CN112574961A - Preparation method of cell vaccine antigen - Google Patents
Preparation method of cell vaccine antigen Download PDFInfo
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- CN112574961A CN112574961A CN202011585398.1A CN202011585398A CN112574961A CN 112574961 A CN112574961 A CN 112574961A CN 202011585398 A CN202011585398 A CN 202011585398A CN 112574961 A CN112574961 A CN 112574961A
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Abstract
The invention discloses a preparation method of cell vaccine antigen, which comprises the steps of healthy cell passage, virus inoculation and liquid replacement, antigen harvesting and the like, wherein when the cells are subjected to passage digestion, a shaker is used for replacing manual bottle throwing, so that the labor intensity can be greatly reduced; when the liquid is changed, a plurality of glass bottles filled with fresh maintenance liquid are connected in series, and the maintenance liquid in the glass bottles is pumped into the cell culture bottles through the peristaltic pumps, so that the liquid changing process is semi-automatic, the number of operators can be reduced, the labor intensity in the production process is reduced, the production efficiency is improved, and the pollution rate of semi-finished products is also reduced; when the antigen is harvested, cells in a cell culture bottle are crushed mainly by a method of converting energy of ultrasonic waves, and the method is used for scraping seedlings, so that the defects of the traditional method for scraping seedlings are avoided, the step of freezing and thawing the cells is also saved, the harvesting process is completed in one step, and the purposes of simplicity, safety, labor saving and shortening of the antigen production time are achieved.
Description
Technical Field
The invention belongs to the field of biological products, and particularly relates to a preparation method of a cell vaccine antigen.
Background
The general cell viroid spinner flask culture comprises the following steps: healthy cells are passaged, inoculated with virus and changed liquid and harvested.
1. Healthy cell passage: when a healthy roller bottle cell grows full of a monolayer, the existing passage process is generally as follows: opening the bottle mouth stopper of the rotary bottle, pouring out the original nutrient solution, adding a small amount of PBS for cleaning once, adding a certain amount of pancreatin for digestion, then discarding the pancreatin and adding a small amount of nutrient solution when the cells are in a foggy state with needle points and white dots, then holding the two ends of the rotary bottle by one person, forcibly rotating up and down to throw, and utilizing the rapid rotation of the thrown liquid to scour the cells to enable the cells to fall off and disperse. This method has the following disadvantages: (1) get rid of the in-process intensity of labour of bottle big, waste time and energy, and it is inefficient, be unfavorable for mass production. (2) The bottle is easy to slip off in the swinging process, thereby causing accidents.
2. Virus inoculation and liquid replacement: transferring the digested and subpackaged cell suspension into an inoculation chamber, adding a specified amount of virus seeds, culturing for 24h at 37 ℃ on a rotary bottle machine, removing the nutrient solution, manually holding a 10-liter or 15-liter rotary bottle (with the weight of about 20 kg) filled with fresh maintenance solution, pouring the fresh maintenance solution into the cell bottle one by one, and further culturing at 37 ℃ on the rotary bottle machine. Because the number of bottles for pouring liquid each time is large, the time is long, the physical labor amount of operators is large, and a plurality of male operators are generally required to pour liquid in turn, if the yield is increased, a large amount of labor force is required, and the requirement of mass production cannot be met; on the other hand, the manual liquid pouring method greatly increases the pollution risk due to the open bottle mouth in the operation process.
