FI60407B - SAETT ATT ODLA FOERANKRINGSBEROENDE CELLER I EN MIKTOBAERARKULTUR - Google Patents

Description

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Patent and registration holders .... .... .... .....

__. . . . . (44) Date of issue of the declaration.

Patent- och registerstyrelsen '' Antökan utlagd och utl. , Mass., USA (72) David W. Levine, Somerville, Mass., William G. Thilly, Cambridge,

Mass., Daniel I.C. Wang, Belmont, Mass., Jason S. Wong, Toledo,

Ohio, USA (US) (TM Oy Kolster Ab (5 * 0 Method of culturing attachment-dependent cells in microcarrier culture - Sätt att odla förankringsberoende Celler i en mikrobärar-kultur

The invention relates to a method for culturing attachment-dependent cells in a microcarrier culture for the preparation of cell cultures and optionally cell culture products such as viruses, hormones or interferon, forming a suspension of positively charged microcarriers and attachment-dependent cells in a suitable culture medium. are maintained under conditions that promote the formation of cell culture products, which are then recovered.

It is important that mammalian cells be able to be cultured on both laboratory and factory scales. On a laboratory scale, the amount of test raw material that is often available is a limiting factor in cell or viral studies. Much effort has been made on an industrial scale to develop pharmaceutical preparations based on mammalian cell products. These are primarily vaccines against human and animal viruses, but also include human growth hormone and other body hormones, as well as biochemical agents for medical applications.

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Certain mammalian cell types are suitable for culturing in suspension cultures. Examples of such cell types are HeLa (human), BHK (hamster kidney kidney) and L cells (mouse). Such cells generally do not have abnormal hereditary complements, i.e., too many or too few chromosomes or abnormal chromosomes. Often, these cells cause a tumor when they are injected into that cell. species.

To date, no other mammalian cell types have been used for culture in suspension cultures and will only grow if they are allowed to adhere to a suitable surface. Such cell types are commonly referred to as "attachment-dependent" and include 3T3 mouse fibroblasts, mouse bone marrow epithelial cells, murine fibroblast Muridae anticancer virus producing strains, primary and secondary chicken CCroblasts, WI-38 human fiboflasts, and normal human fibroblasts. · Some of the attachment-dependent cells that cause tumors have been cultured; other cultured attachment-dependent cells, on the other hand, have been found to be non-tumorigenic. Certain adhesion-dependent cells, such as WI-38 and HEL-299j, which are hereditary normal, can also be cultured.

Although considerable progress has been made in large-scale mammalian cell culture using cell lines capable of growing in suspension culture, progress has been very limited with respect to large-scale culture of attachment-dependent mammalian cells. Previous working methods used for large-scale culture of attachment-dependent cells are based on a straightforward extension of small-scale methods. Cell culture plants use a large number of low-yield batch reactors in the form of plates, vials, rotating culture tubes, and culture flasks. Each of these is a stand-alone unit or an isolated batch reactor that requires individual environmental control. However, for economic reasons, these adjustments are very rudimentary in quality. The change in nutrients is corrected by changing the medium, a procedure that requires two steps, i.e., removal of the medium and addition of the medium. Because it is not uncommon for a moderately sized plant to simultaneously process hundreds of such batch reactors, even a single medium change requires hundreds of work steps, all of which must be performed accurately and under demanding sterile conditions. Each multi-step job, such as cell switching or recovery, complicates the problem accordingly. Therefore, in this type of facility, the cost is high in terms of equipment, space and manpower.

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There are alternative methods that have been proposed for a linear increase in small batch crops. Such alternatives that have been described in the literature include plastic bags, stacked plates, helical membranes, culture with glass beads, artificial capillaries, and microcarriers. Of these, microcarrier systems have certain significant and most unique advantages. For example, the achievable ratio between culture area and vessel volume (S / V) can be significantly improved by using microcarriers, compared to both conventional and recently developed alternative methods. The increase in achievable ratio S / V as a separate unit allows the construction of a homogeneous or pseudo-homogeneous batch or semi-batch multiplier for high volume yield. Thus, a single agitated tank with a simple return feed control for pH and pC 2 creates a homogeneous environment for a large number of cells, eliminating the need for expensive and space-consuming environmentally controlled incubators. Also, the total number of work steps required per unit of cells produced is greatly reduced. In summary, microcarriers appear to be very economical in terms of capital, space, and manpower in the production of adhesion-dependent cells, compared to conventional manufacturing methods.

