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  • 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
  • C12N2533/00—Supports or coatings for cell culture, characterised by material
  • C12N2533/70—Polysaccharides
  • C12N2533/78—Cellulose
• • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
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  • F02B—INTERNAL-COMBUSTION PISTON ENGINES; COMBUSTION ENGINES IN GENERAL
  • F02B61/00—Adaptations of engines for driving vehicles or for driving propellers; Combinations of engines with gearing
  • F02B61/04—Adaptations of engines for driving vehicles or for driving propellers; Combinations of engines with gearing for driving propellers
  • F02B61/045—Adaptations of engines for driving vehicles or for driving propellers; Combinations of engines with gearing for driving propellers for marine engines

Definitions

• This invention is related to a method for growing cells, especially diploid cells and in particular diploid cells of mammals, such as human cells, e.g. fibroblast cells, in a growth medium in the presence of cellulose fibres of a special type.
• Said cellulose fibres consist of cellulose carrying positive charges and/or"anion exchanging cellulose.
• This type of cellulose is well-known and certain types of such cellulose are disclosed e.g. in "Techniques in Protein Chemistry", Elsevier Publishing Company, 1967, I. Leggett, pages 290-304. Suitable materials are commercially available e.g. from Sigma Chemical Co., St. Louis, MO, USA.
• amino groups which may be substituted in various ways, are usually bonded to the cellulose molecule and give anion exchange characteristics.
• the cells which are grown according to this invention preferably consist of so-called anchorage depending cells which are normally grown— ._g. in suspension.
• ECTEOLA epichlorohydrin triethanol amine
• Cell strains Human diploid fibroblasts were used in all experiments. These cells were derived from embryonic lung tissue and passed for various numbers of passages before being frozen and stored in liquid nitrogen. They are dependent cells in that they require a suitable surface onto which they attach, spread out and eventually divide. Two cell strains were used: (1) MRC-5 obtained from England and (2) a cell strain established In the inventors' laboratory designated Lu(S). The growth characteristics of both these cell strains have been thoroughly examined and have been found to be similar. They both have an in vitro life time of between 70 to 90 population divisions.
• Procedure for growing human fibroblasts Frozen ampules of cells were thawed rapidly and seeded into plastic Roux bottles containing 150 ml Eagle's medium. When the cell layer became confluent the cells were suspended in 50 ml 200 ⁇ g/ml crystalline trypsin in phosphate buffered saline without calcium and magnesium, pH 7.8, and the cells were passed into new Roux bottles with fresh medium. Eventually the harvested cells were used to seed a "spinner bottle", a bottle containing a suspended magnetic bar which is used as a stirring device, containing Eagle's medium and a suspension of cellulose fibres. The cell inoculum used was 1x10 cells/mg cellulose.
• the cellulose was used at a concentration of about 3 mg/ml. Most experiments were performed with QAE cellulose. The quantity needed for the spinner culture was centrifuged at 3,000 X g for 10 minutes, the supernatant sucked off and the cellulose resuspended in the growth medium and added to the spinner bottle.
• the QAE cellulose fibres varied in length between 6 microns to 8.00 microns or more and their width varied between 10 microns to 35 microns in diameter.
• the cells added"to the cellulose suspension attached to some of the fibres but did not spread out and grow over the fibres ' surface in the normal manner expected of anchorage dependent cells. They grew, instead, as cell masses into which cellulose fibres of different sizes were incorporated. These aggregates of cells and fibres would reach sizes of 150 to 200 microns or more in diameter after 7 to 8 days incubation.
• the fibres incorporated in these aggregates varied in length between 100 microns or less to 200 microns, longer fibres were very seldom observed.
• the width of the fibres appeared to be between 10 microns to 20 microns in diameter.
• the remainder and majority of the cellulose fibres were completely free of cells.
• the cells appeared to select a population of fibres best suited for their attachment and growth. This effect may be due to the size of the fibres or an unequal distribution of charged chemical groups.
• Cell strains Human embryonic diploid lung fibroblasts were used In all experiments. Most experiments were performed with a cell strain isolated in the inventor's laboratory designated Lu(S) at a population doubling level between 20 to 30 divisions. Some experiments were performed with the MRC5 cell strain at a population doubling level between 30 to 35 divisions. The results were similar with both strains.