3. Harvesting: when the cells are cultured for a specified time or the pathological change degree meets the requirement, the cell culture bottle is moved into a refrigeration house with the temperature of-15 ℃ from a greenhouse for freezing, and then the virus is completely released by a seedling scraping freeze-thawing method. The frequently used seedling scraping method is that the seedlings frozen into ice blocks are rotated by manpower to ensure that the seedlings frozen into ice blocks puncture cells on the bottle wall and release viruses remained in the cell bodies. Meanwhile, by a freeze-thaw method, cells are torn and broken in the process of freezing and thawing, and viruses in the cells are released. Finally transferring the freeze-thawed antigen into an inoculation harvesting room for harvesting, and placing the antigen in a refrigeration house for freezing and storing. The method has the following defects: (1) when the antigen is scraped, a bottle of the antigen needs to be independently operated, and a large amount of labor and time are needed. (2) The cell on the wall of the rotating bottle can be punctured by the continuous rotation of the ice block in the rotating bottle during seedling scraping to obtain the virus in the cell on the wall of the rotating bottle, and the action is particularly light and stable. The ice block is easy to break the rotary bottle with too much force, which causes loss. If the ice is too light, the cells cannot be completely punctured by the ice, and the virus is obtained. Furthermore, the method cannot ensure that the scraping of the seedlings can ensure that all cells on the bottle wall are punctured and the virus is completely released. (3) The temperature of the wall of the vaccine bottle is very low when the vaccine is scraped, the seedling scraping process is long in time consumption, and certain influence is brought to the health of production personnel.
Disclosure of Invention
The invention aims to provide a preparation method of a cell vaccine antigen, which can save labor, is convenient to operate, is flexible to use and reduces the probability of cell pollution.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for preparing cell vaccine antigen comprises the following steps
1) Passage of healthy cells
When the healthy spinner bottle cells grow to be full of a single layer, opening a plug at the opening of the spinner bottle, discarding original nutrient solution in the spinner bottle, cleaning the cells to be digested in the spinner bottle by PBS, adding pancreatin to digest the cells, then discarding pancreatin, adding the nutrient solution when the cells are in a fogging state with needle points and white dots, covering the plug of the spinner bottle, starting an oscillator, placing the spinner bottle on a tray of the oscillator, pressing the spinner bottle by hands, shaking up the liquid in the spinner bottle by oscillation so as to enable the cells on the wall of the spinner bottle to fall off, and subpackaging the cell suspension after digestion and falling into N cell culture bottles;
2) virus inoculation and liquid replacement:
transferring the cell culture bottle filled with the cell suspension into an inoculation chamber, adding a specified amount of virus seeds, culturing at 37 ℃ for 24h in a bottle rotating machine with the speed of 10-15r/h, and removing the nutrient solution;
connecting a plurality of glass bottles filled with fresh maintenance liquid in series, pumping the maintenance liquid in the glass bottles into cell culture bottles through a peristaltic pump, adding sufficient maintenance liquid, replacing the cell culture bottles until sufficient maintenance liquid is added into N cell culture bottles, and then placing the N cell culture bottles on a bottle rotating machine of 10-15r/h for continuous culture at 37 ℃;
because first glass bottle connects partial shipment cover to the cell culture bottle that treats the liquid of trading, all glass bottles are established ties, maintain the liquid in with the glass bottle through the peristaltic pump and take out first cell culture bottle, wait to add enough maintenance liquid alright stop the peristaltic pump operation, change the cell culture bottle that treats the liquid feeding next, arrange partial shipment cover in new cell culture bottle on, open the peristaltic pump and continue to begin the liquid feeding, so the circulation goes on, not only greatly reduced the labour, reduced the intensity of labour of production process, still improved production efficiency, also reduced pollution risk simultaneously and protected
3) Antigen harvesting
When the cells are cultured for a specified time or the pathological change degree meets the requirement, the cell culture bottle is moved into an operation room, and the ultrasonic wave is adopted to harvest the antigens: a bottle rotating machine is arranged in an ultrasonic generator, cell culture bottles are transversely clamped between rotating shafts of the bottle rotating machine bottle by bottle side by bottle, one side surface of each bottle rotating machine is just contacted with the water surface in an ultrasonic device, meanwhile, each cell culture bottle is fixed by a clamping ring, a power switch of the ultrasonic generator and a power switch of the bottle rotating machine are turned on, the rotating speed of the bottle rotating machine is set to be 5-40r/h, the working frequency of ultrasonic waves is set to be 20-25KHz, at the moment, the cell culture bottles are vibrated in water by the ultrasonic generator while rotating, cells in the cell culture bottles and in cell suspension are shaken off and crushed, after the cells are crushed on the whole surfaces of the cell culture bottles by the ultrasonic generator, the power supply of the bottle rotating machine and the ultrasonic power supply are sequentially turned off, when the cell culture bottles stop rotating, the clamping ring is opened, the cell culture bottles are taken out, the crushed antigens are transferred, and (5) placing the mixture in a refrigeration house for freezing and storing. By adopting the method, the cell vaccine does not need to be scraped and repeatedly frozen and thawed to obtain virus in the cell, thereby saving time and labor.