Microcarriers also have the advantage that the environment is continuous because the cells are cultured in a single controlled environment. Thus, by using microcarriers, it is possible to culture attachment-dependent mammalian cells under controlled environmental conditions to achieve unchanged, optimal cell growth. One of the most promising microcarrier systems today, comprising diethylaminoethyl (DEAE) substituted dextran beads in a stirred tank, is disclosed in A.L. van Wezel, "Growth of Cell Strains and Primary Cells on Microcarriers in Homogeneous Culture", Nature 216: 6U (1967); D. van Hemert, D.G. Kilbrun and A.L. van Wezel, "Homogeneous Cultivation of Animal Cells for the Production of Virus and Virus Products", Biotechnol. Bioeng. 11: 875 (1969); and A.L. van Wezel, "Microcarrier Cultures of Animal Cells in Tissue Culture, Methods and Applications, PF, Academic Press, New York, page 372 (1973). These beads are commercially manufactured by Pharmacia Fine Chemicals, Inc., Piscataway, New Jersey, under the tradename DEAE-Sephadex A50, ion exchange system Chemically, these beads are formed from a cross-linked dextran matrix with diethylaminoethyl groups covalently bonded to dextran chains. (in which case the weight of the associated DEAE units is not taken into account 11 60407) Other ani or ht oh art, such as DEAE-Sephadex A25, QAE-Sephadex A50 and QAE-Sephadex A25, have also been shown to enhance cell growth.

The system proposed by Van Wezel combines multiple surfaces with moving surfaces, allowing for innovative manipulation and with advantages in terms of increased production and environmental control. Despite this possibility, these proposed methods have not been significantly exploited, as researchers have had difficulty producing cells due to certain adverse effects caused by beads. These include the high percentage of cell death in the inoculum and insufficient cell growth even in those cells that adhere. The reason for these adverse effects is not fully understood; it has been suggested that they may be due to bead toxicity or nutrient adsorption. See. van Wezel, A.L. (1967), Nature 216: 6U-65; van Wezel, A.L. (1973), Tissue Culture Methods and Applications, pp. 372-377, Academic Press, New York; van Hemert. P., Kilhurn, D.G. and van Wezel, A.L. (1969), Biotechnol. Bioeng. 11: 875-885; Horng. C. and McLimans, W. (1975), Biotechnol. Bioeng. 17: 713-732.

It may be that the detrimental effects of these commercially available ion exchange resins are due to the way they are prepared. Some of these methods are described in connection with the preparation of a polyhydroxy material in U.S. Patents 3,277,025; 3,275,576; 3 O + 2 667 and 3 208 99 ^; whatever the reason, however, the substances currently commercially available are simply not sufficient for the good cultivation of numerous different cell types.

One solution to overcome these detrimental difficulties in attempting to use commercially available microcarriers to grow cells is described in Levine et al., U.S. Patent No. 0,366,693. Although this method has proven successful, it would of course be more advantageous if the beads could be made to have properties originally intended for the successful cultivation of attachment-dependent cells.

It has now been found that the charge capacity of microcarriers must be adapted and / or adjusted to a certain range in order to obtain good growth of several different attachment-dependent cell types at reasonable microcarrier concentrations11a. Based on this finding, microcarrier beads with controlled charge capacities have been prepared and such beads have been used to obtain good growth in a number of adhesion-dependent cells. Cells cultured using such microcarrier systems can be harvested or used in the manufacture of animal or plant viruses, vaccines, hormones, interferon, or other cell culture products.

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Examples of improved microcarriers are those prepared using polymers with hydroxyl groups, such as cross-linked dextran beads. These beads can be treated with an aqueous solution of a tertiary or quaternary amine such as diethylaminoethyl chloride: chloride and an alkaline agent such as sodium hydroxide. The specific charge capacity of the beads is controlled by varying the absolute amounts of dextran, the tertiary amine salt and the early substance, the proportions of these substances and / or the treatment time and temperature.

