriers.
  • Cells were grown to confluence on microcarriers in initial seed culture, then washed several times with at least 30 bead-volumes of phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the beads were then suspended in five volumes of trypsin solution in PBS.
  • the pH of the trypsin solution ranged from about 8.9 to 9.0. In later experiments, it was found that the pH can be reduced to 8.2.
  • the microcarriers were allowed to settle and drain in a glass column or a sintered glass funnel.
  • a continuous monkey kidney cell line (Vero) was also cultivated serially on microcarriers using the detachment technique.
  • the detachment technique is believed applicable to the serial cultivation of many anchorage-dependent cells including chicken embryo fibroblast cells as well as cells which can grow both on a surface and in suspension, such as Chinese hamster ovary cells and baby hamster kidney cells.
  • cells serially propagated on microcarriers using the detachment process of the invention were capable of product formation.
  • serially cultivated FS-4 cells produced Beta-interferon and Vero cells grown in the same manner supported production of vesicular stomatis virus. This indicates that the high pH trypsin treatment does not impair the productive capabilities of cells.
  • cells can be grown in large scale culture entirely on microcarriers, and the cells can be used for the production of viruses, vaccines, hormones, lymphokines or other cellular growth by-products.
  • FS-4 cells were maintained in a roller bottle for up to six weeks after having been inoculated from the previous passage.
  • the medium was replenished weekly with DME supplemented with 2% FCS and 8% newborn calf serum (NCS).
  • the monkey kidney cell line, Vero was obtained from Flow Laboratories (McLean, VA). The cultivation conditions were the same as those for FS-4 cells except that the DME medium was supplemented with 10% horse serum (HS) and each confluent roller bottle was used to inoculate eight new roller bottles per passage. All media used were supplemented with penicillin G (100 units/ml) and streptomycin (100 ug/ml). Staining of Cells for Microscopic Examination
  • Microcarriers were prepared by the procedure of Levine et al., Biotechnol. Bioeng. 21, 821-845
  • the desired bead size was obtained by sieving SephadexTM G-50-80 or
  • the microcarriers regularly used in this study were SephadexTM beads having diameters from 53 to 75 u. Larger SephadexTM beads having diameters ranging from 90 to 105 u were used in some experiments.
  • Diethylaminoethylchloride-hydroxychloride (DEAE Cl-HCl; Sigma Chemical Co., St. Louis, MO) was first recrystalized from methylene chloride before being used to prepare microemulsions.
  • DEAE Cl-HCl Diethylaminoethylchloride-hydroxychloride
  • To prepare charged microcarriers 40 g of sieved dry Sephadex beads were suspended in 480 ml of distilled water and placed in a two-liter round-bottom flask. Two hundred and forty ml of 2 M DEAE.HCl were added and the temperature of the reaction mixture was increased to 50°C by rotating the flask in a water bath. After 30 minutes, the reaction was started by the addition of 240 ml of prewarmed 3 N NaOH.
  • the reaction was carried out at 50°C while rotating the round bottom flask in a water bath. The reaction was allowed to proceed for one hour and then quenched by the addition of one liter of distilled water. The beads were poured into two 2-liter sintered glass funnels and were washed with 20 1 of water followed by 12 1 of 0.1 N HCl and 24 1 of 0.0001 N HCl.
  • microcarriers The charge density of microcarriers was quantified immediately after the washings. To measure the charge density, the microcarriers were washed with 10% (w/w) sodium sulfate (75 ml/g Sephadex G-50) and the effluent was collected. The effluent was then titrated with 1.0 N silver nitrate in the presence of potassium chromate indicator. Microcarriers prepared by this procedure typically have an ion exchange capacity of 1.8 to 2.1 meq/g dextran.
  • microcarrier stock suspension To prepare a microcarrier stock suspension, titrated microcarriers were washed with 25 volumes of distilled water followed by 40 volumes of calcium- and magnesium-free phosphate buffered saline (PBS). Washed beads were resuspended in PBS at 10 g/l, autoclaved, and maintained under sterile conditions until use.