• Cellulose Cell growth Occurred only on positively charged cellulose fibres (anion exchangers), no growth was observed on negatively charged fibres (cation exchangers). All celluloses were purchased from Sigma Chemical Co., St. Louis, MO, USA. The following anion exchangers were tried:
• the fibres incorporated in these aggregates varied in length between less than 100 microns to 200 microns, longer fibres were seldom observed.
• Cell-fibre aggregates can reach sizes of 150 to 200 microns in diameter after 7 to 8 days incubation. Occasionally, larger aggregates are observed.
• the cells appeared to select a population of fibres best suited for their attachment and growth. This selection appears to be related to the size of the fibres.
• a cellulose suspension filtered through a 200 mesh stainless steel screen was inoculated with cells.
• most of the fibres which were about 100 microns or less in length became associated with cells.
• Preliminary attempts to grow fibroblasts on TLC (thin layer chromatography) prep ⁇ arations of QAE and DEAE cellulose which have fibre lengths of 100 microns or less suggest that these preparations are better adapted for cell-fibre aggregates than the coarse grade celluloses.
• the anion exchanging cellulose used according to this invention preferably carries amino groups and especially an anion exchange capacity of at least 0.01 meq/g, optionally at least 0.05 meq/g or at least 0.1 meq/g.
• the anion exchange capacity is preferably up to 5 meq/g, optionally up to 3 meq/g or up to 1.5 meq/g or even up to 1 meq/g, determined with common methods.
• the anion exchanging cellulose used preferably consists of fibres, of which, based on the weight, at least 20%, preferably at least 50% and especially at least 75% or at least 90% have a fibre diameter of at most 200 microns, especially at most 100 microns, preferably at most 50 microns or at most 25 microns.
• the fibre length is preferably at most 1000 microns, especially at most 200 microns or at most 100 microns. Also fibre length values of at most 50 microns or at most 20 microns have shown to be important.
• the fibre length is also preferably at least 0.1 micron, especially at least 1 micron, preferably at least 5 microns or at least 10 microns. Said fibre length values are calculated on at least 50%, preferably at least 75%, at least 90% or at least 99% of the weight of the cellulose material.
• the anion exchanging cellulose which is used preferably belongs to one of the types:
• DEAE diethylamino ethyl cellulose, preferably with an ion exchange capacity of about 0.7-0.9 meq/g,
• TEAE triethylamino ethyl
• TEAE triethylamino ethyl
• amino ethyl cellulose preferably wit an ion exchange capacity of 0.33 meq/g
• benzyl-DEAE cellulose benzoylated-naphthoylated DEAE.
• diploid cells of mammals are grown, preferably human cells, such as fibroblast cells, and said growth can be performed also for the growth of virus or for the production of vaccine.
• the cells are grown at the fibre or fibres as cell aggregates, especially of essentially spherical or cylindrical shape and especially to a diameter of up to 1000 microns, optionally up to 500 microns or up to 300 microns or even up to 200 microns or 100 microns. Growth of the cell aggregates is preferably performed until at least 10%, at least 50% or at least 90% of the weight of the cell material is present as such cell aggregates with a diameter of at least 10 microns, preferably at least 25 microns or at least 50 microns, optionally at least 100 3 microns.
• the cells of said cell aggregates form an aggregate of more than one monolayer of cells.
• Said cell aggregates include preferably at least a part of two or more fibres within the cell aggregate.
• Said fibres preferably have fibre length and fibre diameter values within the limit mentioned above, e.g. a fibre length of at most 500 microns and especially at most 300 microns and e.g. a diameter between 3 and 50 microns, especially between 5 and 30 microns, e.g.
• Suitable values of the upper limit of the concentration * of cells are e.g. at most 10 9 , at most 108 or at most 107 cells/ml dispersion.
• the cell growth time can be varied, e.g. amount to at least 1 day, at least 2 days or at least 3 days or at least 5 days.
• Suitable upper limits of the cell growth time can be e.g. up to 14 days, up to 10 days, up to 7 days or up to 5 days.
• Suitable concentrations of cellulose fibres in the growth medium can be at least 0.01 mg/ml, at least 0.1 g/ml or at least 1 mg/ml, optionally at least 5 mg/ml. Suitable upper limits were found to be e.g. at most 500 mg/ml, at most 100 mg/ml, at most 50 mg/ml or at most 10 mg/ml.
• Suitable growth media and other growth conditions for various cell types are well-known to those skilled In the art and can be used for this invention.
• reference can be made e.g. to the publication "Microcarrier Cell Culture Principles & Methods” from Pharmacia Fine Chemicals and to the literature references mentioned in said publication " .
