SE460906B - CELLKULTURMIKROBAERARE - Google Patents

Description

4 4xcrigagaceae "and include 3T3 mouse fibroblasts, mouse bone marrow epithelial cells; Muridae 1eukemia virus-producing species of mouse fibroblasts, primary and secondary chicken fibroblasts; WI-38 human fibroblast cells, C, and normal human fibroblast cells; tumors that cause tumors, but others have been cultured and found to be non-tumorigenic, and some anchorage-dependent cells, such as WI-38 and HEL-299, may also be cultured which are genetically normal.

Although significant progress has been made in large-scale mammalian cell culture using cell lines capable of growing in suspension culture, progress has been very limited in large-scale anchorage-dependent mammalian cell culture.

Previous work techniques used for culturing anchorage-dependent cells on a large scale were based on linear expansion of processes on a small scale. Cell culture plants utilized a large number of batch reactors with low yield, in the form of bowls, medicine vials, rotating culture tubes and culture vials. Each of these was a stand-alone unit or insulated batch reactor that required individual environmental controls. However, these controls were of a very primitive type due to economic reasons. Change of nutrients was corrected by a change of the medium, an operation that requires two steps, ie removal of medium and addition of medium. Since it was not uncommon for a medium-sized plant to simultaneously operate hundreds of these batch reactors, even a single change of medium required hundreds of operations, all of which had to be performed carefully, and under demanding sterile conditions. Each flBrS fi ê9S ° P @ fati0n, SåS0m cell transfer unites sköraning compound problem 1 accordingly. Thus, the cost of equipment, space and manpower was high for this type of facility.

There are alternative methods that have been proposed for linear magnification from small batch cultures. Among the alternatives that have been reported in the literature are plastic bags, stacked bowls, spiral films, culture on glass beads, artificial capillaries and microcarriers. Among these, microcarrier systems offer certain outstanding and unique advantages. For example, major improvements in the achievable ratio between culture area and vessel volume (S / V) can be obtained using microcarriers, compared to both traditional and recently developed alternative techniques. The increase in the achievable S / V ratio allows the construction of a single unit 460 906 * homogeneous or quasi-homogeneous batch or semi-batch propagator for high volumetric productivity. Thus, a single agitated container with simple return feed control for pH and pO 2 provides a homogeneous environment for a large number of cells, thereby eliminating the need for expensive and space-consuming incubators with controlled environment. The total number of required operations per unit of cells produced is also drastically reduced. In summary, microcarriers seem to offer good economy in terms of capital, space and manpower in the production of anchorage-dependent cells, 1 in relation to current production methods.

Microcarriers also offer the benefit of continuous environment because the cells are grown in a controlled environment. Thus, microcarriers offer the ability to grow anchorage-dependent mammalian cells under a series of ambient conditions that can be regulated to achieve constant, optimal cell growth. One of the currently most promising microcarrier systems has been reported by van Wezel and involves the use of diethylaminoethyl (DEAE) -substituted dextran beads in a stirred tank. A. L. van wezel, 'Growth of Cell Strains and Primary Cells on Microcarriers in Homogeneous Culture', Nature 216: 64 (l967); D. van Hemert, DG Kilburn and AL van Wezel, "Homogeneous Cultivation of Animal Cells for the Production of Virus and Virus 2roducts', Biotechnol. Bioeng. Ll: 87S (l969); and AL van Wezel," Microcarrier Cultures of Animal Cells ", Tissue Culture, Methods and Applications, PF Kruse and MK

Patterson, eds., Academic Press, New York, p. 372 (1973). These beads are manufactured commercially by Pharmacia Fine Chemicals, Inc., Piscataway, New Jersey, under the tradename DEAE-Sephadex A50, an ion exchange system. Chemically, these beads are formed by a crosslinked dextran matrix having diethylaminoethyl groups covalently bonded to the dextran chains. The commercially available DEAE-Sephadex A50 beads are believed to have a particle size of 40-200 microns and a positive charge capacity of about 5.4 meq / g of dry crosslinked dextran (ignoring the weight of connected DEAE units). Other anion exchange resins, such as DEAE-Sephadex A25, QAE-Sephadex A50 and QAE-Sephadex A25, were stated by van Wezel to also support cell growth.

