CN111057675A - Sheep testis immortalized cell domestication method adapting to full suspension culture - Google Patents
Sheep testis immortalized cell domestication method adapting to full suspension culture Download PDFInfo
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Abstract
The invention discloses a method for domesticating immortalized cells of sheep testis adapting to full suspension culture, which comprises the following steps: (1) recovering the unacclimated sheep testicular cells in a water bath and then centrifuging; (2) resuspending cells with DMEM culture solution containing 5% newborn calf serum, and inoculating to a cell culture bottle for culture; (3) subculturing and digesting; (4) performing rotary suspension culture; (5) transferring the goat testis into a suspension culture bottle for culture until cell mass disappears, wherein the cell density can be stable when repeated culture is carried out, and the goat testis immortalized cell suitable for full suspension is obtained. Under the suspension culture condition, the shape of the domesticated and cultured generation cells is spherical, transparent and in a single or small-ball suspension shape; the 10-generation suspension domesticated cells can be stably cultured for 96 hours to 2-3 multiplied by 106Cells/ml; the virus culture adaptability shows that the virus titer reaches a peak value 72 hours after the domesticated suspension cells are inoculated with the orf virus, and is similar to the virus production level of the cells cultured by adherence.
Description
Technical Field
The invention relates to a method for domesticating immortalized cells of sheep testis adapting to full suspension culture, belonging to the field of suspension culture of immortalized sheep testis cell lines.
Background
Compared with the traditional spinner flask culture technology, the cell suspension culture technology has the advantages of simple and easy culture, safe operation, uniform batch, capability of automatic and large-scale preparation and the like, and can reduce the labor, cost, field and production period in the actual production process.
As most cells for vaccine virus production are in adherent growth, the key for restricting the application of the cell suspension culture technology is to adapt to cell strains in full suspension culture. The suspension culture of the cell carrier is similar to the adherent culture, the seed cells need to be provided by a rotary bottle, a high-cost microcarrier is used, complicated steps such as pancreatin digestion are carried out, and a culture medium with high serum content is needed, so that the method is not beneficial to industrial production. The cell full suspension culture technology can be used for culturing by serum-free or low-serum, has low cost and safety, and is easy to amplify the production process.
The testis Caprae Seu Ovis cell DST-3 is immortalized cell prepared in the laboratory of the present inventor, is suitable for the replication of orf virus (ORFV), grows adherent in vitro, and can be continuously subcultured.
Therefore, the domestication method of the testis Caprae Seu Ovis immortalized cells adapting to full suspension culture is provided, and the obtained testis Caprae Seu Ovis immortalized cells adapting to full suspension culture are of great significance for the production of large-scale ORFV vaccine.
Disclosure of Invention
The invention mainly aims to provide a method for domesticating sheep testis immortalized cells adapting to full suspension culture;
the main purpose of the invention is realized by the following technical scheme:
a sheep testis immortalized cell domestication method adapting to full suspension culture comprises the following steps:
(1) reviving the immortalized sheep testicle cell DST-3 growing by adherence in water bath and then centrifuging;
(2) suspending the cells again by DMEM culture solution containing 5% newborn calf serum, and inoculating the cells in a common cell culture bottle for culture;
(3) subculturing the cultured cells, and digesting;
(4) carrying out rotary suspension culture on the digested cells;
(5) and transferring the cells into a suspension culture bottle for culture after the cell density is increased, and obtaining the sheep testis immortalized cells suitable for suspension culture when the cell mass disappears and the cell density can be stabilized by repeated culture.
As a preferred embodiment of the invention, the recovery mode in the step (1) is to put the unacclimated cells into a water bath at 37 ℃ for recovery; the centrifugation is preferably carried out at a rotation speed of 1000r/min for 1 min.
As a preferred embodiment of the present invention, the cultivation in the step (2) is carried out at a cultivation temperature of 37 ℃ and 5% CO2Culturing under the conditions of (1); the culture time is preferably 72 h.
As a preferred embodiment of the present invention, the subculture in step (3) is a subculture at a ratio of 1: 3, and the subculture time is preferably 72 hours.