• the use of human diploid fibroblasts for polio vaccine production has been limited in the past because of the technical difficulties in growing these cells on currently available microcarriers.
• the cells grew to relatively low concentrations and the culture was so sensitive to manipulation that a large number of cells on the microcarriers could be lost in the various treatment procedures a culture undergoes.
• the use of positively charged cellulose micro ⁇ carriers according to this invention has eliminated many of these technical problems.
• the human diploid fibroblast cells grew well as cell-fibre aggregates with several of cellulose fibres tried, e.g. diethyl 2-hydroxypropylamino ethyl (QAE), diethylamino ethyl (DEAE), triethylamino ethyl (TEAE).
• Cellulose anion exchanger fibres Most of the experiments were done with QAE or DEAE cellulose, particularly preparations with short fibre lengths made for thin layer chromatography (TLC) . Other celluloses used were TEAE and benzyl-DEAE. All the celluloses had a capacity of about 0.9 meq/g. The celluloses were used at a concentration of 3 mg/ml.
• the medium used for cell growth was Eagle's minimum essential medium supplemented with 10% calf serum, 4 mM glutamine, 20 mM TRICINE buffer, pH 7..8 and 1 rnM Na pyruvate.
• the cells were first grown to a confluent monolayer in plastic Roux bottles, suspended with trypsin (200 ⁇ g/1 crystalline trypsin (Sigma) in phosphate buffered saline solution containing 20 mM TRICINE, pH 7.7-7.8 and 0.08% sodium bicarbonate), centrifuged, washed once and added at a l ⁇ concentration of about 1.5x10 cells/mg cellulose. After 5-7 days incubation the cell-fibre mass was washed three times with phosphate buffered saline solution, pH 7.4, resuspended to the original volume in Parkers 199 medium without serum and infected with polio virus.
• trypsin 200 ⁇ g/1 crystalline trypsin (Sigma) in phosphate buffered saline solution containing 20 mM TRICINE, pH 7.7-7.8 and 0.08% sodium bicarbonate
• polio virus type 1 (Brunender) was used and type 2 polio virus was used in one experiment.
• the virus infectivity was measured by inoculating the virus preparations at ten-fold dilutions in tissue culture tubes and the dilution where 50% of the cultures would be infected was calculated and referred to as "Tissue Culture Infectious Dose", 50% end point (TCID--). All titers are given in log-, 0 TCID-.-.
• Table II shows the results from 5 independent 100 ml suspension cultures of Lu(S) cells with 3 mg/ml QAE cellulose infected with 6.3 TCID-- type 1 polio virus.
• Table III describes the results from five 100 ml suspension cultures of MRC 5 cells with 3 mg/ml of the celluloses listed. The cultures were infected with 6.0 TCID 5Q /ml type 1 polio virus. TABLE III
• Polio virus type 2 was inoculated at a concentration of 5.0 TCID gQ /ml into a 5 litres suspension culture of MRC 5 with 3 mg/ml DEAE-TLC cellulose. After 24 hours the titer had risen to 6.5 and after 48 hours to 6.8 TCID gQ /ml. Although these titers were low, presumably because the inoculum was too low, it showed that the virus was capable of increasing almost 100-fold which is about the same increase obtained with type 1 polio virus.
• Human diploid fibroblasts are considered the best available cell substrate for human virus vaccines because the cell strains are easily controlled and standardized, extremely well investigated, support the growth of most human viruses and they are completely normal eliminating the risk of oncogenic DNA coming into the vaccine preparation. It has, however, been difficult to use these cells for vaccine production because of the difficulty of growing these cells in large scale on microcarriers.
• human diploid fibroblasts can grow to sufficient cell concentrations together with cellulose microcarriers (e.g. to between 1.5 to g 2x10 cells/ml) so that when infected with polio virus, titers can be obtained which are significantly greater than those obtained in monolayer cultures of these cells or e.g. monkey kidney cells.
• the cells which were grown consisted of human diploid fibroblast cells, "strain MRC5". This cell strain is described e.g. in an article by Jacobs, Jones, Bailie: "Characteristics of a human diploid cell designated MRC5" in Nature, Vol. 227, pages 168-170, 1970, and was obtained from National Institute for Biological Study and Control.
• the growth medium consisted of "Eagle's minimum essential medium” in all experiments, supplemented with 10% calf serum, 20 mM Tricine pH 7.8, 4 mM glutamine, 1 mM Na pyruvate and 0.08% bicarbonate.
