NO860180L - - Google Patents

Description

The present invention relates to a method for growing cells, especially diploid cells and especially diploid cells from mammals such as human cells, including fibroblast cells, in a culture medium in the presence of cellulose fibers of a special type. The cellulose fibers consist of cellulose with positive charges and/or anion exchange cellulose. This type of cellulose is well known, and certain types of such cellulose are e.g. described in "Techniques in Protein Chemistry", Elsevier Publishing Company, 1967, I. Leggett, pp. 290-304. Suitable material is carried in the trade, e.g. by Sigma Chemical Co., St. Louis, MO, USA. In these cellulose anion exchangers, common amino groups, which can be substituted in various ways, are bound to cellulose molecules and provide anion exchange properties.

The cells cultivated according to the invention preferably consist of so-called anchoring-dependent cells which are normally cultivated e.g. in suspension ion.

The following description of experiments according to the invention forms part of the present description.

Attempt 1

Medium: "Eagle's minimum essential medium" with the addition of 4 mM glutamine, 1 mM sodium pyruvate, 20 mM TRICINE (N-tris(hydroxymethyl)-methylglycine), pH 7.8, 10% calf serum and 0.1% sodium bicarbonate.

Cellulose: Cell growth is only achieved on positively charged cellulose fibers (anion exchangers), no growth was observed on negatively charged fibers (cation exchangers). All cellulosic materials were from Sigma Chemical Co., St. Louis, MO, USA. The following cellulose fibers were tested:

(1) QAE (diethyl-(2-hydroxypropyl)-aminoethyl) 0.9 meq/g

(2) DEAE (diethylaminoethyl) 0.9 meq/g +0.7 meq/g

(3) TEAE (triethylaminoethyl) 0.9 meq/g

(4) Aminoethyl 0.33 meq/g

(5) Benzyl-DEAE

(6) Benzoylated-naphthoylated DEAE

(7) ECTE0LA (epichlorohydrin-triethanolamine) 0.3 meq/g

(8) PEI (Polyethyleneimine) 1.07 meq/g

Likewise, good growth was obtained with QAE, DEAE and TEAE and weaker growth was obtained with the other types of cellulose. The fibers were slurried in distilled water at a concentration of 30 mg/ml and autoclaved for 30 minutes.

Cell lines: Human diploid fibroblasts were used in all experiments. These cells were derived from embryonic lung tissue and underwent various numbers of passages before being frozen and stored in liquid nitrogen. They are anchorage-dependent cells because they require a suitable surface to which they can attach, spread and eventually divide. Two cell strains were used: (1) MRC-5 obtained from England and (2) a cell strain obtained in the inventor's laboratory called Lu(S). The growth properties of both of these cell strains have been thoroughly investigated and have been shown to be similar.

They both have a lifespan in vitro of between 70 and 90 population divisions.

Method of preparation for cultivation of human fibroblasts: Frozen vials of cells were rapidly thawed and inoculated into plastic Roux flasks containing 150 mm of Eagle's medium. When the cell layer became confluent, the cells were resuspended in 50 ml of 200 ug/ml crystalline trypsin in phosphate-buffered saline without calcium and magnesium, pH 7.8, and the cells introduced into new Roux bottles with fresh medium. Eventually, the cells were used to inoculate a "spinner bottle", a bottle containing a suspended magnetic rod used as a stirring device, containing Eagle's medium and a suspension of cellulose fibres. The cell seeding used was 1x10 5 cells/mg cellulose. The cellulose was used in a concentration of approx. 3 mg/ml. Most experiments were carried out with QAE cellulose. The required amount of the stirred culture was centrifuged at 3000 X g for 10 minutes, the supernatant liquid was aspirated and the cellulose resuspended in the culture medium and added to the flask equipped with stirring.

The QAE cellulose fibers (coarse quality) had a length varying between 6 µm and 800 µm or more, and their width varied between 10 µm and 35 µm in diameter.