The system proposed by van Wezel combines multiple surfaces with moving surfaces and provides an opportunity for innovative cellular manipulations as well as offering advantages in scaling and environmental controls. Despite this possibility, these proposed techniques have not been significantly exploited, as researchers have encountered 460,906 difficulties in cell production due to certain deleterious effects caused by the beads. Among these, initial cell death is among a high percentage of the cell inoculum and inadequate cell growth even for the cells that get trapped. The reason for these harmful effects is not fully understood, although it has been suggested that they may be due to bead toxicity or nutrient absorption. See van Wezel, A. L. (1967), Nature 216: 64-65; van Wezel, A. L. (1973), Tissue Culture Methods and Applications. Kruse, P. R. and Patterson, M. R. (eds.), Pp. 372-377, Academic Press, New York; van Hemert, P., Kilburn, D. G., and van Wezel, A. L. (1969), _ Biotechnol. Bioeng. ll: 875-885; Horng, C. and McLimans, W. (1975), Biotechnol. Bioeng. 17: 713-732.

The harmful effects of these commercially available ion exchange resins may be due to their production method. Certain patents in the patent such as: U.S. Patents 3,277,025; 3,275,576; 3,042,667 and 3,208,994; all by Flodin et al. Whatever the reason, however, the currently commercially available materials are simply not sufficient for good cell culture of a variety of cell types.

A solution to overcome some of the deleterious effects of trying to use such commercially available microcarriers for cell growth has been described in U.S. Patent 4,036,693 to Levine et al. In said patent, a method has been proposed for treating these commercially available ion exchange resins with macromolecular polyanions, such as carboxymethylcellulose. Although this method has proven to be successful, it would of course be more advantageous if the beads could be manufactured so that they initially have properties intended for excellent culture of anchorage-dependent cells. It has now been found that the charge capacity of microcarriers must be adjusted and / or controlled within a certain range in order to result in good growth of a variety of different anchorage dependent cell types at reasonable microcarrier concentrations. Based on this discovery, microcarrier beads have been produced with controlled charging capacities, and such beads have been used to obtain good growth of a variety of anchorage-dependent cells. Cells grown using such microcarrier systems can be harvested or used in the production of animal or plant viruses, vaccines, hormones, interferons or other cell culture by-products. An example of the improved microcarriers is then provided using polymers with protruding hydroxyl group. such as crosslinked 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 material, such as sodium hydroxide. The specific charge capacity of the beads is controlled by varying the absolute amount of dextran, tertiary amine salt and alkaline material, the conditions of these materials and / or the time and temperature of the treatment.

Microcarriers prepared according to the present invention can be used in cultures without the high initial cell loss hitherto experienced with commercially available microcarriers.

In addition, the attached cells spread and grow to confluence on the beads, whereby extremely high cell concentrations are reached in the suspension medium. The concentration of microcarriers in suspension is not limited to very low levels, as was customary with the prior art materials, and cell growth occurs, being limited only by factors which do not appear to be associated with the microcarriers. It follows that large increases in the voluetric productivity of cell cultures can be obtained. In short, the potential opportunity offered by the use of microcarriers in cell culture, and especially anchorage-dependent cells, can now be realized.

Fig. 1 is a graph showing normal, diploid, human embryonic lung fibroblast cells (HEL299) growth characteristics at a microcarrier concentration of 2 g dry, crosslinked dextran / liter for both commercially available DEAE-treated dextran microcarriers and DEAE-treated microcarriers. the present invention; Figure 2 graphically shows the growth characteristics of both normal, diploid, human embryonic lung fibroblast cells (HEL299) and secondary chicken embryo fibroblasts at a microcarrier concentration of S g dry, crosslinked dextran / liter using, improved DEAE-treated microcarriers according to the present invention.