The microcarriers prepared in accordance with this invention can be used in cultures without the high cell losses that have hitherto occurred with commercially available microcarriers. In addition, the adherent cells proliferate and grow together with beads, achieving very high cell concentrations in the suspension medium. The concentration of microcarriers in the suspension is not limited to very low levels, as has been the case with previously known substances; cell growth is limited only by factors that do not appear to be related to microcarriers. As a result, the volume productivity of cell cultures is greatly increased. In short, the potential offered by the use of microcarriers in cell culture, and in particular in the attachment-dependent cells, can now be exploited.

Figure 1 is a graph depicting the normal, diploid growth characteristics of human fetal lung fibroblast cells (HEL299) at a microcarrier concentration of 2 g dry, cross-linked, dextran / L and commercially available DEAE-treated dextran microcarriers according to the invention and DEAE-treated Figure 2 shows graphically the growth characteristics of both normal, diploid, human fetal lung fibroblast cells (HEL299) and secondary chick fetal fibroblast cells at a microcarrier concentration of 5 g dry, cross-linked dextran / l using the improved DEAE-treated microcant of this invention.

As used herein, the terms "microcarrier" and "cell culture microcarrier" refer to small, free particles suitable for cell attachment and culture. Often, though not always, microcarriers are porous beads formed from polymers. Usually, the cells attach and grow on the outer surface of such beads.

As previously described, it has been found that the amount of charge capacity on cell culture microcarriers must be adjusted and / or adjusted to be within a certain range to achieve suitable cell growth at a reasonable microcarrier concentration. Suitable ranges of action and preferred ranges will vary depending on factors such as the cells being cultured, the nature of the microcarriers, the concentration of the microcarriers, and the other culture parameters, including the composition of the medium. In all cases, however, the amount of suitable charging capacity has been found to be significantly less than the amounts believed to be present in commercially available anion exchange resins previously proposed for microcarrier cell cultures. For example, the charge capacity of the DEAE-Sephadex A50 beads proposed by van Wezel is likely to be about 5 x meq / g dry, untreated (without DEAE) crosslinked dextran. In contrast to this relatively high charge capacity, microcarriers have been prepared and found to be suitable for cell culture in accordance with the present invention, having a charge capacity of about 0.1 to about 1.5 meq / g of dry, untreated microcarrier. Below about 0.1 meq / g, it is assumed that the cells are difficult to adhere to the microcarrier. Above N. i +, 5 meq / g, losses occur due to the stem cell lymph, and the surviving cells do not grow well, especially at relatively high microcarrier concentrations.

For culturing normal, diploid human fibroblasts with cross-linked dextrar microcarriers, it has been found that the preferred range for the charge capacity provided by the DEAE groups is about 1.0-2.8 meq / g dry, untreated, cross-linked dextran. Although the preferred range may vary with different cell types or different culture conditions, it is believed that the preferred ranges under all conditions will be in the range of 0.1 to 0.5 meq / g. Optimum conditions can be determined by one skilled in the art by routine experimentation.

It should of course be noted that there are certain shortcomings in determining the charging capacity of microcarriers on a unit weight basis only. For example, two beads that are similar in all other respects except that they are formed of different materials with different densities and the same charge distribution would give different values for the charge capacity per unit weight. In the same way, two beads with the same charge capacities per unit weight may have a completely different charge split.

Alternative charge capacity can be determined by presenting a range of suitable charges expressed as charge capacity per unit weight of microcarriers in their final functional form. In this case, factors such as the weight of the associated DEAE groups or other positively charged groups and the hydration of the beads, etc. have been taken into account, whereas the previous definition is based on dry, cross-linked dextran and does not take such factors into account. In an aqueous cell culture medium, the density of the microcarriers should be selected to be 1.0 g / ml so that the microcarriers can be easily dispersed throughout the culture. Based on this, it has been determined that the range of suitable charging capacities of the microcarriers determined in this way according to the invention is about 0.012-0.25 meq / g.

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The weight-based limits of suitable charge capacities defined above apply provided that the microcarriers have a substantially similar charge distribution throughout the mass. If the charge distribution is uneven, microcarriers with charge capacities outside these limits could be used. An important criterion, of course, is that the charging capacity is adjusted and / or adjusted to a value sufficient for good cell growth on the microcarriers.