  • PBS calcium- and magnesium-free phosphate buffered saline
  • the impeller was placed 1 cm above the bottom of the flask to avoid destruction of the microcarriers by shear.
  • the spinner flasks were siliconized with 1% Prosil (VWR Sci. Co.) prior to use to prevent microcarriers from sticking to the vessel wall.
  • the medium used for microcarrier culture was Dulbecco's Modified Eagle (DME) medium.
  • DME Dulbecco's Modified Eagle
  • FCS fetal calf serum
  • Vero cells Vero cells
  • a microcarrier concentration of 5 g/l was normally used.
  • PBS Phosphate buffered saline
  • the microcarriers were then reconstituted to 80 ml with serum-supplemented medium and transferred to a spinner flask. The spinner flask was placed in a humidified carbon dioxide incubator to allow equilibration of both temperature and pH before inoculation with cells.
  • the inoculum was obtained by trypsinizing cells from seed roller bottles with a 0.2% trypsin solution in PBS (no calcium or magnesium) containing 0.02% ethylenediamine tetraacetic acid (EDTA). After trypsinization, cells were pelleted by centrifugation at 800 x g for ten minutes. The pelleted cells were resuspended in the culture medium and the concentration of viable cells was determined by the dye exclusion method using a hematocytometer. The dye employed was 0.1% trypan blue in PBS. The volume of the inoculum was then increased to 20 ml with prewarmed medium and inoculated into the microcarrier suspension. An impeller speed of 65 to 75 rpm was used for agitation.
  • PBS calcium or magnesium
  • EDTA ethylenediamine tetraacetic acid
  • the impellers of the spinner were modified. Two 45 degree pitched blades (2.2 x 22 cm) were installed onto the paddle to permit a lower agitation rate (45 rpm).
  • the 500 ml cultures were carried out in one liter vessels. Eighty cm of silicone rubber tubing (0.058 in. i.d., 0.077 in. in o.d., silastic tubing, Dow Corning) were placed in the vessel. A surface aerator was used for the cultivation of Vero cells to improve oxygen transfer. For FS-4 cells, the dissolved oxygen level could be maintained at about 40% of the saturation level by aeration without the use of silicone rubber tubing.
  • DME medium the pH of the culture ranged from 7.15 to 7.35 for FS-4 cells and from 7.00 to 7.35 for Vero cells. However, when a DME/F12 mixture was used for the FS-4 cells, pH decreased significantly in the absence of pH control.
  • pH was regulated by activating a controller which allowed air to pass through silicone rubber tubing, thereby reducing the dissolved CO 2 concentration.
  • the air flow rate through the silicone rubber tubing was about 100 ml/min.
  • Cell growth on microcarriers was monitored according to the method of van Wezel, supra, which is based on the original technique of Sanford et al., J. Natl. Cane. Inst. 11, 772 (1949).
  • a 2 ml sample of the culture was withdrawn from a wellmixed spinner vessel and then centrifuged at 1,000 rpm for 3 minutes.
  • the supernatant was decanted and pelleted microcarriers were resuspended in 2 ml of 0.1% (w/w) crystal violet in 0.1 M citric acid. After incubation for one hour at 37°C, the suspension was mixed with a Pasteur pipette to detach cell nuclei from the microcarriers. Stained nuclei were then counted in a hematocytometer. Detachment of Cells from Microcarriers
  • Microcarriers were withdrawn from a spinner flask and placed in a 250-ml plastic centrifuge tube. After the supernatant medium was removed, cells and microcarriers were washed extensively with 30 volumes (ml/ml beads) of PBS.
  • a trypsin solution was prepared by diluting a concentrated stock solution ten-fold in a saline solution containing 30 mM HEPES buffer, 4 mM glucose, 3 mM KCl, 130 NaCl, 1 mM Na 2 HOP 4 and 0.0033 mM phenol red (McKeehan et al., 1977).
  • the diluted trypsin solution contained 0. 2% trypsin and 0.02% EDTA.