By adopting the technical scheme, the invention has the following beneficial effects:
1. when the cell is subjected to passage digestion, the manual bottle throwing is replaced by a machine, so that the labor intensity can be greatly reduced, the operation is convenient and fast, and the product is more stable.
2. When the liquid is changed, a plurality of glass bottles filled with fresh maintenance liquid are connected in series, and the maintenance liquid in the glass bottles is pumped into the cell culture bottles through the peristaltic pumps, so that the liquid changing process is semi-automatic, the number of operators can be reduced, the labor intensity of the production process is reduced, the production efficiency is improved, and the pollution rate of semi-finished products is also reduced.
3. Harvesting: the invention mainly depends on the method of converting energy of ultrasonic waves to break the cells in the cell culture bottle, and the method is used for scraping the seedlings, thereby avoiding the defects of the traditional method for scraping the seedlings, saving the step of freezing and thawing the cells, leading the harvesting process to be in place in one step, and achieving the purposes of simplicity, safety, labor saving and shortening the antigen production time.
Detailed Description
Preparation method of cell vaccine antigen
1) Passage of healthy cells
When the healthy spinner bottle cells grow to be full of a single layer, opening a plug at the opening of the spinner bottle, discarding original nutrient solution in the spinner bottle, cleaning the cells to be digested in the spinner bottle by PBS, adding pancreatin to digest the cells, then discarding pancreatin, adding the nutrient solution when the cells are in a fogging state with needle points and white dots, covering the plug of the spinner bottle, starting an oscillator, placing the spinner bottle on a tray of the oscillator, pressing the spinner bottle by hands, shaking up the liquid in the spinner bottle by oscillation so as to enable the cells on the wall of the spinner bottle to fall off, and subpackaging the cell suspension after digestion and falling into N cell culture bottles;
2) virus inoculation and liquid replacement:
transferring the cell culture bottle filled with the cell suspension into an inoculation chamber, adding a specified amount of virus seeds, culturing at 37 ℃ for 24h in a bottle rotating machine with the speed of 10-15r/h, and removing the nutrient solution;
connecting a plurality of glass bottles filled with fresh maintenance liquid in series, pumping the maintenance liquid in the glass bottles into cell culture bottles through a peristaltic pump, adding sufficient maintenance liquid, replacing the cell culture bottles until sufficient maintenance liquid is added into N cell culture bottles, and then placing the N cell culture bottles on a bottle rotating machine of 10-15r/h for continuous culture at 37 ℃;
because first glass bottle connects partial shipment cover to the cell culture bottle that treats the liquid of trading, all glass bottles are established ties, maintain the liquid in with the glass bottle through the peristaltic pump and take out first cell culture bottle, wait to add enough maintenance liquid alright stop the peristaltic pump operation, change the cell culture bottle that treats the liquid feeding next, arrange partial shipment cover in new cell culture bottle on, open the peristaltic pump and continue to begin the liquid feeding, so the circulation goes on, not only greatly reduced the labour, reduced the intensity of labour of production process, still improved production efficiency, also reduced pollution risk simultaneously and protected