Since the charge distribution of the outer surface can be important, it is desirable that a suitable charge capacity range can be determined in the form of a probable surface change. This can be done by assuming that only the active surface of the microcarriers is formed by the outer surface of the bead to a depth of about 20. Assuming further that the charged groups in the aforementioned cases are evenly distributed throughout the beads, the former regions can be converted to the charging capacity in this outer shell. When the problem is approached in this way, the preferred range of charging capacity has been found to be about 0.012 to 0.25 meq / cm 2. This approach takes into account changes in microcarrier volume due to different charge densities.

Microcarriers with the required charge capacity can be prepared by treating microcarriers formed from polymers containing hydroxyl groups with an aqueous solution of an alkaline substance and a tertiary and quaternary amine. The beads may initially be allowed to swell in an aqueous medium without other ingredients, or they may simply be contacted with an aqueous medium containing the required base and amine. This method of using an alkaline agent to catalyze the attachment of positively charged amino groups to hydroxyl-containing polymers is described in U.S. Patent 1,777,970.

Examples of suitable polymers containing hydroxyl groups include polysaccharides such as dextran, dextrin, starch, cellulose, polyglucose and substituted derivatives thereof. Certain synthetic polymers such as polyvinyl alcohol and hydroxy-substituted acrylates or methacrylates such as hydroxyethyl methacrylate are also suitable. Dextran, and especially cross-linked dextran in the form of small spheres or beads, is preferred, especially because it is commercially available, relatively inexpensive, and provides microcarriers that are excellent aids in cell growth.

Any alkaline substance can be used in the reaction. However, alkali metal hydroxides such as sodium or potassium hydroxide are the most preferred alkaline substances.

Tertiary or quaternary amines are suitable for giving positively charged groups which can be incorporated into hydroxyl-containing polymers. Particularly preferred substances are chlorine- or bromo-substituted tertiary amines or their salts, such as diethylaminoethyl chloride, diethylaminoethyl-bromide, dimethylaminoethyl chloride, dimethylaminoethyl bromide, dimethylaminoethylminethyldiethylaminoethyl chloride, diethylamino-methylchloride, diethylaminoethyl chloride; di- (hydroxyethyl) aminomethyl chloride, di- (hydroxyethyl) aminomethyl bromide,.

Reaction temperature and reaction time ranges can be wide. It is preferred to carry out the reaction at temperatures of about 18-65 ° C. However, other temperatures can be used. The reaction kinetics naturally depend to a large extent on the reaction temperature and the concentrations of the reactants. Both time and. temperature affect the final anion exchange capacity.

The reason why the storage capacity of microcarriers is so crucial in cell culture is not fully known. Without wishing to be bound by any theory, it is possible that the surface charge capacity causes certain local irregularities in the composition of the substance, which are the main factors in regulating microcarrier cell culture.

Naturally, there may be certain beads that are not suitable for good cell culture, despite their charge capacity in a defined range. This may be due to side chains of the charge-providing group that are toxic or otherwise detrimental to cell growth, the presence of harmful compositions or compounds adsorbed or absorbed, or this may be due to the porosity of the beads or for other reasons. If such beads are not suitable for cell culture in terms of their properties other than the amount of charge capacity, the beads are not considered to be "cell culture microcarriers".

The invention is illustrated by the following examples.

Example 1

Manufacture of improved microcarriers

Improved microcarriers were prepared as follows. Dry, uncharged, cross-linked dextran beads were screened to obtain a diameter of about 75. One gram of this fraction was added to 10 ml of distilled water and the beads were allowed to swell. A suitable commercial dry, crosslinked dextran is Sephadex G ~ 50 manufactured by Pharmacia Fine Chemicals, Piscatavay, New Jersey.

A 10 ml aqueous solution containing 0.01 moles of diethylaminoethyl chloride: chloride recrystallized twice from methylene chloride and 0.015 moles of sodium hydroxide was formed. This aqueous solution was then added to a suspension of swollen dextran beads, followed by vigorous stirring in a vibrating water bath for 1 hour at 60 ° C. After 1 hour, the beads were separated from the reaction mixture by filtration through Whatman filter paper no. 595 and washed with 500 ml of distilled water.