  • the trypsin solution could also be prepared in PBS with pH adjustment. The pH was adjusted with 2 N aaOH to 8.9 to 9.0. Solutions of pH 8.2 to 8.4 were also used to detach cells successfully.
  • Trypsinization was performed in a sintered glass funnel (50 ml) or in a chromatographic column (2.5 x 10 cm, Biorad Co., NY) to facilitate the removal of excess trypsin solution.
  • a sintered glass funnel 50 ml
  • a chromatographic column 2.5 x 10 cm, Biorad Co., NY
  • the microcarrier suspension was then transferred to the sintered glass funnel or the chromatographic column, the microcarriers were allowed to settle and the excess trypsin to flow through the bed formed by microcarriers. A portion of the microcarriers was withdrawn periodically for microscopic examination. When cells became more spherical, residual trypsin was suctioned from the top of the microcarrier bed.
  • microcarriers were suspended in prewarmed medium. Cells so treated could be detached by repeatedly pipetting the medium if the volume was small. For larger volumes, the microcarrier suspension was passed through a 30 cm high, 1.5 cm diameter column packed with glass beads having a diameter of about 3 mm. The bed height of glass beads was 20 cm. Typically more than 90% of the cells on the microcarriers were released under such conditions. If a significant portion of cells remained attached, the microcarrier suspension was repeatedly passed through the column. The cell suspension, along with the used microcarriers, was then inoculated into a new culture vessel. The inoculation procedure was the same as described above in the microcarrier culture section. Under the conditions used, cells reattached to microcarriers within half an hour after inoculation into the new vessels. Measurement of Trypsin Activity
  • the proteolytic activity of trypsin was measured with a chromogenic substrate, azocollagen (Azocoll, Sigma Chemical Co.). Solutions of Azocoll (10 mg/ml) of different pH were prepared in 30 mM buffer. Trypsin solutions of various pH's were prepared in 30 mM HEPES buffer. To measure trypsin activity, 4 ml of the trypsin solution were added to test tubes containing an equal volume of substrate solution at the same pH. The mixtures were incubated at 37°C for 15 minutes. One ml samples were withdrawn from each tube periodically and filtered through filter paper. The color intensity of the filtrate determined with a spectrophotometer (520 nm). Vesicular Stomatitis Virus (VSV) Production
  • VSV Vesicular Stomatitis Virus
  • a 500 ml culture of Vero cells in a one-liter vessel was used for production of VSV as described by Giard et al. (1977), "Virus Production with a Newly Developed Microcarrier System” Appl. Environ, Microbiol. 34, 668-672.
  • the multiplicity of infection (moi) defined as the ratio of viral plaqueforming units (PFU) to cell number, was 0.1.
  • microcarriers were allowed to settle and 250 ml of the medium were removed to reduce the culture volume. For one hour, the pH was maintained between 6.5 and 6.8, and the culture was agitated occasionally (about one minute in every ten) to ensure even adsorption of virus onto the cells.
  • the VSV titer was determined by a plaque assay, using secondary chicken embryo fibroblasts.
  • Ten-day-old chick embryos were used to establish the primary culture. Two days prior to the plaque assay, confluent primary chicken or embryo fibroblasts were trypsinized and inoculated into 6 cm diameter Petri dishes (5 x 10 5 cells per dish) to start secondary cultures. The medium for secondary cultures was DME supplemented with 1% chicken serum, 2% tryptose phosphate broth and 1% calf serum. To perform the plaque assay, serial ten-fold dilutions of the virus were made in DME supplemented with FCS (10%) and .2 ml of diluted sample was inoculated onto each dish. Each sample was assayed in duplicate.
  • the virus was allowed a one-hour adsorption period at 37°C in a humidified 10% CO 2 atmosphere. After the adsorption period, 2 ml of 1% agar overlay (consisting of DME medium + 10% FCS) was added to each dish, and then the dishes were incubated at 37°C for two days. Plaques were scored after the dishes were stained with a 1:2, 500 dilution of neutral red in PBS. Interferon Production
  • Interferon production by FS-4 cells was measured by assay for the inhibition of virus-induced cytopathic effect (CPE) as described by Havell and vilcek, supra. Samples were assayed in duplicate using 96-well microplates. One hundred microliters (ml) of medium (DME supplemented with 2% FCS) was added to each well. Serial two-fold dilutions of prediluted samples were performed in each row of twelve wells. To each well 5 x 10 4 FS-4 cells in
  • FS-4 cells grown to confluence on microcarriers were washed extensively and resuspended in 0.2% trypsin solution in 20 mM HEPES buffered at various pH's. After an exposure time of three minutes, a profound change in cell morphology was observed.