The cells added to the cellulose suspension adhered to some of the fibers but did not spread and grew over the surface of the fibers in the normal manner expected of anchorage-dependent cells. Instead, they grew as a mass of cells, in which cellulose fibers of different sizes were incorporated. These aggregates of cells and fibers reached a size of 150 to 200 µm or more in diameter after 7 to 8 days of incubation. The estimated number of cells obtained after this time ranged between 1.2x10<6> and 1.8x10<6>cells/ml. After 8 to 10 days of incubation, the number of cells in the suspension decreased. The medium was drained at intervals of 2 to 3 days by allowing the fiber cell masses to settle on the bottom of the bottle, half of the medium was sucked off and an equal volume of fresh medium added.

The fibers that were built into these aggregates had a length that varied between 100 µm or less and 200 µm, longer fibers being rarely observed. The width of the fibers appeared to be between 10 µm and 20 µm in diameter. The rest and the main part of the cellulose fibers were completely free of cells. The cells appeared to select a population of fibers that were best suited for cell attachment and growth. This effect may be due to the size of the fibers or an uneven distribution of charged chemical groups.

Attempt 2

Materials and methods

Medium: "Eagle's minimum essential medium" with the addition of 4 mM glutamine, 1 mM sodium pyruvate, 20 mM TRICINE (N-tris(hydroxy-methyl)-methylglycine), pH 7.8, 10% calf serum and 0.1% sodium bicarbonate.

Cell lines: Diploid lung fibroblasts from human embryos were used in all experiments. Most experiments were performed with a cell strain isolated in the inventor's laboratory called Lu(S) with a population doubling rate between 20 and 30 divisions. Certain experiments were performed with the cell strain MRC5 with a population doubling rate between 30 and 35 divisions. The results were similar for the two strains.

Cellulose: Cell growth was only obtained on positively charged cellulose fibers (anion exchangers), no growth was observed on negatively charged fibers (cation exchangers). All cellulose was from Sigma Chemical Co., St. Louis, MO, USA. The following anion exchangers were investigated:

(1) QAE - 2-Hydroxypropylaminoethyl 0.9 meq/g

(2) DEAE - diethylaminoethyl 0.9 meq/g

(3) TEAE - triethylaminoethyl 0.9 meq/g

(4) Aminoethyl 0.33 meq/g

(5) Benzy1-DEAE

(6) Benzoylated-naphthoylated DEAE

(7) ECTEOLA - epichlorohydrin-triethanolamine 0.3 meq/g

(8) PEI - polyethyleneimine 1.07 meq/g

Microscopic examination of the cellulose cell suspension (figure 4) showed that the cells did not adhere to and were spread on the fibers forming a monolayer, which is the most common form of cell growth in vitro. Instead, aggregates of cells and cellulose fibers were formed and increased in diameter as the incubation progressed.

The cellulose preparations used at the beginning of these experiments were classified as "coarse grade" (coarse grade) by the manufacturer and the fibers had a length varying between 6 pm and 800 pm or more, and their width varied between 10 pm and 35 pm in diameter . As can be seen from figure 4a, b, c, and f, a large part of the fibers were not connected to the cell fiber aggregates. The fibers that were built into these aggregates had a length varying between less than 100 pm and 200 pm, longer fibers were rarely observed. The width of the fibers appeared to be between 10 µm and 20 µm in diameter. The cell-fiber aggregates can reach sizes of 150 to 200 pm in diameter after 7 to 8 days of incubation. Larger aggregates were sometimes observed.

The cells appeared to select a population of fibers that are best suited for cell attachment and growth. This selection seems to be related to the size of the fibers. A cellulose suspension filtered through a 200 mesh stainless steel mesh was inoculated with the cells. As can be seen from Figure 4d, e, larger parts of the fibers with a length of approx. 100 pm or less attached to cells. Preliminary attempts to grow fibroblasts on TLC (thin layer chromatography) preparations of QAE and DEAE cellulose, having fiber lengths of 100 pm or less, indicate that these preparations are better suited for cell-fiber aggregates than coarse grade cellulose materials.

The ability of diploid human fibroblasts to grow in the form of their cell-fibrous aggregates has not previously been reported to the inventor's knowledge.

Folkman & Greenspan have shown that spheroids of cell masses can grow to a diameter of several millimeters, but at a diameter of 1 mm the cells in the center became necrotic. However, as the cell-fibre aggregates were much smaller and permeated by fibres, which could possibly help to conduct medium to the inner parts of the aggregates, it is possible that the largest part of the cells were alive in the aggregates that were formed during at least 8 days' incubation. Longer incubation, however, often resulted in a reduction of the cell protein, even with frequently repeated changes of medium, and there must therefore be a limit to the size and viability of the cells in these aggregates.