3) Antigen harvesting
When the cells are cultured for a specified time or the pathological change degree meets the requirement, the cell culture bottle is moved into an operation room, and the ultrasonic wave is adopted to harvest the antigens: a bottle rotating machine is arranged in an ultrasonic generator, cell culture bottles are transversely clamped between rotating shafts of the bottle rotating machine bottle by bottle side by bottle, one side surface of each bottle rotating machine is just contacted with the water surface in an ultrasonic device, meanwhile, each cell culture bottle is fixed by a clamping ring, a power switch of the ultrasonic generator and a power switch of the bottle rotating machine are turned on, the rotating speed of the bottle rotating machine is set to be 5-40r/h, the working frequency of ultrasonic waves is set to be 20-25KHz, at the moment, the cell culture bottles are vibrated in water by the ultrasonic generator while rotating, cells in the cell culture bottles and in cell suspension are shaken off and crushed, after the cells are crushed on the whole surfaces of the cell culture bottles by the ultrasonic generator, the power supply of the bottle rotating machine and the ultrasonic power supply are sequentially turned off, when the cell culture bottles stop rotating, the clamping ring is opened, the cell culture bottles are taken out, the crushed antigens are transferred, and (5) placing the mixture in a refrigeration house for freezing and storing. By adopting the method, the cell vaccine does not need to be scraped and repeatedly frozen and thawed to obtain virus in the cell, thereby saving time and labor.
The comparison of the labor efficiency of the virus inoculation and liquid replacement method of the invention with the original method is as follows:
taking 120 cells in 15L flasks per time, the total amount of maintenance fluid required to be replenished is 180L.
| Original method | The method of the invention | |
| Bear the weight of each bottle | 20kg | ___ |
| All in all, takes time | About 120min | About 80min |
| Number of persons | 8-9 persons (at least 3 men) | 5 persons (unlimited male and female) |
The contamination risk of the virus inoculation and liquid replacement method of the invention is compared with the original method as follows:
in the original method, the bottle mouth is always exposed to the air when the maintenance liquid is replenished until the maintenance liquid in the bottle is poured out, and in order to avoid the maintenance liquid from rushing to the cells on the bottle wall, the maintenance liquid can only be slowly added into the cell bottle by manually holding the bottle filled with about 20kg of maintenance liquid, the air exposure time of the maintenance liquid in the process is about 10min/15L, the requirement on labor force is high, and the bottle mouth is hardly exposed to the air after the method is applied, so that the probability of cell pollution is reduced, and the labor is saved.
In the step of antigen harvesting, a circle of rubber sleeve is sleeved on a rotating shaft of the bottle rotating machine to prevent the cell culture bottles from slipping, the thickness of the rubber sleeve is made to be adjustable, and the distance between the rotating shafts can be adjusted to be 5L, 10L, 15L and other sizes of the cell culture bottles with different specifications by replacing the rubber sleeves with different specifications so as to meet different production requirements.