  • Figure 1 cells are shown before trypsinization ( Figure 1A), after trypsinization at pH 7.4 ( Figure IB) and after trypsinization at pH 8.6. With increasing pH of the trypsin solution, cells changed from an elongated polarized shape to a more retracted, round form. The retraction of the elongated shape did not occur at the pH range typically used in cell culture (7.4 - 7.0) even after fifteen minutes trypsinization.
  • FS-4 cells grown to confluence on roller bottles were trypsinized at pH 9.0 and subsequently inoculated. into microcarrier culture. Cells so . treated grew normally to confluence (data not shown). Thus, short exposure to a high pH did not appear to have any observable detrimental effect on the growth of FS-4 cells.
  • FS-4 cells grown in microcarriers were trypsinized at various pH values above 7.0 to determine the optimum range of pH for detachment. After treatment, cells could be detached from microcarriers by mild mechanical manipulation, such as repeated pipetting. However, at the culture volume used in this study, 100 to 500 ml, it was more convenient to remove cells by passing the trypsinized microcarrier suspension through a small conduit or through a column packed glass beads as described above. To quantify the effect of pH on cell detachment, confluent cells on microcarriers were treated with a 0.2% solution of trypsin at different pH values and subsequently passed through the packed glass-bead column. As shown in Figure 2, cell detachment by trypsinization improved progressively with increasing pH.
  • Multiplication ratio is the ratio of the total surface area available for cell growth in the newly inoculated culture to that of the seed microcarrier culture.
  • a 100 ml culture at 5 g/l microcarrier concentration used to inoculate a 400 ml culture at the same microcarrier concentration yields a multiplication ratio of four.
  • the resulting 400 ml culture contains of 1.5 g of new microcarriers and 0.5 g of carried-over used microcarriers.
  • a microcarrier suspension was inoculated with FS-4 cells grown in roller bottles. At the end of the period of exponential cell growth, 100 ml of the culture were withdrawn and trypsinized at pH 8.6 as described above. About 90% of cells were detached from microcarriers. The detached cells, along with cells remaining on microcarriers, were inoculated into a 400 ml culture (Culture II in Table 2). After cell attachment, a sample was withdrawn for cell enumeration. The resulting cell concentration in the 400 ml culture was 3.4 x 10 5 cells/ml. There was no apparent loss of cells.
  • proteolytic activity of trypsin solution was assessed at different pH with a chromogeni ⁇ substrate, Azocoll. As shown in Figure 4, the proteolytic activity of the enzyme did not change much over the tested pH range. Activity increased with increasing pH to a maximum at pH 7.9-8.2. Further increase in pH resulted in a decrease on the proteolytic activity. The increase in the proteolytic activity coincided with the observed improvement in cell detachment shown in Figure 2. The subsequent reduction in proteolytic activity at pH higher than 8.4, however, did not impede cell detachment. Furthermore, the difference of proteolytic activity over the tested pH range was too small to account for the effect on cell detachment. The insensitivity of trypsin activity has been reported by Northrop and Kunitz, J. Gen.
  • Trypsinization was allowed to proceed at room temperature for five minutes after which microcarriers were resuspended in 50 ml of PBS and then passed through the packed column of glass beads. In the other two cases, the trypsinization was also carried out at pH 7.2 for five minutes but, after trypsinization, cells were incubated in buffered solutions at different pH values. Before incubation, the microcarriers were suspended in 50 ml of PBS and centrifuged at 200 x g for one minute. The supernatant was discarded and the microcarriers were mixed with the HEPES buffers containing 50 mg/l of soybean trypsin inhibitor (Sigma Chemical Co.).