The method described has proven beneficial for large-scale production of e.g. virus vaccines with human diploid fibroblasts.

The anion exchange cellulose used according to the invention preferably contains amino groups and preferably has 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. Preferably, the anion exchange capacity is up to 5 meq/g, optionally up to 3 meq/g or up to 1.5 meq/g or also up to 1 meq/g, determined by conventional methods.

The anion exchange cellulose used is preferably in the form of fibers of which, in relation to the weight, at least 20%, preferably at least 50% and in particular at least 75% or at least 90% have a fiber diameter of a maximum of 200 µm, preferably a maximum of 100 µm, especially a maximum of 50 µm or a maximum of 25 pm. The fiber length is preferably a maximum of 1000 pm, in particular a maximum of 200 pm or a maximum of 100 pm. Also fiber length values of a maximum of 50 µm or a maximum of 20 µm have been found to be significant. At the same time, the fiber length is preferably at least 0.1 µm, in particular at least 1 µm, especially at least 5 µm or at least 10 µm. The fiber length values are hereby calculated at at least 50%, preferably at least 75%, at least 90% or at least 99% of the cellulose material weight.

The cellulose anion exchanger used preferably belongs to one of the types: QAE (diethyl-(2-hydroxypropyl)-aminoethyl) cellulose, preferably with an ion exchange capacity of approx. 0.9 meq/g,

DEAE (diethylaminoethyl) cellulose, preferably with an ion exchange capacity of approx. 0.7 to 0.9 meq/g,

TEAE (triethylaminoethyl) cellulose, preferably with an ion exchange capacity of approx. 0.9 meq/g,

aminoethyl cellulose, preferably with an ion exchange capacity of 0.33 meq/g,

benzyl DEAE cellulose,

benzoylated-naphthoylated DEAE.

According to the invention, one preferably cultivates diploid cells from mammals, preferably human cells, such as fibroblast cells, and this cultivation can also be carried out for virus cultivation or vaccine production.

According to one aspect of the invention, the cells are made to grow on fibers or the fibers as cell aggregates, preferably of spherical or cylindrical shape and preferably with a diameter of up to 1000 pm, optionally up to 500 pm or up to 300 pm or also up to 200 or 100 pm. The cell aggregates are preferably grown until at least 10%, at least 50% or at least 90% of the cell material weight exists in the form of such cell aggregates with a diameter of at least 10 pm, preferably at least 25 pm or at least 50 pm, possibly at least 100 pm. The cells in such cell aggregations form an accumulation of more than one monolayer of cells. These cell collections preferably include at least parts of two or more fibers within the cell collection. These fibers preferably have fiber lengths and fiber diameters within the above-mentioned limits, e.g. a fiber length of a maximum of 500 pm and in particular a maximum of 300 pm and e.g. a diameter between 3 and 50 pm, in particular between 5 and 30 pm, e.g. 10-20 pm. Growth is preferably carried out until the number of cells per ml of the dispersion reaches at least 10<4>, at least 10<5> or at least 10<6> cells per ml dispersion. Appropriate values for the upper limit of the cell amount are e.g. maximum 10 9 , maximum 10 8 or maximum 10 7 cells/ml dispersion. The cell culture time can be varied, e.g. go up for at least 1 day, at least 2 days or at least 3 days or at least 5 days. Appropriate Upper limit values for cell culture time can e.g. be up to 14 days, up to 10 days, up to 7 days or up to 5 days.

Suitable concentrations of cellulose fibers in the culture medium can be at least 0.01 mg/ml, at least 0.1 mg/ml or at least 1 mg/ml, possibly at least 5 mg/ml. Appropriate upper limit values have been shown, e.g. to be a maximum of 500 mg/ml, a maximum of 100 mg/ml, a maximum of 50 mg/ml or a maximum of 10 mg/ml.

Suitable culture media and culture conditions otherwise for different cell types are well known to a person skilled in the art and can be used according to the invention. In this respect, it can e.g. refer to the publication "Microcarrier Cell Culture Principles & Methods" from Pharmacia Fine Chemicals and to the literature references indicated in this publication.

