IE57767B1 - A process for obtaining cell cultures - Google Patents

Abstract

The cell cultures are obtained from the kidneys of pig carcasses by digestion with a proteinase. Cell cultures of this type can be used advantageously for growing viruses.

Description

Processes for the preparation of cell cultures, including those of pig kidneys, for obtaining virus sus5 pensions as vaccine antigens are known. They are based, for example, on the use of pig embryo kidneys, piglet kidneys, permanent pig kidney cell lines or kidneys from slaughtered pigs.

The disadvantages of the processes of the state of the art for industrial production are: pig embryo kidneys are not obtainable in adequate quantity and at the desired times for production on the industrial scale. Piglet kidneys are relatively small, which means that, while the cell growth is good, the resulting cell yield is only low. In addition, as a rule, the carcass from which these kidneys have been removed cannot be used as a foodstuff. Although permanent pig kidney cell lines, such as PK 15, IBRS 2, 3TA or SK 6, can be propagated in large amounts, they are frequently contaminated with foreign agents, such as mycoplasma or viruses, so that their industrial utilisation for the preparation of immuno biological products entails increased risk. Moreover, the effort associated w i t h maintaining the cell line, including the continuous checks of quality, is considerable.

An additional factor is that many viruses do not multiply on permanent cell lines, because of their genetic changes, or they multiply only poorly compared with primary cultures of organ cells.

Kidneys from slaughtered pigs are a favorable basis for cells in terms of cost However, compared with embryonal or piglet kidneys, kidneys from slaughtered adult pigs result in only relatively few cells having mitotic activity. If, moreover, kidneys from slaughtered pigs are disintegrated in the conventional manner using the digestive enzyme trypsin, which damages cells, there is a further drastic reduction in the number of vital cells in a cell culture thus set up. It is evident from ) this that suspensions of kidney cells from slaughtered pigs which have been disintegrated by fermentation in the conventional Banner lead only with considerable effort, such as by changing the medium, increased addition of serum to the culture medium or special culture me di a, and after a long culture time, to cell cultures, which are only partially complete in most cases.

Thus, there are difficulties in respect of the readiness to grow of cultures of kidney cells from slaugh10 tered pigs which have been prepared by the customary trypsin disintegration of the organs. In particular, these difficulties with growth emerge when the culture vessels containing the kidney cells from slaughtered pigs which have been disintegrated by trypsin are rolled for optimal utilization of the culture surface.

Thus, there has continued to be a requirement for a process, which is also industrially satisfactory, for obtaining cultures of pig kidney cells.

Surprisingly, & process permits kidney cells from si augh dense cell cultures without special and without changing the medium, stationary or rolled cultures, the use of collagenase solution neys to give single cells. has now been found which tered pigs to grow to a d d ΐ t i ves to the me di um i n 5 to 8 days in This process comprises to disintegrate the kidThus the invention relates to a process for obtaining a cell culture by disintegration of kidneys of slaughtered pigs using an enzyme, wherein the enzyme is collage n a s e, from, used i A collagenase which can be used can be obtained ;or example, Clostridium histolyticum, and it is ; a concentration of 10-15, preferably 12.5, mg (corresponding to 6,,250 Mandi units) per 100 ml o‘ phosphate-buffered saline (PBS, pH 7.0 to 7.5).

It is particularly important in order to obtain large amounts of vital single cells having mitotic activity to expose the comminuted kidneys to the collagenase solution for a period of 12 to 24 hours, while agitating at 16 to 220C . are then purified Tne single cells thus obtained in a known manner by centrifugation at about 800 x g and washing with PBS. The centrifuged cell sediment is subsequently resuspended in the ratio of, preferably,, 1:250 to 1 :350 in known culture media,, such ©s Eagles minimal essential medium (E a ales HER) or in T C R 199 with the addi· tion of 100 ml/l calf ss um, and divided into portions in culture vessels which are kept stationary^ or are rolled,, at 37°C. Hany of the kidney cells sedimented on the glass surface start to divide within 3 to 16 hours. This process cen be observed under a microscope and it shows that there is a significant difference in the number of kidney cells which ire a ble to divide,, and in the speed of the sequence of cell division,, compared with batches of cells using conventional culture processes. The mitotic activity of the pig kidney cells treated with collagenase rapidly leads to dense growth of the cell cultures without any additional auxiliary measures for fhe cell culture.

By this aeans, it is possible, as illustrated in the examples which follow, advantageously to prepare, in rapid sequence, large amounts of cell cultures for the industrial culturing of viruses.