As a preferred embodiment of the present invention, the conditions of the rotary suspension culture described in the step (4) include:at 37 ℃ 5% CO2Performing rotary suspension culture; wherein the rotating speed of the shaking table is preferably 100r/min, the pH value is 7.0-7.4, and NaHCO is used every day3The pH was adjusted 2 times.
As a preferred embodiment of the present invention, the cell concentration in step (5) is repeatable means 96h from 0.5X 106Cells/ml grow to 2-3X 106Cells/ml.
The invention carries out morphology, bacteria, mycoplasma and identification on the DST-3S F1 generation cells obtained by the domestication method:
under the suspension culture condition, the DST-3S F1 generation cells are spherical, transparent and in a single or small-ball suspension shape; the 10 generations of suspension domesticated cells can be stabilized for 96 hours and can grow to 2-3 multiplied by 106Cells/ml, the maximum growth density of 10 generations of cells has already stabilized as the number of acclimated generations of cells increases.
The results of the sterility test and the mycoplasma test show that F1 DST-3S suspension cells have no bacteria and mycoplasma contamination.
The invention cultures the suspension cells of DST-3S F1 generation and F10 generation for 12h, inoculates ORFV AH-GY13 strain to 100TCID50The virus content was determined every 24h per ml sample. According to virus culture adaptability test, the virus content reaches the peak value after inoculation for 72h, and the peak value is 10 respectively-6.5TCID50A ratio of 10 to 10-6.4TCID50/ml。
The sheep testicular cell DST-3 is an immortalized cell prepared in the laboratory of the inventor, supports the replication of ORFV, is cultured in vitro to grow adherent, and can be subjected to continuous subculture; in order to adapt to large-scale ORFV vaccine production, the invention domesticates the sheep testicular cell DST-3 adherent cell in a suspension culture bottle, and the cell can completely adapt to suspension culture after 10 generations of culture. Through detection, the cell density can reach 2-3 multiplied by 106Cells/ml. The cell line is named as sheep testicular cell DST-3S, the established cell line is ORFV adaptive cell line, and a stable cell environment and a standardized culture method are provided for the development and production of the virus attenuated vaccine and the inactivated vaccine.
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FIG. 1DST-3 adherent monolayer cells (A) and domesticated (B) DST-3S suspension cells.
Detailed Description
The invention is further described below in conjunction with specific embodiments, the advantages and features of which will become apparent from the description. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.
Experimental example 1 domestication and identification of immortalized cells of sheep testis adapted to full suspension culture
1 materials of the experiment
1.1 cells sheep testis cells DST-3 are immortalized cells prepared in the laboratory of the inventor.
1.2 Medium for sterility test thioglycollate Medium (T.G), TSB and Mycoplasma Medium were purchased from Mediterranean organisms; DMEM cell culture medium containing 5% newborn bovine serum, the raw material was purchased from GIBCO company; cell serum-free medium was purchased from Shanghai culture Bio.
1.3 suspension culture flasks Wheaton, USA.
1.4 Virus ORFV strain AH-GY13, Huifeng by the university of agriculture, Anhui.
2 method of experiment
2.1 cell acclimation and shaking culture
Taking the unacclimated cells out of the liquid nitrogen tank, putting the unacclimated cells in a water bath at 37 ℃ for resuscitation, and centrifuging the cells at 1000r/min for 1 min. Discarding the frozen stock solution, resuspending the cells in DMEM culture containing 5% newborn calf serum, inoculating in a common cell culture flask, placing at 37 deg.C and 5% CO2And (5) culturing. After 72h, subculture at a ratio of 1: 3, culturing for 72h, digesting and collecting cells, inoculating into 250ml triangular flask, and culturing at 37 deg.C with 5% CO2And (5) performing rotary suspension culture. The rotating speed of the shaking table is 100r/min, the pH value is 7.0-7.4, and NaHCO is used every day3The pH was adjusted 2 times. When the cell density is increased to a certain amount, transferring the cells into a special suspension culture bottle for culture and passage. Culturing until cell mass disappears, and culturing for 96h when cell density is repeated,steadily from 0.5X 106The cell/ml is increased to 2-3 × 106Cells/ml, cell acclimation was completed, and the cells after suspension acclimation were DST-3S (passage F1).