The use of diploid human fibroblasts for polio vaccine production has previously been limited due to technical difficulties in culturing these cells on previously available microcarriers. The cells grow to relatively low concentrations, and the cultures have been so sensitive to handling that a large number of cells on the microcarriers could be lost in the various treatment steps to which a culture is subjected. The use of microcarriers of positively charged cellulose according to the invention for cell cultivation has excluded many of these problems. The diploid human fibroblast cells have been cultured with good results as cell-fiber aggregates with several cellulose fibers that were tried, as mentioned above, e.g. diethyl-2-hydroxypropyl-aminoethyl (QAE), diethylaminoethyl (DEAE), triethylaminoethyl (TEAE).

In the following, results obtained with suspension cultures of such cell-fibrous aggregates from diploid human fibroblast cells infected with poliovirus are given.

Materials and methods:

Cell strains: Two strains of embryonic human lung fibroblasts were used: (1) MRC5 obtained from The National Institute for Biological Standards and Control, England, and (2) Lu(S) isolated from embryonic human lung tissue in the inventor's laboratory.

Cellulose anion exchange fibers: Most experiments were performed with QAE or DEAE cellulose, especially preparations with short fiber lengths made for thin layer chromatography (TLC). Other cellulosic materials used were TEAE and benzyl-DEAE. All cellulose materials had an ion exchange capacity of approx. 0.9 meq/g. The cellulose was used in a concentration of 3 mg/ml.

Medium and cell culture: The medium for the cell culture was "Eagle's minimum essential medium" supplemented with 10% calf serum, 4 mM glutamine, 20 mM TRICINE buffer, pH 7.8 and 1 mM Na-pyruvate.

The cells were first grown to a confluent monolayer in plastic Roux flasks, slurried with trypsin (200 µm/ml crystalline trypsin (Sigma) in phosphate-buffered saline containing 20 mM TRICINE, pH 7.7-7.8 and 0.08 % sodium bicarbonate), was centrifuged, washed once and added to a concentration of approx. 1.5x10^ cells/mg cellulose. After 5-7 days' incubation, the cell-fiber mass was washed three times with phosphate-buffered saline, pH 7.4, resuspended to the original volume in "Parker's 199 medium" without serum and infected with poliovirus. In most of these experiments, poliovirus type 1 (Brunender) and poliovirus type 2 (MEF-1) were used in one experiment. The infectivity of these viruses was measured by inoculating the virus preparations in tenfold dilution in tissue cultures and the dilution at which 50% of the cultures were infected was calculated and termed "tissue culture infection dose", 50% endpoint (VKID5Q). All strength values are given in log1QVKID5Q.

Result:

Table II shows the results of 5 independent 100 ml suspension cultures of Lu(S) cells with 3 mg/ml QAE cellulose infected with 6.3 VKIDC_ poliovirus type 1.

YOU

Table III shows the results from five 100 ml suspension cultures of MRC 5 cells with 3 mg/ml of the specified cellulose materials. The cultures were infected with 6.0 VRID^/ml poliovirus type 1.

Suspension cultures with volumes greater than 100 ml also gave rise to similar viral strength values. Table IV describes some results of poliovirus type 1 grown in MRC 5 cells together with 3 mg/ml DEAE-TLC cellulose. The virus inoculation amounted to 6.0 VKID50</ml.>

Type 2 poliovirus was inoculated at a concentration of 5.0 VKID5Q/ml in a 5 liter suspension culture of MRC 5 with 3 mg/ml DEAE-TLC cellulose. After 24 hours the strength value had risen to 6.5 and after 48 hours to 6.8 VKIDc_/ml. Although these potency values were low, presumably because the inoculation was so low, they show that virus could increase in the vicinity of a hundredfold, which is about the same increase achieved with poliovirus type 1.

Diploid human fibroblasts constitute the best available cell substrate for human virus vaccine production, as the cell lines can be easily regulated and standardized, are very well studied, support growth of most human virus types, and are completely normal, which excludes the risk of oncogenic DNA entering in the vaccine preparation. However, it has been difficult to use these cells for vaccine production because of the difficulties in growing the cells on a large scale on microcarriers. According to the present invention, it has been shown that diploid human fibroblasts can be grown to sufficient cell concentrations together with cellulose micro-carriers (e.g. between 1.5 and 2x10^ cells/ml), so that in case of infection with viruses, e.g. . poliovirus, strength values can be achieved which are significantly higher than what can be achieved with monocultures of these cells on e.g. monkey kidney cells.