As used herein, the terms "microcarrier", "cell culture microcarrierP" and "cell culture microcarrier" refer to small, free-standing particles suitable for cell anchoring and culture. Often, though not always, microcarriers are porous beads formed from polymers. cells grow on the outer surfaces 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 controlled so that it is within a certain range for adequate cell growth at reasonable microcarrier concentrations. Suitable working areas and preferred areas vary with such factors as § the specific cells to be grown, the nature of the microcarriers, the concentration of the microcarriers and other cultural parameters including the composition of the medium. In all cases, however, the amount of charge capacity found to be suitably significant is below the amounts believed to be present on commercially available anion exchange resins previously proposed for microcarrier cell cultures. For example, the DEAE-Sephadex A50 beads, proposed by van Wetzel, are believed to have a loading capacity of about 5.4 meq / g of dry, untreated (without DEAE), crosslinked dextran. In contrast to this relatively high charging capacity, microcarriers have been prepared and found suitable for good cell culture according to the present invention, which have between about 0.1 and about 4.5 meq / g of dry, untreated microcarriers. Below about 0.1 meq / g, it is believed that cells would have difficulty attaching to the microcarriers. above about 4.5 meq / g, losses occur due to the initial cell inoculum, nor do the - * "<surviving cells grow well, especially at relatively high microcarrier concentrations. M For culturing normal, diploid human fibroblasts on cross-linked dextran microcarriers, found that a preferred range for the charge capacity provided by DEAE groups is from about 1.0 to 2.8 meq / g of dry, untreated, crosslinked dextran, although the preferred range may vary with different cell types or culture conditions, it is believed that the preferred ranges for any given series of conditions will be in the range of 0.1 - 4.5 meq / g.

The preferred and optimal conditions can be determined by one skilled in the art for each series of conditions by routine experimentation.

It should, of course, be appreciated that there are some shortcomings in trying to define the charge capacity of microcarriers only on a unit weight basis. For example, two beads that are identical in any other way than that they are formed from different materials with different densities and with the same charge distributed thereon would lead to different values for their charge capacity per unit weight. In the same way, two beads with identical charging capacities per unit weight can have a completely different charge distribution thereon.

An alternative definition can be made by specifying re- 7. _S 4sg eos Council of suitable charges expressed as charge capacity per unit weight of microcarriers in their final functional form. This basis would take into account such factors as the weight of attached DEAE or other positively charged groups, as well as hydration of the beads, etc., whereas the previous definition is based on dry, crosslinked dextran and does not take into account such factors. In an aqueous cell culture medium, the density of microcarriers should be selected to 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 charge capacities for microcarriers according to the invention defined in this way is from about 0.012 to about 0.25 meq / g. the microcarriers have a substantially uniform charge distribution throughout their main mass.

If the charge distribution is uneven, it is possible to have suitable microcarriers with charging capacities outside said ranges. The important criterion is, of course, that the charging capacity is adjusted to and / or controlled at a value that is sufficient to allow good cell growth on the microcarriers. A Since it may be the charging pattern on the outer surface that is important, it is also desirable to be able to: define the appropriate charging capacity range in the form of a probable surface pattern. This can be done by assuming that the active part of the microcarriers consists only of the outer surface of the bead to a depth of about 20 Å. If it is also assumed that the charged groups in the previously mentioned cases are evenly distributed throughout the beads, the former areas may converted to a charge capacity in this outer shell. When problems are approached in this way, the suitable range for the charging capacity has been found to be from about 0.012 meq / cm3 to about 0.25 meq / cm3.

This method takes into account the changes in the microcarrier volume due to different charge densities.

Microcarriers with the required charge capacity can be prepared by treating microcarriers formed from polymers containing protruding hydroxyl groups with an aqueous solution of an alkaline material and a tertiary or quaternary amine. The beads may initially swell in an aqueous medium without the other constituents, or they may simply be contacted with an aqueous medium containing the required base and amine.

This method of using alkaline material to catalyze the anchoring of positively charged amino groups on hydroxyl-containing 46 polymers has been described in Hartmann, U.S. Patent No. 1,777,970.