2.2 cell cryopreservation
Placing well-grown suspension cells into a centrifuge bottle, centrifuging at 1000r/min for 10min, discarding supernatant, adding cell freezing medium (final concentration of newborn calf serum is 50%, final concentration of DMSO is 10%), and adjusting cell concentration to 5 × 106The cells/ml, re-prepared into cell suspension, and subpackaged in cryopreservation tubes, 1.8ml per tube.
2.3 subculture of suspension cells
The frozen cells were taken out of the liquid nitrogen, immediately thawed in a 37 ℃ water bath, and added to a suspension flask containing DMEM growth medium containing 5% newborn bovine serum at 37 ℃ with 5% CO2In (3), suspension culture was performed, and the culture was expanded to F10 generation. The morphology and growth status of the cells, as well as the suitability of the cells for infection and replication of ORFV, were examined.
2.4 preliminary characterization of suspension cells
And performing morphology, bacteria, mycoplasma and identification on the domesticated F1 generation cells.
2.4.1 cell morphology and enumeration
The cells were observed under a microscope to confirm the consistency of their microscopic characteristics and morphology. And a certain number of cells were counted.
2.4.2 suspension cell purity test
2.4.2.1 sterility test
The test is carried out according to the appendix of three parts of Chinese animal pharmacopoeia 2015 edition, and no bacteria grow.
2.4.2.2 Mycoplasma assay
The test is carried out according to the appendix of three parts of Chinese animal pharmacopoeia 2015 edition, and no mycoplasma grows.
2.5 suitability test of cells for ORFV culture
200ml of DST-3S F1 generation and F10 generation suspension cells are taken respectively, and the suspension cells are mixed with the solution at 0.5X 106Inoculating the cells/ml into a 500ml suspension culture flask for culture, and inoculating ORFV AH-GY13 strain to 100TCID after 12-16 h50Sampling every 24h for each mlAnd (5) determining the virus titer to 96 h.
3 results of the experiment
3.1 growth of cells in suspension flasks
The cells after suspension domestication are named as DST-3S, and the cells are in a transparent spherical shape, single or small-ball suspension under the suspension culture condition (figure 1). The 10-generation suspension domesticated cells can stably grow to 2-3 multiplied by 10 hours in 96 hours6Cells/ml (Table 1). With the increase of the domestication generations of the cells, the highest growth density of the cells of the F10 generation already tends to be stable.
TABLE 1 cell suspension acclimation Process
3.2 sterility test and Mycoplasma test results
The F1 DST-3S suspension cells are free from bacteria and mycoplasma pollution.
3.3 Virus culture Adaptation test
Culturing DST-3S F1 and F10 suspension cells for 12h, inoculating ORFV AH-GY13 strain to 100TCID50The virus content was determined every 24h per ml sample and the results are shown in Table 2. The virus content reaches the peak value after 72h of virus inoculation, and the peak value is 10 respectively-6.5TCID50A ratio of 10 to 10-6.4TCID50/ml。
TABLE 2DST-3-S F1 and F15 cell virus adaptability test results
4 conclusion of the experiment
4.1 the invention establishes a method for domesticating sheep testis immortalized cells adapting to full suspension culture.
4.2 the invention adopts the domestication method to domesticate the sheep testicular cell line which is suitable for full suspension culture, and the cell state is stable after 10 passages. This cell line was designated DST-3S.
4.3 full suspension sheep testis cell line is ORFV adapted cell, ORFV AH-GY13 strain is inoculated on F1 generation and F10 generation, and harvest is carried out for 72hWhen the virus has a virus valence of 10-6.5TCID50A ratio of 10 to 10-6.4TCID50/ml。
Claims (10)
1. A sheep testis immortalized cell domestication method adapting to full suspension culture comprises the following steps:
(1) reviving the immortalized sheep testicle cell DST-3 growing by adherence in water bath and then centrifuging;
(2) suspending the cells again by DMEM culture solution containing 5% newborn calf serum, and inoculating the cells in a common cell culture bottle for culture;
(3) subculturing the cultured cells, and digesting;
(4) carrying out rotary suspension culture on the digested cells;
(5) and transferring the cells into a suspension culture bottle for culture after the cell density is increased until large cell masses disappear, and obtaining the sheep testis immortalized cells suitable for suspension culture when the cell density is stable through repeated culture.