• microcarriers were used:
• Microcarrier according to the invention DEAE cellulose fibres (small fibre) intended for thin layer chromatography, capacity 0.96 meq/g.
• Biosilon which is a plastic bead microcarrier and was added to the medium as a sterile powder.
• Cytodex and Gelibeads were prepared according to the manufacturers' direction.
• the cellulose according to the invention was used at a concentration of 3 mg/ml, biosilon in a quantity of 60 mg/ml, cytodex and gelibeads at a concentration of 4 mg/ml.
• the experiment comprises determining titer values for polio virus type 1 obtained from the cell cultures on microcarriers stated above.
• MRC5 passage 21 was inoculated into suspension cultures at a concentration of 4x10 cells/ml. The cultures were stirred at a rate of about 40 revolutions per minute. The medium was changed once after 4 days incubation by allowing the cell microcarrier mass to settle to the bottom of the flask, suck off the old medium and adding an equal volume of fresh medium.
• the cells which are grown according to this invention consist preferably of normal cells, i.e. cells with a restricted ability to undergo division, e.g. usually in average at most 150, at most 100 or at most 70 divisions (so-called population divisions) . Definitions of such cells can be found in literature, e.g. Haeflick, Moorhead: "The serial cultivation of human diploid strains", Experimental Cell Research, Vol. 25 (1961), pages 585-621, and Haeflick: "The limited in vitro lifetime of human diploid cell strains". Experimental Cell Research, Vol. 37 (1965), pages 614-636.
• animal cells in general, vertebrate cells, e.g. cells of birds, e.g. gallinaceous birds, mammal cells, e.g. cells of rat, mouse, ape, cattle, horse, swine, sheep, dog, cat, and especially human cells.
• the cells may also come from special organs, e.g. lung, skin, foreskin, epitelium cells.
• embryo cells chicken embryo fibroblasts may also be mentioned.
• the cell culture can be used for producing various products, e.g. enzymes, hormones, e.g. interpheron, as a basis for growing virus and for preparing vaccines against these, e.g. polio, rabies, rubella, influenza, measles, herpes, pseudo-rabies, foot and mouth disease, virus causing aids, etc. It is suitable to use cell cultures which are accepted for vaccine production by WHO.
• Suitable dimensions of the cellulose fibres are stated above.
• a fibre length of at least 20 microns, at least 40 microns or at least 60 microns is frequently suitable.
• the upper limit of the fibre length may be around the previously mentioned limit values, e.g. at most 200 microns or at most 150 microns or even at most 100 microns.
• the fibre length values are then calculated, as the previously mentioned fibre length values, on at least 30%, preferably at least 50%, especially at least 75%, at least 90% or at least 99% of the weight of the cellulose material.
• Cellulose which is used as a basis for the anion exchanging cellulose materials used according to this invention normally consists of a polymer of glucose with about 2000-4000 or more, e.g. 3500 or more repeated units in a chain.
• the glucoside bond is at ⁇ , the cellulose can be defined as a polymer of ⁇ -D-glucose.
• Figure 1 The growth of Lu(S) cells in 100 ml suspension cultures containing 3 mg/ml QAE, DEAE or TEAE cellulose and iinnooccuullaatteedd wwiitthh 2200xx1100 cceellllss.. TT]he cell number was estimated from the measured protein value.
• Figure 2 The effect of different QAE cellulose concentration on the growth of Lu(S) cells in 100 ml suspension cultures iinnooccuullaatteedd wwiitthh 2200xx1100 cceellllss.. TT!he cell number was estimated from the measured protein value.
• Figure 3 The effect of cell inoculum on the growth of Lu(S) cells in 100 ml suspension cultures containing 3 mg/ml QAE cellulose. The cell number was estimated from the measured protein value.
• Figure 4 The morphological appearance of human diploid fibroblast cell fibre aggregates (see arrow).
• a and B 4 days MRC 5 culture with DEAE cellulose, 38OX.
• C 13 days MRC 5 culture with 3 mg/ml QAE cellulose, 380X.
• D 8 days Lu(S) culture with about 1 mg/ml QAE cellulose filtered through a 200 mesh stainless steel screen, 48X.
• E 8 days Lu(S) culture with about 1 mg/ml QAE cellulose filtered through a 200 mesh stainless steel screen, 960X.
• F 13 days MRC 5 culture with 3 mg/ml QAE cellulose, 120X.

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