In the following, comparative experiments which were carried out with the method according to the invention and with conventional cell culture substrates are described. The cultured cells consisted of diploid human fibroblast cells, strain MRC5. This cell strain is described e.g. in an article by Jacobs, Jones, Baille: "Characteristics of a human diploid cell designated MRC5" in Nature, vol. 227, pp. 168-170, 1970, originating from the National Institute for Biological Study and Control.

The culture medium consisted of "Eagle's minimum essential medium" in all experiments and this was supplemented with 10% calf serum, 20 mM Tricine, pH 7.8, 4 mM glutamine, 1 mM Na-pyruvate and 0.08% bicarbonate.

The following microcarriers were used:

1. Microcarriers according to the invention: DEAE cellulose fibers (small fibers) for thin layer chromatography, capacity 0.96 meq/g. 2. Biosilon, which consists of microcarriers of plastic balls and was added to a medium as a sterile powder. 3 and 4. Cytodex and Gelibeads (thiogelatin beads) prepared according to the manufacturer's instructions.

The cellulose according to the invention was used in a concentration of 3 mg/ml, biosilon in an amount of 60 mg/ml, cytodex and gelibeads in a concentration of 4 mg/ml.

Result

The experiment concerns the determination of potency values for poliovirus type 1 obtained from the above-mentioned cell cultures on microcarriers.

MRC5 , passage 21, was seeded in suspension cultures at a concentration of 4x10 5 cells/ml. The cultures were stirred at a speed of approx. 40 revolutions/minute. The medium was replaced once after 4 days' incubation by allowing the cell-microcarrier mass to settle to the bottom of the flask, the old medium was aspirated and an equal volume of fresh medium added.

After 7 days' incubation at 37°C, the cell-microcarrier mass was washed three times with phosphate-buffered saline pH 7.4 and serum-free Parker's 199 medium added. The cultures were then infected with poliovirus type 1 (inoculum amount about 10 5 - 5 TCID 4 Q/ml). The results are shown in Table V. Previous experiments have shown that the optimal time to obtain antigenic material for poliovirus vaccine is 72 hours after infection.

The cells cultivated according to the invention preferably consist of normal cells, i.e. cells with limited divisibility, e.g. on average, often a maximum of 150, a maximum of 100 or a maximum of 70 divisions (so-called population divisions). Definitions of such cells can be found in the literature, e.g. Haeflick, Moorhead: "The serial cultivation of human diploid strains" Experimental Cell Research, vol. 25 (1961), pp. 585-621, and Haeflick: "The limited in vitro lifetime of human diploid cell strains", Experimental Cell Research, vol. 37 (1965), pp. 614-636.

However, one can also grow cells of cancer type or cells that have been modified for combination of cells of cancer type or by introducing genetic material from cancer cells or into cancer cells.

Examples of cell origin include animal cells in general, cells from vertebrates such as cells from birds, e.g. chicken birds, mammalian cells, e.g. cells from rats, mice, monkeys, rodents, horses, pigs, sheep, dogs, cats and especially human cells. The cultured cells often consist of embryo cells (fetal cells) of the above-mentioned origin, for example male cells or female embryos. The cells can also originate from special organs, e.g. lung, skin, foreskin, epithelial cells. Chicken embryo fibroblasts can also be mentioned as examples of embryo cells.

The cell culture can be used for the production of various products, e.g. enzymes, hormones, e.g. interferon, as a substrate for virus cultivation and the production of a vaccine against these, e.g. polio, rabies, rubella, influenza, measles, herpes, pseudorabies, foot-and-mouth disease, AIDS-causing virus, etc. Cell cultures that have been approved for vaccine production by the WHO are often used.

Appropriate dimensions of the cellulose fibers are indicated beforehand.