Examples of suitable hydroxyl-containing polymers 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, are preferred, especially because it is commercially available, relatively inexpensive and provides microcarriers that support excellent cell growth. Any material that is alkaline can be used for the reaction.

However, the alkali metal hydroxides, such as sodium or potassium hydroxide, are the preferred alkaline substances.

Either tertiary or quaternary amines are suitable sources of positively charged groups that can be coupled to the hydroxyl-containing polymers. Particularly preferred materials are chlorine or bromine-substituted tertiary amines or salts thereof, such as diethylaminoethyl chloride, diethylaminoethyl bromide, dimethylaminoethyl chloride, dimethylaminoethyl bromide, diethylaminomethyl chloride, diethylaminomethylbromyl (hydroxymethylbromyl) hydroxyaminobromethylbromide - (hydroxyethyl) -aminomethyl chloride, di- (hydroxyethyl) -aminomethyl bromide, β-morpholinoethylethyl chloride, β-morpholinoethyl bromide, 5-morpholinomethyl chloride, β-morpholinomethyl bromide and salts thereof, for example the hydrochlorides.

A wide range of reaction temperatures and times can be used. It is preferred to carry out the reactions at temperatures of between about 18 ° C and 6S ° C. However, other temperatures can be used. The reaction kinetics, of course, depend largely on the reaction temperature and the concentrations of the reactants. Both the time and the temperature affect the final exchange capacity obtained.

The reason why the microcarriers' charging capacity is so critical in cell culture is not fully understood. Although it is not a shame to be bound by this theory, it is possible that the contact capacity of the surface causes certain local irregularities in the composition of the medium, which are the most important factors in controlling microcarrier culture. Notwithstanding this, this does not exclude other possibilities. 'There may of course be some beads that will not be suitable for good cell culture even though they have a charging capacity within one of the specified areas. This may be due to side chains on the device that pre-charge the capacity, which are harmful or otherwise harmful to cell growth, the presence of adsorbent or absorbing harmful compositions or compounds or this may be due to the porosity of the bead or other causes. If such beads are not suitable for cell culture except for the amount of charge capacity, the beads are not considered to be 'cell culture microcarriers'.

The invention is further illustrated by the following examples.

EXAMPLE 1 PREPARATION OF IMPROVED MICROCARRIERS Improved microcarriers can be prepared in the following manner.

Dry, uncharged, crosslinked dextran beads were sieved to obtain those with a diameter of about 75 .mu.m. One gram of this fraction was added to 10 ml of distilled water and the beads were allowed to swell. An adequate commercial source of dry, crosslinked dextran is Sephadex-G-50 from Pharmacia Fine Chemicals, Piscataway, New Jersey.

An aqueous solution containing 0.01 moles of diethylaminoethyl chloride 2 chloride, twice recrystallized from methylene chloride, and 0.015 moles of sodium hydroxide was formed in a volume of 10 ml. This aqueous solution was then added to the suspension of swollen dextran beads, after which it was stirred vigorously in a shaking water bath for 1 hour at 60 ° C. After 1 hour, the beads were separated from the reaction mixture by filtration on Whatman No. 595 filter paper and washed with 500 ml of distilled water. Beads produced in this way have about 2.0 meq charge capacity per gram of dry, untreated crosslinked dextran.

This charge capacity can be characterized by measuring the anion exchange capacity of the beads as follows. The bead preparations are washed thoroughly with 0.1 normal H01 to saturate all exchange sites with Cl 'ions. They are then rinsed with 10-4 normal H01 to remove unbound chloride ions. The beads are then washed with a 10% w / w sodium sulfate solution to saturate the exchange sites with SO 4 =. The effluent of the sodium sulfate wash is collected and contains liberated 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 process yields hydrated beads with a diameter of about 120-200 Fm, which have a charge capacity of about 2.0 meq / g of dry, untreated crosslinked dextran. 4 ßßnægß A CULTIVATION OF ANCHOR-DEPENDENT CELLS WITH MICROCARRIERS ACCORDING TO THE INVENTION IN COMPARISON WITH COMMERCIAL 'AVAILABLE ION CHANGER RESIN All cells were grown in Dulbecco's Modified Eagle's Medium.