Example 1 Kidneys from slaughtered pigs were disintegrated to give single cells by the conventional process using 2 g/l trypsin solution, centrifuged at about 800 x g, washed with PBS and again centrifuged. The cell sediment was suspended in the ratio 1:300 in Eagles HER, to which 2.5 g/1 I acta I bumin hydrolysate (LAH) and 100 ml/l calf serum had been added. 600 ml quantities of this suspension were placed in sterile 5 I glass flasks. tures were incubated at 3 7°C, tially complete cell cultures had formed within 10 to 14 days, After changing iVue^edi um to serum-free medium (Eagles MEH with 2.5 g/ 1 LAH), the cultures were infected wifh reoT h e cul· while rolling. Only parvirus of serotypes 1 and 3, and kept 37°C for 3 to 5 The days until the culture was ready for harvesting, virus content of the harvested cultures tested, after removal of the cell residues by centrifugation followed by filtration, in the customary manner by inoculation of serial dilutions onto cell cultures.

For comparison, sterile, comminuted kidneys of slaughtered pigs were treated with collagenase solution which contained 12.5 mg of collagenase per I (corresponding to 6,250 Mandi units/100 ml)- After exposure for a period of 18 hours at 18°C, the kidney cells which had been disintegrated into single cells were further processed r I I, in the sai manner as betors The culture media- the cuI· ture vessels, the quantities of cell suspensions placed therein, and th® incubation were the same, in contrast to the cell cultures by the conventional process, mitoses of the kidney cells disintegrated by the process according to the invention were detectable under a microscope after only a few hour: Moreover, the individual cells with :o one mother, since mitotic activity were very close the number of damaged, and thus amitotic, cells was greatly reduced compared with the conventional disintegration process- Because the mitotic processes started very early 20 and were very numerous, densely closed tissue cultures which were capable of infection were present 5 to, at the most, 8 days after setting up the cell culture. The infection of th® tissue cultures ©nd the harvesting and testing of the viruses were carried out in a manner ana25 logous to that for the cultures prepared by the conventional process5 culture batches were prepared on the industrial seel® by each of the two processes described, snd 150 to I 300 I of reovirus of serotypes 1 and 3 were cultured on 30 each of these. The virus contents of the comparison batches were determined on permanent monkey kidney cells (vero cells). The titers found with the process according to the invention in place of the process of the state of the art were up to 14-5 times higher for reovirus of sero35 type 3, ©nd 5-6 to 6-3 times higher for serotype 1,.

Example 2 To prepare 40 I of a suspension of rhinopneumonitis virus (cause of abortion in mares) on piglet kidney cells, young piglets were necessary, while only two pairs of I - 6 kidneys from slaughtered pigs are used in the process, according to the invention, of Example 1. Wo deficiency in the quality of the virus prepared by the process according to the invention was observed» Example 3 As a herpes virus, the Aujeszky virus is a poor antigen, for which reason it is very important to have a high virus content in the vaccine. Comparison cultures of Aujeszky virus were carried out on cultures of kidneys from slaughtered pigs which had been prepared, on the one hand, by the conventional process using kidney cells disintegrated with trypsin and, on the other hand, by the process according to the invention (Example 1). While the virus titer/ml for the cultures prepared by the conventional process was 10"^ *5 z £ he mean for the cultures prepared by the process according to the invention was 10o,?z which means that the virus synthesized by the tissue cultures, which were younger (5-8 days old) and dense, was about ten times that synthesized by the cultures of the state of the art, which were older (10-14 days old) and, as a rule, not dense.

Example 4 Pig parvovirus (PPV) is regarded as the cause of the SMEDI syndrome in breeding sows, the characteristics of which are abortion, stillbirths, birth of piglets of low viability, and sterility in sows. PPV multiplies only in young cultures of pig tissue which are still growing, preferably in pig kidney cultures. Trials of the cultivation of PPV in cultures, prepared by the process according to the invention, tissue from slaughtered Dias have shown that cell cultures of this type are particularly well suited for the propagation of this virus. Hemagglutination titers of from 1:2048 to 1:16,384 were found, while virus titers of only 1:1024 are regarded as being good for PPV propagated according to the state of the art in embryonal pig kidney cell cultures or permanent pig kidney lines. itatmur)

Claims (3)

1. Patent claims:

1. A process for obtaining a cell culture by disintegration of the kidneys of slaughtered pigs using en enzyme, which comprises exposing the comminuted kidneys 5 to a stirred collagenase solution with a concentration of 100-150 mg/1 for 12 to 24 hours at IS to 22 fl C and allowing the pig kidney cells obtained in this way to grow to cell cultures in a known manner.

2. A process as claimed in claim 1, substantially 10 as hereinbefore described and exemplified.

3. A cell culture whenever obtained by a process claimed in a preceding claim. F. R. KELLY & CO. AGENTS FOR THE APPLICANTS