9 60407 The charge capacity of the beads prepared in this way was about 2.0 meq / g dry, untreated crosslinked dextran. This charge capacity can be characterized by measuring the anion exchange capacity of the beads as follows:

The bead preparation is washed thoroughly with 0.1 N HCl for all ion exchange conditions. . -I4 to saturate sites with Cl ions. They are then rinsed with 10N HCl to remove unbound chloride ions. The beads are then washed with 10- $ (w / w) sodium sulfate solution to saturate the exchange sites with SO 2. The sodium sulfate wash liquor is collected and contains the released chloride ions. This solution is titrated with 1 M silver nitrate using dilute potassium chromate as indicator.

After titration, the beads are washed thoroughly with distilled water, rinsed with phosphate buffered saline (PBS), suspended in PBS and autoclaved. This method gives hydrated beads with a diameter of about 120-200 [mu] m and a charge capacity of about 2.0 meq / g of dry, untreated cross-linked dextran.

Example 2

Culturing of attachment-dependent cells on microcarriers according to the invention compared to a commercially available ion exchange resin

All cells were cultured in "Dulbecco's Modifield Eagle's Medium" medium. To culture normal, diploid fibroblasts, 10% fetal calf serum was added to the medium. To culture primary and secondary chicken fibroblasts, 1% chicken serum, 1% calf serum, and 2% tryptose phosphate broth (Difco Laboratories Detroit, MI) were added to the medium. The bases were sieved onto 100 mm plastic plates (Falcon Plastics, Inc., Oxnard, CA).

Primary fetal fibroblasts from chickens were prepared by cutting enamel into 10-day-old fetuses and then treating them with trypsin. Secondary chicken fetal fibroblasts were prepared from primary confluence on the first day by trypsinization. Cells cultured in plastic plates had a doubling time of about 20 hours.

Diploid, human fibroblasts taken from the fetal lung (HEL2999 ATCC No. CCL 137) were obtained from the American Type Culture Collection, Rockville, MD. The doubling time of these cells was 19 hours in plastic plates.

Microcarrier cultures were initiated simply by combining cells and beads in a mixed culture. 100 ml culture volumes were used in 250 ml beakers (6.5 cm in diameter) equipped with a h, 5 cm magnetic-driven Teflon (D-coated stir bar (Wilbur Scientific, Inc. Boston, MA)). The cultures were immediately sampled and examined microscopically and photographed.

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Cells were counted by counting nuclei using a modified method of Sanford et al. (Sanford, KK, Earle, WR, Evans, VJ, Waltz, HK, and Shannon, JE (1951) J. Natl Cancer Inst. 11: 773 ·) by van Wezel reported by van Wezel, AL (1973). Tissue Culture, Methods and Applications, pages 372-377, Academic Press,

New York).

The beads with adherent cells were separated from the culture medium by allowing the beads to settle at 1 g for a few minutes and then the supernatant was aspirated off. This procedure greatly facilitates medium exchange as well as facilitating the separation of cells from microcarriers after trypsin treatment.

The commercial DEAE Sephadex A-50 was used as a microcarrier for diploid human fibroblasts and compared to carriers prepared synthetically and titrated as described in Example 1. For each type of bead, the carrier content was 2 g dry, untreated, cross-linked dextran / l. The charge capacity for DEAE-Sephadex A-50 was 5 μM meq / g dry, crosslinked dextran, while for newly synthesized beads it was 2.0 meq / g. The results are shown in Figure 1.

For this cell type, the losses of the original inoculum using A-50 microcarriers were significant. Fibroblasts, in turn, adhered, proliferated, and reached confluence with the microcarriers of the invention in 6 days. This behavior is very consistent with the behavior shown for this cell type on standard plates. As shown in Figure 1, the new microcarriers at a concentration of 2 g of dry, cross-linked dextran / l achieved a final cell density of 1.2 x 10 6 cells / ml.

Cultures containing these new carriers showed no initial cell loss or inhibition of confluence. More importantly, cell cultures normally grew at higher microcarrier concentrations. Figure 2 shows by way of example that human fibroblasts and chicken secondary fetal fibroblasts reach a saturation concentration close to. H χ 10 cells / ml using 5 g of dry, cross-linked dextran / l, with a new carrier charge capacity of 2.0 meq / g dextran. As can be seen, there is no significant seed loss even at this relatively high concentration of microcarrier.