For culture of normal diploid fibroblasts, the medium was increased to% with fetal calf serum. For culture of primary and secondary chicken fibroblasts, the medium was supplemented with 1% chicken serum, 1% calf serum, and tryptophosphate broth (Difco Laboratories, Detroit, MI). Base substances were passed on 100 mm plastic trays (Falcon Plastics, Inc., Oxnard, CA).

Primary chicken embryo fibroblasts were prepared by chopping and subsequent trypsin treatment of 10 day old embryos. Secondary chicken embryo fibroblasts were prepared on the first day of primary confluence by trypsin treatment. For cells grown in plastic dishes, the doubling time was about 20 hours.

Diploid, human fibroblasts derived from embryonic lung (HEL299, ATCC No. CCL 137) were obtained from the American Type Culture Collection, Rockville, MD. These cells had a doubling time of 19 hours in plastic bowls.

Microcarrier cultures were started simply by bringing cells and beads together in stirred culture. 100 ml of culture volumes in 250 ml glass flasks (6.5 cm in diameter) equipped with a 4.5 cm magnetically driven Tefloga coated stir bar (wilbur Scientific, Inc., Boston, MA) were used. The stirring speed was about 90 r / v. Samples of the cultures were taken directly and the samples were examined microscopically and photographed. The cells were counted by counting nuclei using the modified method of Sanford et al. (Sanford, KK, Earle, WR, Evans, VJ, Waltz, HK, and Shannon, JE (1951) J. Natl Cancer Inst. 11: 773.) as described by van Wezel (van Wezel, AL (1973) Tissue Culture, Methods and Applications Kruse, PF and Patterson, MR (eds.), Pp. 372-377, Academic Press, New York).

Beads with attached cells were separated from the culture medium by allowing the beads to sink at 1 g for a few minutes and then aspirating the overlying layer. This process facilitates much exchange of the medium and facilitates the separation of cells from microcarriers after trypsin treatment.

Commercial DEAE Sephadex A-S0 was used as a microcarrier for the diploid human fibroblasts and was compared to carriers synthesized and titrated as described in Example 1. For both types of beads, the carrier concentration was 2 g of dry, untreated, crosslinked dextran per liter. The charge capacity of.DEAE Sephadex A-50 was 5.4 meq / g of dry, cross-linked dextran, whereas that of the newly synthesized beads was 2.0 meq / g. The results are shown in Fig. 1.

For this cell type, the loss of original inoculum on A-50 microcarriers was pronounced, while the fibroblasts adhered, proliferated and reached confluence on the microcarriers of the invention in 6 days. This behavior is in good agreement with the reported behavior of this cell type on standard dishes. As Fig. 1 shows - the final cell density achieved with the new microcarriers at 2 g dry, crosslinked dextran / liter was 1.2 x 10 6 cells / ml.

--- ~ ~ Xultures containing the new bars showed neither initial cell loss nor any inhibition upon reaching confluence. Even more important was that the cell cultures grew normally at higher microbial concentrations. Figure 2 shows, for example, that human fibroblasts and secondary chicken embryo fibroblasts at saturation concentrations close to 4 x 10 6 cells / ml when 5 g of dry, crosslinked dextran / liter were used, the new carriers having a loading capacity of 2.0 meq / g dextran. As can be seen, there was no significant loss of inoculum even at this relatively high microcarrier concentration.

Secondary chicken embryo fibroblasts were grown even at microcarrier concentration of 10 g / liter. Under the conditions described above, a saturation concentration of 6 x 10 6 cells / ml was achieved; upon addition of the medium with an additional 1% fetal calf serum, a saturation concentration of 8 x 10 6 cells / ml was achieved. There was no significant loss of cell inoculum.