Claims (3)
1. A method for preparing a cell vaccine antigen, which is characterized by comprising the following steps: which comprises the following steps:
1) passage of healthy cells
When the healthy spinner bottle cells grow to be full of a single layer, opening a plug at the opening of the spinner bottle, discarding original nutrient solution in the spinner bottle, cleaning the cells to be digested in the spinner bottle by PBS, adding pancreatin to digest the cells, then discarding pancreatin, adding the nutrient solution, covering the plug of the spinner bottle, starting an oscillator, placing the spinner bottle on a tray of the oscillator, pressing the spinner bottle by hands, shaking up the liquid in the spinner bottle through oscillation, so that the cells on the wall of the spinner bottle fall off, and subpackaging the cell suspension after digestion and shedding into N cell culture bottles;
2) virus inoculation and liquid replacement:
transferring the cell culture bottle filled with the cell suspension into an inoculation chamber, adding a specified amount of virus seeds, culturing for 24h on a rotary bottle machine, and removing the nutrient solution;
connecting a plurality of glass bottles filled with fresh maintenance liquid in series, pumping the maintenance liquid in the glass bottles into cell culture bottles through a peristaltic pump, adding sufficient maintenance liquid, replacing the cell culture bottles until sufficient maintenance liquid is added into N cell culture bottles, and then placing the N cell culture bottles on a bottle rotating machine for continuous culture;
3) antigen harvesting
When the cells are cultured for a specified time or the pathological change degree meets the requirement, the cell culture bottle is moved into an operation room, and the ultrasonic wave is adopted to harvest the antigens: a bottle rotating machine is arranged in the ultrasonic generator, the cell culture bottles are transversely clamped between rotating shafts of the bottle rotating machine bottle by bottle side by side, one side surface of each rotating bottle just contacts with the water surface in the ultrasonic device, meanwhile, each cell culture bottle is fixed by a clamping ring, a power switch of an ultrasonic generator and a power switch of a bottle rotating machine are turned on, setting rotation speed and ultrasonic working frequency, vibrating the cell culture bottle in water through an ultrasonic generator while rotating, shaking and breaking the cells in the cell culture bottle and the cell suspension, after the whole surface of the cell culture bottle breaks the cells through the ultrasonic generator, sequentially turning off a bottle rotating machine power supply and an ultrasonic power supply, when the cell culture bottle stops rotating, and opening the snap ring, taking out the cell culture bottle, transferring the crushed cell vaccine antigen into an inoculation harvesting room, harvesting, and placing in a refrigeration house for freezing and storing.
2. The method for producing a vaccine antigen according to claim 1, wherein: in the step 2), the rotating speed of the bottle rotating machine is 10-15r/h, and the culture temperature is 37 ℃.
3. The method for producing a vaccine antigen according to claim 1, wherein: in the step 3), the rotating speed is 5-40r/h, and the ultrasonic working frequency is 20-25 KHz.
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| CN202011585398.1A CN112574961A (en) | 2020-12-28 | 2020-12-28 | Preparation method of cell vaccine antigen |
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| CN202011585398.1A CN112574961A (en) | 2020-12-28 | 2020-12-28 | Preparation method of cell vaccine antigen |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1840647A (en) * | 2006-01-11 | 2006-10-04 | 中国人民解放军第三军医大学第三附属医院 | Liquid changing device for artificial skin production and cell culture engineering |
| CN101406699A (en) * | 2008-11-19 | 2009-04-15 | 长春长生生物科技股份有限公司 | Method for preparing lyophilized hepatitis A attenuated live vaccine using cell factory |
| CN109810952A (en) * | 2019-04-11 | 2019-05-28 | 郑州伊美诺生物技术有限公司 | The method of human respiratory syncytial virus's large-scale culture |
| CN109913443A (en) * | 2019-03-24 | 2019-06-21 | 罗火生 | A kind of method and apparatus of ultrasonic wave harvest cell |
| CN210765348U (en) * | 2019-10-08 | 2020-06-16 | 孟明耀 | Large-scale umbilical cord mesenchymal stem cell liquid conversion and liquid conversion system |
-
2020
- 2020-12-28 CN CN202011585398.1A patent/CN112574961A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1840647A (en) * | 2006-01-11 | 2006-10-04 | 中国人民解放军第三军医大学第三附属医院 | Liquid changing device for artificial skin production and cell culture engineering |
| CN101406699A (en) * | 2008-11-19 | 2009-04-15 | 长春长生生物科技股份有限公司 | Method for preparing lyophilized hepatitis A attenuated live vaccine using cell factory |
| CN109913443A (en) * | 2019-03-24 | 2019-06-21 | 罗火生 | A kind of method and apparatus of ultrasonic wave harvest cell |
| CN109810952A (en) * | 2019-04-11 | 2019-05-28 | 郑州伊美诺生物技术有限公司 | The method of human respiratory syncytial virus's large-scale culture |
| CN210765348U (en) * | 2019-10-08 | 2020-06-16 | 孟明耀 | Large-scale umbilical cord mesenchymal stem cell liquid conversion and liquid conversion system |
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