Secondary chicken fetal fibroblasts were also cultured at a microcarrier concentration of 10 g / L. Under the conditions described above, a saturation concentration of 6x10 6 cells / ml was obtained; adding 1 more to the medium! fetal calf serum was achieved at a saturation concentration of 8 x 10 6 cells / ml. No significant cell inoculum loss occurred.

Primary chicken fetal fibroblasts were cultured at microcarrier concentrations of 5 and 10 g / L and the growth characteristics were similar to those of secondary 60407 chicken fibroblasts, although negligible inoculum losses were observed and somewhat longer residence times were obtained.

Attempts were also made to culture secondary chicken fetal fibroblasts under the same conditions used above, with the exception that DEAE-Sephadex A-50 microcarriers were used at concentrations of 1 and 5 g / L. No cell growth was observed and inoculum losses were significant.

Example 3

Preparation of microcarriers using varying amounts of reaction components

Microcarrier batches were prepared by dissolving diethylaminoethyl chloride. : chloride and sodium hydroxide in 20 ml of distilled water. The solution was then poured onto dry Sephadex-G-50 beads, after which the beads were placed on a water vibrator table maintained at 60 ° C. One batch of beads was treated with a solution containing 0.01 moles of amine and 0.015 moles of sodium hydroxide, while the other set was treated with a solution containing 0.03 moles of amine and 0.05 moles of sodium hydroxide. The reaction time was varied to obtain different meq / g values for each series.

Diploid human fibroblasts (HEL299) were cultured in suspension cultures at a microcarrier concentration of 5.0 g dry, untreated, cross-linked dextran / l, following the procedures of Example 2, using microcarriers selected from each batch with varying meq / g values.

g

Productivity (10 cultured cells / h) was then calculated and plotted against meq / g for each bead batch prepared as above. The curves plotted using the values obtained for both series had the same shape, they were predominantly clock-shaped, but the curves plotted from those batches treated with higher reactant concentrations had a somewhat steeper rise and fall. Carriers were prepared from each batch to give excellent cell growth.

Example b

Preparation of microcarriers with varying amine / alkali ratios This example illustrates other changes in charge capacity that can be obtained by varying the DEAE chloride: chloride / NaOH ratios. In this example, the procedures of Example 3 were followed with the exception that sodium hydroxide concentrations over a wide range were used, while diethylaminoethyl chloride: chloride concentration was maintained at 0.01 mol / 20 ml. The concentrations used for sodium hydroxide were 0.01; 0.011; 0.012; 0.013; 0,01U; 0.015; 0.02; 0.03; 0.05; 0.75; 0.10 mcolia / 2C ml.

A curve for meq / g was made after 1.25 hours at 60 ° C against the concentration of sodium hydroxide. It can be read from the curve that sodium hydroxide concentrations below about 0.01 do not give a demonstrable charge capacity. However, the charge capacity increased rapidly with increasing concentration and reached a maximum of about 2.3 meq / g dry, cross-linked dextran at a sodium hydroxide concentration of about 0.01 .mu.mol. The charge capacity then decreased almost linearly to about 1.1 meq / g at a sodium hydroxide concentration of about 0.10 moles. Thus, a change in reaction kinetics occurred when the ratio of DEAE chloride: chloride to sodium hydroxide was varied while the concentration of DEAE chloride: chloride and crosslinked dextran remained unchanged.

Example 5

Production of Human Interferon by Culturing with Improved Microcarriers The ability of microcarrier cultured cells to produce human interferon is described herein. The cells used to make human interferon were normal, diploid, human foreskin microblasts FS-U. They were cultured in microcarrier cultures using the methods described in Example 2. Microcarriers prepared and titrated according to Example 1 were used at a concentration of 5 g of dry, cross-linked dextran / l. The medium used for this culture was DMEM (Dulbecco's Minimal Essential Medium) supplemented with 10% fetal calf serum.

After 8–10 days, the cultures stopped growing. At this point, the culture medium was removed. Cultures were washed 1-U times with 100 ml of serum-free DMEM. The cells were then ready for interferon induction. This was accomplished by adding 50 ml of serum-free DMEM medium containing 50 ug / ml cyclohexamide and varying amounts of poly I. poly C inducer to the cultures.

after 1 hour, actinomycin D was added to the cultures to give a final concentration of 1 μg / ml.