Primary chicken embryo fibroblasts were grown at a microcarrier concentration of S and 10 g / liter and the growth characteristics were similar to those of the secondary chicken fibroblasts, although insignificant inoculum losses were noted and slightly longer decay times were obtained. Attempts were also made to grow secondary chicken embryo fibroblasts under the same conditions as used above except that DEAE-Sephadex A-50 microcarriers were used at concentrations of 1 and g / liter. No cell growth was recorded and significant inoculum loss occurred. 12. 2 * láåmäåš PREPARATION OF MICROCARRIERS WITH VARIOUS QUANTITIES OF REACTANTS Batches of microcarriers were prepared by dissolving diethylaminoethyl chloride: chloride and sodium hydroxide in 20 ml of distilled water. The solution was then poured over dry Sephadex G-50 beads, after which the beads were placed on a water shaker table maintained at 60 ° C. One series of bead batches was treated with a solution containing 0,01 mol of amine and 0.015 mol of sodium hydroxide, whereas another series of batches was treated with a solution containing 0.03 mol of amine and 0.045 mol of sodium hydroxide.

The reaction time was varied to produce different meq / g within each batch.

Diploid human fibroblasts (HEL299) were grown in suspension cultures at a microcarrier concentration of 5.0 g dry, untreated crosslinked dextran per liter, following the procedures of Example 2, using microcarriers of varying meq / g selected from each batch. Then the productivity was calculated (106 cells grown / liter.h) and plotted against meq / g for each batch of beads prepared as above. Curves plotted using data obtained for both series had the same shape, they had mainly a bell shape, but the curve from the batches treated with the higher concentration of reactants had a slightly steeper rise and fall.

From each batch progenitors that gave excellent cell growth.

EXAMPLE 4 PREPARATION OF MICROCARRIERS BY VARIOUS MHN / ALKALI CONDITIONS This example illustrates further changes in charge capacity, which can be obtained by varying the DEAE chloride / NaOH ratios. In this example, the procedures of Example 3 were followed except that a wide range of sodium hydroxide concentrations were used while maintaining the diethylaminoethyl chloride concentration at 0.01 mol per 20 ml. The concentrations used for sodium hydroxide were 0.01; 0.011; 0.012; 0.013; 0.014; 0.015; 0.02; 0.03; 0.05; 0.75; 0.10 mol per 20 ml.

A curve was made up of meq / g after 1.25 hours at 60 ° C against the concentration of sodium hydroxide. From the curve it could be seen that sodium hydroxide concentrations below about 0.01 gave no detectable charge capacity. However, the charging capacity increased rapidly with increasing concentration and reached a maximum of about 2.3 meq / g dry, crosslinked dextran at a concentration of about 0.014 mol 13 L of sodium hydroxide. The charge capacity then decreased näsâšàpi gêåó linear relationship to a value of about 1.1 meq / g at a sodium hydroxide concentration of about 0.10 mol. Thus, a change in the reaction kinetics occurs when the ratio of DEAE chloride: chloride to sodium hydroxide is varied at a constant concentration of DEAE chloride chloride and crosslinked dextran.

EXAMPLE S HUMAN INTERFERONE MADE IN CELLS CULTIVATED ON IMPROVED MICRO-CARRIERS The ability of microcarrier-grown cells to generate human interferon is described herein. The cells used to make human! interferon were normal, diploid, human foreskin fibroblasts, FS-4. These fibroblasts were grown in microcarrier cultures using the procedures described in Example 2. Microcarriers prepared and titrated according to Example 1 were used at a concentration of S g of dry, crosslinked dextran / liter. The DMEM used for culture culture was augmented with 10% fetal calf serum.

After 8 to 10 days, the cultures stopped growing. At this stage, the culture medium was removed. The cultures were washed 1-4 times with 100 ml of serum-free DMEM. The cells were then ready for interferon induction. This was accomplished by adding to the cultures 50 ml of serum-free DMEM medium containing 50 pg / ml cyclohexamide, and varying amounts of poly I. poly C inducers. After 4 hours, Actinomycin D was added to the cultures to give a final concentration of 1 pg / ml. 5 hours after starting induction, the induction medium was decanted and the cultures were washed 3-4 times with 100 ml of warm, serum-free DMBM.

The cultures were filled with 50 ml of DMEM containing 0.5% human plasma protein. The 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.