2. The method for acclimatizing sheep testis immortalized cell adapted to full suspension culture according to claim 1, wherein the recovery mode in step (1) is to put the unacclimated cell into water bath at 37 ℃ for recovery; the centrifugation is carried out under the conditions that the rotating speed is 1000r/min and the centrifugation time is 1 min.
3. The method for acclimatizing immortalized cells of testis Caprae seu Ovis according to claim 1, wherein the culturing in step (2) is carried out at 37 ℃ and 5% CO2Culturing under the conditions of (1); the culture time is 72 h.
4. The method for domesticating immortalized cells of sheep testis adapted to full suspension culture according to claim 1, wherein the subculture in step (3) is subcultured at a ratio of 1: 3, and the subculture time is 72 h.
5. Immortalized cells of sheep testis adapted to full suspension culture according to claim 1The method of culturing in a rotary suspension as described in the step (4), wherein the conditions for the rotary suspension culture comprise: at 37 ℃ 5% CO2And (5) performing rotary suspension culture.
6. The method for acclimatizing sheep testis immortalized cell adapted to full suspension culture according to claim 1 or 5, wherein the shaking table rotation speed is 100 r/min; the pH value of the rotary suspension culture is controlled to be 7.0-7.4, and NaHCO is used every day3The pH was adjusted 2 times.
7. The method for acclimatizing immortalized cells of testis Caprae seu Ovis according to claim 1, wherein the cell density in step (5) is repeatable from 0.5X 10 to 96h6Cells/ml grow to 2-3X 106Cells/ml.
8. A fully suspended sheep testis immortalized cell obtained by the method for acclimatizing a sheep testis immortalized cell adapted to full suspension culture according to any one of claims 1 to 7.
9. The use of the fully suspended sheep testis immortalized cell of claim 8 for the preparation of orf virus attenuated vaccine.
10. The use of the fully suspended sheep testis immortalized cell of claim 8 in the preparation of an inactivated orf virus vaccine.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104958760A (en) * | 2015-03-27 | 2015-10-07 | 中牧实业股份有限公司 | Goat inactivated vaccine and preparation method thereof |
| CN107058212A (en) * | 2017-03-31 | 2017-08-18 | 金宇保灵生物药品有限公司 | Can Secondary Culture Testis Caprae seu Ovis passage cell and its passage and attenuation cultural method and special cultivating system and application |
| CN107287149A (en) * | 2017-05-09 | 2017-10-24 | 杨凌博德越生物科技有限公司 | A kind of permanent cell line and its method for building up bred for sheep of virus |
| CN107432930A (en) * | 2017-08-08 | 2017-12-05 | 安徽东方帝维生物制品股份有限公司 | A kind of type aviadenovirus DNA vaccination of I group 4 and its preparation method and application |
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- 2018-10-16 CN CN201811205098.9A patent/CN111057675A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104958760A (en) * | 2015-03-27 | 2015-10-07 | 中牧实业股份有限公司 | Goat inactivated vaccine and preparation method thereof |
| CN107058212A (en) * | 2017-03-31 | 2017-08-18 | 金宇保灵生物药品有限公司 | Can Secondary Culture Testis Caprae seu Ovis passage cell and its passage and attenuation cultural method and special cultivating system and application |
| CN107287149A (en) * | 2017-05-09 | 2017-10-24 | 杨凌博德越生物科技有限公司 | A kind of permanent cell line and its method for building up bred for sheep of virus |
| CN107432930A (en) * | 2017-08-08 | 2017-12-05 | 安徽东方帝维生物制品股份有限公司 | A kind of type aviadenovirus DNA vaccination of I group 4 and its preparation method and application |
Non-Patent Citations (1)
| Title |
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| 王帅等: "和田羊睾丸支持细胞的体外培养与分离鉴定", 《西南农业学报》 * |
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