5 hours after the start of the induction, the induction medium was decanted and the cultures were washed 3 ~ b times with 100 ml of warm, serum-free DMEM. 50 ml of DMEM containing 0.5% human plasma protein was added to the cultures. Cultures were incubated under standard conditions for an additional 18 hours. At this time, the cultures were decanted and the decanted medium was analyzed for interferon activity. Interferon activity was analyzed by determining a 50% level of protection for samples and standard solutions, FS-U fibroblasts infected with Vesicular Stomatitis virus (VSV), (Indiana strain). The results of interferon preparation experiments are shown in the following table.

• i V.

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Inducer Scl Concentration Preparation Interferon concentration (μg / ml) during preparation U / 10 ^ cells) (cells / ml) h 2.0 x ^ 06 39 5 2.6 x 106 378 25 2.6 x 106 886 50 2.0 x 106 n 5000

Each of these values is obtained from a different experimental run and does not appear to be related to the concentration of inducer.

Example 6

Cell culture with enhanced microcarriers for the production of viruses

The ability of microcarrier cultured cells to produce the virus is described below. Primary and secondary chicken embryo fibroblasts were cultured in microcarrier culture according to the method described in Example 2, wherein the primary cells were cultured at a microcarrier concentration of 10 g / l and the secondary cells at a microcarrier concentration of 5 g / l. To initiate virus production, the culture medium was removed and the cultures were washed twice with 100 ml of serum-free DMEM. Infection of cells with Sindbis virus occurred in 50 ml of DMEM supplemented with 1% calf serum, 2% tryptose phosphate broth and sufficient Sindbis flow to meet an MOI (multiplicity of infection) of 0.05 ·

Viruses were harvested 2 h after inflation by collecting the broth, clarifying it by gentle centrifugation, and cooling the supernatant. Virus production was examined by plaque formation (PF) in the area of secondary chicken fibroblasts. The results of infecting these microcarrier cultures were:

Cell type Total production PFU / ml PFU / cell (cells / ml) secondary 1 *, 0 x 10 ^ 8, U x 10 ^ 2000 primary 1, U x 10 ^ 2.3 x 10 ^ 16000 primary 6.0 x 10 ^ 2.6 x 10 ^ 5000

Virus production was determined for the following virus / cells with microcarrier combinations: Polio / WI-38i Moloney MuLV / Cl-1 mouse and VSV / chicken fetal fibroblast.

Example 7

Comparative culture of cells in spinner flasks and improved microcarriers to produce muridae leukemia virus proviral PNA 1U 60407

DNA obtained by Moleney blood cancer virus (M-MuLV) reverse transcriptase after infection of mouse bone marrow cells (JLS-Vg) was examined.

The methodology involved culturing the cells in rotating flasks. Cells were grown in rotary flask culture, the medium was removed, and the virus inoculum was added to the flasks. Immediately thereafter, fresh medium was added to the cultures and 8-16 hours later they were extracted for final purification of viral DNA. The cultures were washed with fresh buffer and the cells were lysed with a solution containing sodium dodecyl sulfate. Subsequent cooling of the lysate and addition of salt to one molar resulted in co-precipitation of detergent and high molecular weight DNA.

The low molecular weight DNA remaining on the overlay layer could then be purified from proteins and concentrated for further analysis, with 9 50-flask spinner cultures containing approximately 10 cells. These cells were infected with 6 n 1 l of virus inoculum at a concentration of 3 X 10u bar-forming units / ml. This resulted in a nominal MOI of 1-3 and the infected cells gave 5-20 nanograms of virus-specific DNA.

A simpler method was developed using the improved microcarriers of this invention. A culture containing 10 g of beads in 1 l of culture medium was used. After reaching confluence, 10 cells on the beads were infected by allowing the beads to be immersed and replacing the medium with 1 in 11 virus inocula. For extraction, the beaded cells were washed with buffer and then placed in buffer containing sodium dodecyl sulfate. After co-precipitation of high molecular weight DN.A and detergent, the precipitate was centrifuged together with the beads and the supernatant was separated for further analysis. The yield of viral DNA was comparable to that obtained in rotary flask culture and the work used was 5-10% of that required in rotary flask culture.