The interferon activity was analyzed by determining the 50% level of cell protection for samples and standard solutions, for FS-4 fibroblasts infected with Vesicular Stomatitis Virus (VSV), Indiana strain). The results of the interferon production runs are presented below in tabular form. '14 460 906 Concentration of Cell Concentration during Interferon Inducer Preparation (U / 106 cells) (pg / ml) (cells / ml) 4 2, ox 1ø6 39 2.6 x 106 378 zs 2.6 X 106 sas so 2.0 X 1o6 -sooo Each of these data is from a separate run and is not intended to show any correlation with the concentration of inducers.

EXAMPLE 6 g CULTURE OF CELL BAR ON IMPROVED MICROCARRIERS FOR THE PURPOSE OF PRODUCING VIRUS The ability of microcarrier-grown cells to produce a virus is described below. Primary and secondary chicken embryo fibroblasts were grown in microcarrier culture according to the procedure described in Example 2, the primary cells being grown at 10 g / liter and the secondary at 5 g / liter microcarrier concentration. To initiate virus production, the culture medium was removed and the cultures were washed twice with 100 ml of serum-free DMEM. Infection of the parts with Sindbis virus occurred 1 50 ml DMEM supplemented with 1% calf serum, 2% tryptophosphate broth and enough Sinbis virus to correspond to an MOI (diversity of infection) of 0.05.

The virus was harvested 24 hours after infection by collecting the culture broth, preparing it at low spin and freezing the overlying layer. Virus production was analyzed by plaque formation (PF) in one area of secondary chicken fibroblasts. The results from infection of these microcarrier cultures were: Total concentration for production Cell type (cells / ml) (PFU / ml) PFU / cell Secondary 4.0 x 106 8.4 x 109 2,000 Primary '1.4 x 106. 2.3 x 1o1 ° 16 ooo primary 6.0 x 105 2.6 x 1o1 ° s ooo Virus production was determined for the following virus / cell on the microcarrier combinations: Polio / WI-38; Moloney MuLV / Cl-1 mouse and VSV / - "chicken embryo fibroblasts.

EXAMPLE _, 460 906 COMPARATIVE CULTURE OF CELLS IN ROTARY PISTONS AND WITH IMPROVED MICRO-CARRIERS FOR THE PURPOSE OF PRODUCING MURIDAE LEUKEMIVIRUS PROVIRAL DNA Reversible DNA M , was studied.

The technology involved culturing cells in rotary flasks. Cells were grown in rotary flask culture, the medium was removed, and viral nucleus was introduced into the flasks. Shortly thereafter, the cultures were given fresh medium and 8-16 hours later they were extracted for final purification of viral DNA.

The cultures were washed with fresh buffer and the cell was lysed with a solution containing the detergent sodium dodecyl sulfate. Subsequent cooling of the lysate and addition of salt to a molar caused co-precipitation of the detergent and high molecular weight DNA.

Low molecular weight DNA remaining in the overlying layer could then be deproteinized and concentrated for further analysis.

An SO 50 rotary flask culture containing approximately 109 cells. These were infected with approximately 1 liter of viral inoculum titration at 3 x 10 6 plaque units per ml. This resulted in a nominal fl diversity of infection of 1-3 and the infected cells yielded -20 nanograms of virus-specific DNA.

A simpler method was developed using improved microcarriers according to the present invention. A culture containing g of beads in 1 liter of culture medium was used. Upon reaching confluence, 109 cells were infected on the beads by allowing the beads to sink and the medium was replaced with 1 liter of virus inoculum.

For extraction, the cells on the beads were washed with buffer and then placed in the SDS-containing buffer. After co-precipitation of high molecular weight DNA and the detergent, the precipitate was centrifuged together with the beads and the overlying layer was separated for further analysis. The yield of viral DNA was comparable to that obtained in the rotary flask culture and the work involved was 5fl0 Q of that required by the rotary flask culture.

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IMPROVED MICROCARRIER PRODUCTION WITH DIMETHYLAMINOETHYL CHARGING GROUPS A suitable microcarrier was prepared by using an alternating exchange unit belt for the extranetrix used in Example 1.