  • the composition of the HEPES buffer was the same as that used for trypsin solution.
  • the pH of the HEPES buffer was 8.7 and the other 7.2. After incubation in the buffer solution at room temperature for five minutes, the supernatant was withdrawn, the microcarriers resuspended in 50 ml PBS and subsequently passed through the packed glass bead column.
  • the detached cells were enumerated in a hematocytometer. As shown in Table 2, more than 90% of cells were detached from microcarriers when trypsinization was carried out at a high pH. In contrast, less than 30% of cells were detached at pH 7.2. After the removal of trypsin, further incubation at a physiological pH had little effect on cell detachment, whereas incubation at pH 8.7 facilitated cell detachment significantly.
  • a 500 ml culture was inoculated with roller bottle-grown FS-4 cells at a concentration of 7 x 10 4 cells/ml.
  • a high multiplication ratio was achieved by the use of microcarriers having a median diameter 40% larger than the microcarriers routinely used in this laboratory (270 micron median diameter instead of 190 micron), and the use of DME/F-12 (50:50) mixture supplemented with 5% FCS as the medium.
  • Confluent cells from this culture (40 mis of culture; 1.35 x 10 6 cells/ml) were then trypsinized at pH 8.5 and subsequently detached to inoculate a second culture. In the second culture, cells grew normally until reaching a confluent concentration of 1.30 x 10 6 cells/ml.
  • monkey kidney epithelial cells (Vero) were tested. These cells are commonly used for vaccine production.
  • a culture was inoculated at a concentration of 3.0 x 10 5 cells/ml and grown to confluence. The confluent cell concentration was 3.8 x 10 6 cells/ml. Forty ml of the confluent culture were trypsinized at pH 9.0 with the procedure described above and used for the inoculation of a 500 ml culture at a cell density of
  • Vero cells thus cultivated grew very well with no appreciable lag phase or decrease in growth rate. After cells grew to confluence, they were infected with vesicular stomatitis virus (VSV) to test the affect of direct inoculation on virus production. The results are shown in Figure 7. The production period lasted for about fifteen hours. The titer of virus obtained was comparable to that reported in the literature, Giard et al., supra.
  • VSV vesicular stomatitis virus

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Abstract

Dans un procédé utilisé pour dégager des cellules de micro-porteurs positivement chargés de culture cellulaire, les micro-porteurs chargés de cellules attachées sont exposés à un agent de protéolyse à un pH relativement élevé et ensuite soumis à un cisaillement modéré.
PCT/US1985/001615 1984-08-28 1985-08-27 Procede pour degager de micro-porteurs des cellules dependant d'ancrage WO1986001531A1 (fr)

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0317810A2 (fr) * 1987-11-23 1989-05-31 IMMUNO Aktiengesellschaft Procédé et appareil pour séparer des cultures cellulaires de leurs microsupports
WO1998033886A1 (fr) * 1997-01-31 1998-08-06 Schering Corporation Procedes pour la culture de cellules et la multiplication de virus
US6168944B1 (en) 1997-01-31 2001-01-02 Schering Corporation Methods for cultivating cells and propagating viruses
US6783983B1 (en) 1997-01-31 2004-08-31 Schering Corporation Methods for cultivating cells and propagating viruses
WO2009018847A1 (fr) * 2007-08-09 2009-02-12 Fraunhofer-Gesellschaft Zur Förderung Der Angewandten Forschung_E.V. Dispositif et procédé de détachement de cellules