Example 8

Improved production of microcarriers with dimethylaminoethyl charge groups

A suitable microcarrier was prepared by attaching a second exchange unit to the dextran matrix used in Example 1. Dimethylaminoethyl groups (DMAE) were attached to the dextran matrix by the following method: 1 g of dry dextran beads (Pharmacia G-50) with a diameter of 50-75 μm was placed in 10 ml. distilled water and allowed the beads to swell. A 10 ml aqueous solution containing 0.01 moles of dimethylaminoethyl chloride: chloride (Sigma Chemical Co.) and 0.015 moles of sodium hydroxide was formed. This aqueous solution was added to the swollen dextran beads and this suspension was then stirred vigorously for 1 hour at 60 ° C. After the reaction, the bead mass was titrated as in Example 1. This reaction binds 1.0 meq of dimethylaminoethyl to the dextran mass. To prepare 60 '60407 microcarriers with a higher degree of substitution, the above reaction was carried out and the bead mass was washed thoroughly with water. Excess water was filtered off and the bead mass was weighed to determine the amount of water remaining in the bead mass. To this bead mass was added an appropriate amount of fresh reaction components (i.e., DMAE-C1: C1 and NaOH) so that the final concentration of DMAE-Na and NaOH in these subsequent reaction mixtures was equal to that originally used.

In this way, a microcarrier kit containing 1.0; 2.0; 2.5 and 3.5 meq DMAE / g unreacted dextran. Cells (HEL299) were cultured in microcarrier culture (5 g / l) with these microcarriers according to the methods of Example 2. The results are given in the following table:

Degree of substitution Cell proliferation Net cell growth (meq / g) 1.0 2.0 2.5 + + 3.2 +

As expected, cell growth depends on the degree of substitution in the charged groups. When the degree of substitution is too high, no cell growth occurs, despite attachment and proliferation. At too low a degree of substitution, cell attachment to the surface is not sufficient to allow proper proliferation and growth.

Example 9

Improved microcarriers with positively charged phosphonium groups

Improved microcarriers were also prepared using non-amine exchange groups as follows: 1 g of dry dextran beads were prepared and swollen with water as in Example 1. To the swollen beads was added 5 ml of saturated triethyl (ethyl bromide) phosphonium (TEP), C 2 H 5 C 2 H 5

P

C \ H2 60407 16 aqueous solution and 5 ml of a 3 molar solution of sodium hydroxide were added. This slurry was allowed to react at 65 ° C. A microcarrier kit was prepared at a concentration of 1.1; 1.7 and 2.9 meq / g. Microcarriers at a concentration of 1.1 meq / g were prepared by reaction under the above conditions in k minutes. 1.7 meq / g microcarriers were allowed to react for 1 hour and 2.9 meq / g microcarriers were allowed to react three times in a row as described in Example 7. Each of these carriers was cultured in 5 g / L microcarrier cell with continuous cell type JLS-V9 and compared to the growth of this cell with improved DEAE microcarriers prepared according to Example 3. The results are given in the following table.

DEAE

meq / g Cell attachment Net cell growth and proliferation_ 0.9 + + 1, 7 + + 3.8 +

TEP

1.1 + + 1, 7 + + 2.9 +

Claims (7)

17 60407 A method of culturing attachment-dependent cells in a microcarrier culture to prepare cell cultures and optionally cell culture products such as Viruses or interferon, forming a suspension of positively charged microcarriers and attachment-dependent cells in a suitable culture medium, optionally maintaining such microcarrier cell culture under conditions that promote the formation of cell culture products, which are then recovered, characterized in that positively charged microcarriers with an anion exchange capacity of 0.1-1 +, 5 meq / g of dry, untreated microcarriers are used to form a suspension. Method according to Claim 1, characterized in that microcarriers consisting of cross-linked dextran beads are used. The method according to claim 2, characterized in that the microcarriers consist of cross-linked dextran beads having tertiary or quaternary amino groups. Method according to Claim 2 or 3, characterized in that the microcarriers consist of cross-linked dextran beads with diethylaminoethyl groups. Method according to Claim 1 or 2, characterized in that the microcarriers consist of cross-linked dextran beads with phosphonium groups. Process according to one of Claims 1 to 5, characterized in that positively charged microcarriers with an anion exchange capacity of 1 to 2.8 meq / g of dry, untreated dextran are used. Method according to one of Claims 1 to 6, characterized in that positively charged microcarriers with an average diameter of 120 to 200 μm are used.