Dimethylaminoethyl groups (DMAE) were bonded to a dextran matrix by the following procedure: 1 g of dry dextran beads (Pharmacia G-50) with a diameter of 50-75 Fm were added to 10 ml of distilled water and the V beads were allowed to swell. The aqueous solution, containing 0.01 moles of dimethyl chloride (chloride) (Sigma Chemical Co.) and 0.015 moles of sodium hydroxide, was formed in a volume of 10 ml. 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 microcarriers with higher grade Qistiündon, the above reaction was carried out and the bead mass was thoroughly washed with water. The excess water was filtered off and the bead mass was weighed to determine the amount of water retained by the bead mass. To this bead was added the appropriate amount of fresh reactants (ie, DMAE-Cl: Cl, and NaOH) so that the final concentration of DMAE and NaOH in these subsequent reaction mixtures was identical to those originally used.

In this way, a series of microcarriers were prepared at 1.0; 2.0; 2.5 and 3.5 meq DMAE / g unreacted dextran. Cells (HEL 299) were grown in microcarrier culture (5 g / liter) with these microcarriers according to the procedures of Example 2. The results are given below in tabular form: Degree of substitution (meq / g) \ ce11spr1an1ng net growth 1, o - - 2. o - - 2, 5 + + 3, 2 + - As expected, cell growth is associated with the degree of substitution with charge-bearing groups. With too high a degree of substitution, no cell growth occurs, despite anchoring and proliferation. at a too low degree of substitution, adhesion of cells to the surface is not sufficient to allow proper dispersal and growth. ammar. IMPROVED MICROBARRIERS WITH POSITIVELY CHARGED PHOSPHONIUM GROUP Enhanced microcarriers were also prepared using non-amine substituents 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 a saturated aqueous solution of triethyl CZHS- (ethyl bromide) -phosphonium (TEPÉ, and 5 ml of a 17 460 '906 3 molar solution of sodium hydroxide.This slurry was allowed to react at 65 ° C. A series of microcarriers were prepared at 1.1; 1.7 and 2.9 meq / g. The microcarriers at 1.1 meq / g was prepared by reaction under the above conditions for 4 minutes. 1.7 meq / g microcarriers were allowed to react for 1 h and 2.9 meq / g microcarriers were allowed to react 3 times in succession as described in Example 7. A microcarrier cell culture at g / liter was established for each of these carriers with a continuous cell type, JLS-V9, and was compared with the growth of this cell on improved DEAB microcarriers prepared as in Example 3.

The results are given below in tabular form.

DEAE meq / g Cell anchoring and proliferation Net growth 0.9 + + 1.7 + + 3'8 + _ TEP 1.1 + + 1.7 + + 2.9 + - It will be appreciated by those skilled in the art that there is certain equivalents to the specific techniques, materials, etc. described herein and these are considered part of this invention and are intended to be included within the scope of the appended claims. Although most of this description has been limited to the use of improved microcarriers for culturing anchorage-dependent cells, they can, of course, also be used for culturing other cell types.

Claims (7)

18. 460 906 PATENT CLAIMS 1. Cell culture microcarriers are characterized in that the microcarriers have a degree of substitution thereon with positively charged groups sufficient to give an anion exchange capacity of 0.1-4.5 meq / g of dry, untreated microcarriers, at which good cell growth occurs. Cell culture microcarriers according to Claim 1, characterized in that the microcarriers consist of cross-linked dextran beads. Cell culture microcarriers according to Claim 1 or 2, characterized in that the positively charged groups consist of tertiary or quaternary amino groups. Cell culture microcarrier according to one of the preceding claims, characterized in that the positively charged groups consist of diethylaminoethyl groups. Cell culture microcarrier according to Claim 1 or 2, characterized in that the positively charged groups consist of phosphonium groups. Cell culture microcarriers according to any one of the preceding claims, characterized in that they have an anion exchange capacity of 1-28 meq / g of dry, untreated, crosslinked dextran. Cell culture microcarrier according to one of the preceding claims, characterized in that the positively charged microcarriers have an average diameter of 120-200 μm.