CN101775364A (zh) * 2010-03-05 2010-07-14 广州齐志生物工程设备有限公司 一种动物细胞的分离方法及分离装置
CN109689855A (zh) * 2016-12-27 2019-04-26 富有干细胞株式会社 从三维多孔支架回收培养细胞的方法
CN110272859A (zh) * 2019-04-15 2019-09-24 中国辐射防护研究院 一种贴壁细胞的培养方法
US10801003B2 (en) 2016-06-03 2020-10-13 Lonza Ltd Single use bioreactor
US11559811B2 (en) 2017-02-10 2023-01-24 Lonza Ltd. Cell culture system and method

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JP5350364B2 (ja) * 2007-04-28 2013-11-27 ヒョンジン ヤン， 粉末状の足場を用いた細胞間の信号調節による細胞培養方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4189534A (en) * 1976-11-11 1980-02-19 Massachusetts Institute Of Technology Cell culture microcarriers
GB2059991A (en) * 1979-09-12 1981-04-29 Pharmacia Fine Chemicals Ab Cultivating cells on particulate microcarriers

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4189534A (en) * 1976-11-11 1980-02-19 Massachusetts Institute Of Technology Cell culture microcarriers
GB2059991A (en) * 1979-09-12 1981-04-29 Pharmacia Fine Chemicals Ab Cultivating cells on particulate microcarriers

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Biotechnology-Bioengineering, Volume 23, 1981, John Wiley & Sons, Inc. C.L. CRESPI et al.: "Continuous Cell Progagation using Low-Charge Microcarriers", see page 983, line 12 - page 984, line 11; page 989, lines 10-18 *
Biotechnology-Bioengineering, Volume 23, 1981, John Wiley & Sons, Inc. C.L. CRESPI et al.: "Microcarrier Culture: Applications in Biologiclas Production and Cell Biology", see page 2675, lines 7-17; page 2676, lines 25-36; page 2678, lines 2-18, 23-41; page 2685, lines 5-25 *

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0317810A2 (fr) * 1987-11-23 1989-05-31 IMMUNO Aktiengesellschaft Procédé et appareil pour séparer des cultures cellulaires de leurs microsupports
DE3739649A1 (de) * 1987-11-23 1989-06-22 Immuno Ag Verfahren zum abloesen von zellkulturen von mikrotraegern
EP0317810A3 (fr) * 1987-11-23 1991-02-06 IMMUNO Aktiengesellschaft Procédé et appareil pour séparer des cultures cellulaires de leurs microsupports
US5100799A (en) * 1987-11-23 1992-03-31 Immuno Aktiengesellschaft Method for releasing cell cultures from microcarriers
US7390652B2 (en) 1997-01-31 2008-06-24 Schering Corporation Device for cultivating cells and propagating viruses
US6783983B1 (en) 1997-01-31 2004-08-31 Schering Corporation Methods for cultivating cells and propagating viruses
EP1731598A1 (fr) * 1997-01-31 2006-12-13 Schering Corporation Procédés pour la culture de cellules et la multiplication de virus
WO1998033886A1 (fr) * 1997-01-31 1998-08-06 Schering Corporation Procedes pour la culture de cellules et la multiplication de virus
US6168944B1 (en) 1997-01-31 2001-01-02 Schering Corporation Methods for cultivating cells and propagating viruses
WO2009018847A1 (fr) * 2007-08-09 2009-02-12 Fraunhofer-Gesellschaft Zur Förderung Der Angewandten Forschung_E.V. Dispositif et procédé de détachement de cellules
CN101775364A (zh) * 2010-03-05 2010-07-14 广州齐志生物工程设备有限公司 一种动物细胞的分离方法及分离装置
US10801003B2 (en) 2016-06-03 2020-10-13 Lonza Ltd Single use bioreactor
US11371002B2 (en) 2016-06-03 2022-06-28 Lonza Ltd Single use bioreactor
CN109689855A (zh) * 2016-12-27 2019-04-26 富有干细胞株式会社 从三维多孔支架回收培养细胞的方法
CN109689855B (zh) * 2016-12-27 2021-10-26 富有干细胞株式会社 从三维多孔支架回收培养细胞的方法
US12049641B2 (en) 2016-12-27 2024-07-30 Fullstem Co., Ltd. Method for harvesting cultured cells from three-dimensional porous scaffold
US11559811B2 (en) 2017-02-10 2023-01-24 Lonza Ltd. Cell culture system and method
CN110272859A (zh) * 2019-04-15 2019-09-24 中国辐射防护研究院 一种贴壁细胞的培养方法

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