IE850932L - 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

7? 6 7 PATENTS ACT, 1964 COMPLETE SPECIFICATION I V. i .. - - n A PROCESS FOR OBTAINING CELL CULTURES r> ^ /s ,, &7,~s Processes for the preparation ot cell cultures, T . . \ including those of pig k i dneys, for obtaimng virus sus- 5 pensions as vaccine antigens are known. They are based, for example, on the use of pig embryo kidneys, piglet ki dneys, permanent pig kidney cell lines or kidneys from slaughtered pigs.

The disadvantages of the processes of the state 10 of the art for industrial product i on are: pig embryo kidneys are not obtainable in adequate quantity and at the desi red tines for product i on on the industrial scale., Piglet ki dneys are relatively sma 11, which aeans that, while the cell growth is good, the resulting cell yield 15 is only low. In addition, as a rule, the carcass from which these ki dneys have been removed cannot be used as a foodstuffAlthough permanent pig kidney cell lines, such as PK 15, I3RS 2, 3TA or SK 6, can be propagated in large amounts, they are frequently contaminated with 20 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 with maintaining the cell line, including the continuous checks of quality, is considerable-25 An additional factor is that saany 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 t 30 basis for cells in term?, of cost- However, compared with I embryonal or piglet ki dneys, kidneys from slaughtered f adult pigs result in only relatively few cells having mitotic activity. If, moreover, kidneys from slaughtered pigs are disintegrated in the conventional manner using 55 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 haws been disintegrated by fermentation in the conventional wanner lead only with considerable effort, such as by changing the medium,, increased addition of 5 serum to the culture medium or special culture medi a, and after a long culture time,, to cell cultures, which are only I partially complete in most cases. 1 Thus, there are difficulties in respect of the ^ readiness to grow of cultures of kidney cells from s laugh-10 tered pigs which nave 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 15 utilisation 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, a process has now been found which 20 permits kidney cells from slaughtered pigs to grow to dense cell cultures without special additives to the medi urn, and without changing the medium, in 5 to 8 days in stationary or rolled cultures. This process comprises the use of col lagenase solution to disintegrate the kid-25 neys to give single cells.

Thus 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 0. 30 A col lagenase which can be used can be obtained from, for example, Clostridium histolyti cum, and it is used at a concentration of 10-15, preferably 12.5, ag (cor respond! ng to 6,25 0 Hand I units) per 100 «l of phosphate-buffered saline (PBS, pH 7»Q to 7). 35 It is particularly important in order to obtain 1 large amounts of vital single cells having ai tot i c activity to expose the comminuted kidneys to the col lagenase solution for a period of 12 to 24 hours, while agitating at 16 to 22°C, Tne single cells thus obtained are then purified in a known manner by centrifugation at about 800 x g and washing with PBS. The centrifuged cell sedi went is subsequently re suspended in the ratio of, preferably^ 1:250 5 to 1 :35 0 in known culture snedi a , such as Eagles minimal essential medium (Eagles MEM) or in TCh 199 with the addition of 100 tnl/l calf serurn, and divided into portions in culture vessels which are kept stationary, or are rolled, at 37°C. Many of the kidney cells sedi ment ed on the 10 glass surface start to divide within 3 to 16 hours. This process can be observed under a microscope and it shows that there is a significant difference in the number of kidney cells which are able to divide, and in the speed of the sequence of cell division, compared with batches 15 of cells using conventional culture processes. The Mitotic activity of the pig kidney cells treated with collagenase rapid ly leads to dense growth of the cell cultures without any additional auxiliary measures for the cell culture. By this means, it is possible, as illustrated in the 20 examples which fo I low, advantageous ly to prepare, in rapid sequence, large amounts of cell cultures for the industrial culturing of viruses.

Example 1 Kidneys from slaughtered pigs Mere disintegrated 25 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 MEM, to which 2.5 g/l I a c t a I bum i n hydrolysate (LAH) and 100 ml/l calf 3 0 serum had been added. 600 mI quantities of this suspension were placed in sterile 5 I glass flasks. The cultures were incubated at 37°C, while rolling. Only partially comp lete cell cultures had formed within 10 to 14 days. After changing Th^ymldi um to serum-tree medium (Eagles MEM 35 with 2.5 g/ 1 LAH), the cultures were infected with reo-vi rus of serotypes 1 and 3, and kept at 37°C for 3 to 5 days until the culture was ready for harvesting. The virus content of the harvested cultures tested, after removal of the cell residues by cent r i fugat i on followed by filtration, in the customary manner by inoculation of serial dilutions onto cell cultures.

For comparison, steri I e, comminuted kidneys of slaughtered pigs were treated with co I lagenase solution which contained 12.5 tag of collagenase per I (corresponding to 6,25 0 Mandl units/100 ml). After exposure for a period of 18 hours at 1 8°C , the kidney cells which had been disintegrated into single cells were further processed in the same manner as before. The culture media,, the culture vessels, the quantities of cell suspensions placed therein^, and the incubation were the same. In contrast to the cell cultures by the conventions I process, mitoses of the kidney cells disintegrated by the process according to the invention were detectable under a microscope after only a feu hours. Moreover, the individual cells with mitotic activity were very close to one another, since the number of damaged, and thus arai toti c, cells was greatly reduced compared with the conventional di sintegration process. Because the Mitotic processes started very early 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 the tissue cultures and the harvesting and testing of the viruses were carried out in a taanner analogous to that for the cultures prepared by the conventional process. 5 culture batches were prepared on the industrial scale by each of the two processes described,, and 150 to I 300 I of reovi rus of serotypes 1 and 3 were cultured on each of these. The virus contents of the comparison batches were determined on permanent laonkey 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 tisaes higher for reovi rus of serotype 3, and 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, 20 young piglets were necessary, while only two pairs of 5 i 'i 1 0 15 20 25 30 J 35 - 6 - kidneys from slaughtered pigs are used in the process, according to the invention, of Ex amp Ie 1. No deficiency in the quality of the virus prepared by the process according to the invention was observed.

Example 3 As a herpes vi rus, the Auj eszky virus is a poor antigen, for which reason it is very important to have a high virus content in the vaccine., Cornea r i son cultures of Auj e sz ky 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 ot her hand, by the process according to the invention (Example 1). While the virus titer/nl for the cultures prepared by the conventional process was 10~7-5 r the nean for the cultures prepared by the process according to the invention was 10*~® , which means that the virus synthesized by the tissue cuItures, which were younger £5-8 days old) and dense, was about ten tines that synthesized by the cultures of the state of the art, which we re older (10-14 days old) and, as a rule, not dense.

Example 4 Pig parvovirus (PPV) is regarded as the cause of the SHEDI syndrome in breedi ng sows, the characteristics of which are abortion, stillbirths, birth of piglets of low viabi lity, end sterility in sows. PPV multiplies only in young cultures of pig t issue which are still growi ng, preferably in pig kidney cultures™ Trials of the cultivation of PPV in cultures, prepared by the process according to the invention, of tissue frosn slaughtered pigs have shown that cell cultures of this type are particularly well suited for the propagat i on of this virus,. Hemagglutination titers of from 1:2048 to 1:16,384 were found, while virus titers of only 1:1024 are regarded a$ being good for PPV propagated according to the state of the art in embryonal pig kidney cell cultures or permanent pig kidney lines-

Claims (3)

Patent claims:

1. A process for obtaining & cell culture by disintegration of the kidneys of slaughtered pigs using an enzyme, which comprises exposing the comminuted kidneys 5 to a stirred collagenase solution with a concentration of 100-150 aig/I for 12 to 24 hours at 16 to 22*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