A fiber length of at least 20 µm, at least 40 µm or at least 60 µm has often been found to be suitable. At the same time, the fiber length's upper limit can lie on the previously mentioned limit values, e.g. maximum 200 um or maximum 150 um, or also maximum 100 pm. The fiber length values are calculated below in the same way for the aforementioned fiber length values of at least 30%, preferably at least 50%, especially at least 75%, at least 90% or at least 99% of the cellulose material weight.

Cellulose, which is used as a substrate for the anion-exchange cellulose materials used according to the invention, normally consists of a polymer of glucose with approx. 2000-4000 or more, e.g. 3500 or more repeating units in a chain. The glycosidic bond sits at 0, cellulose can be defined as a polymer of (3-D-glucose.

Figure 1 - Growth of Lu(S) cells in 100 ml suspension cultures containing 3 mg/ml QAE, DEAE or TEAE cellulose and seeded with 20x10<6> cells. The cell count is estimated from measured protein values. Figure 2 - The effect of different QAE cellulose concentrations on the growth of Lu(S) cells in 100 ml suspension cultures seeded with 20x10<6> cells. The cell number was determined from measured protein values. Figure 3 - The effect of cell seeding on the growth of Lu(S) cells in 100 ml suspension cultures containing 3 mg/ml QAE cellulose. The cellulose amount was estimated from the measured protein value. Figure 4 - Morphological appearance of diploid human fibroblast cell fiber aggregates (see arrow).

A and B: 4-day MRC 5 cultivation with DEAE-cellulose, 380 times.

C: 13 day MRC 5 cultivation with 3 mg/ml QAE cellulose, 380 times. D: 8-day Lu(S) cultivation with approx. 1 mg/ml QAE cellulose filtered through a 200 mesh stainless steel mesh, 48 times.

E: 8-day Lu(S) cultivation with approx. 1 mg/ml QAE cellulose filtered through a 200 mesh stainless steel mesh, 960 times.

F: 13-day MRC 5 cultivation with 3 mg/ml QAE cellulose, 120 times.

Claims (7)

1. Procedure for growing normal diploid cells in suspension in a culture medium, characterized in that the growth is carried out in a suspension comprising fibers of cellulose with positive charges and/or anion exchange capacity. 2. Method according to claim 1, characterized in that the cellulose consists of an anion exchange cellulose, preferably with amino groups and especially with an anion exchange capacity of at least 0.01 meq/g and possibly up to 5 meq/g. 3. Method according to claim 1 or 2, characterized in that fibers of anion exchange cellulose are used, of which, in relation to the weight, at least 20% have a fiber diameter of a maximum of 100 µm, and preferably an amount of a maximum of 1000 µm and at least 1 µm. 4. Method according to each of claims 1 to 3, characterized in that the anion exchange cellulose belongs to one of the types QAE (diethyl-(2-hydroxypropyl)aminoethyl)-cellulose, preferably with an ion exchange capacity of approx. 0.9 meq/g, DEAE (diethylaminoethyl) cellulose, preferably with an ion exchange capacity of approx. 0.7-0.9 meq/g, TEAE (triethylaminoethyl) cellulose, preferably with an ion exchange capacity of approx. 0.9 meq/g, aminoethyl cellulose, preferably with an ion exchange capacity of 0.33 meq/g, bensy1-DEAE-cellulose, benzoylated-naphthoylated DEAE. 5. Method according to each of claims 1 to 4, characterized in that it comprises the cultivation of diploid cells from mammals, especially human cells, especially fibroblast cells, possibly for the cultivation of viruses or vaccine production. 6. Method according to each of claims 1 to 5, characterized by growing the cells on fibers or the fibers as cell aggregates, preferably with an essentially spherical or cylindrical shape and especially to a diameter of up to 1000 pm and preferably with a diameter of at least 10 pm in an amount that exceeds a monolayer of cells, the cell aggregate enclosing at least parts of two or more fibers within the cell aggregate, preferably fibers with a length of at most 500 pm and preferably a diameter of between 3 and 50 pm , as the cultivation is preferably carried out until the number of cells per ml in the dispersion reaches at least 10 4 cells per ml dispersion and preferably to a maximum of 10 g cells per ml dispersion, especially over a period of at least 1 day and preferably up to 14 days. 7. Method according to each of claims 1 to 6, characterized in that the concentration of cellulose fibers in the culture medium is at least 0.01 mg/ml and preferably a maximum of 500 mg/ml.