CN115340975A

CN115340975A - Method for preparing Vero cell suspension

Info

Publication number: CN115340975A
Application number: CN202211270782.1A
Authority: CN
Inventors: 王晓丽; 徐秀芝; 李研宇; 刘利国; 郝然; 刘发明; 赵雪
Original assignee: Changchun Zhuoyi Biological Co ltd
Current assignee: Changchun Zhuoyi Biological Co ltd
Priority date: 2022-10-18
Filing date: 2022-10-18
Publication date: 2022-11-15

Abstract

The invention relates to the technical field of biology, in particular to a method for preparing Vero cell suspension. The invention provides a method for preparing Vero cell suspension by using a WAVE swing bed reactor and a flaky carrier. The WAVE swing bed reactor has an independent control system, the production process is simple and convenient, the number of required passage cells is small, the culture process can be completed without a cell culture chamber, the pollution risk is reduced, and the space and the cost are saved. The WAVE swing bed reactor is matched with a cell culture bag, contains a flaky carrier, has large cell proliferation space, and is more favorable for the growth and proliferation of cells by setting the most suitable cell growth environment by using a control system. The used sheet-shaped carrier can effectively improve the utilization rate of Vero cells, and the prepared cell suspension has high density.

Description

Method for preparing Vero cell suspension

Technical Field

The invention relates to the technical field of biology, in particular to a method for preparing a Vero cell suspension.

Background

The Vero cell is a cell approved by WHO for vaccine production, has clear genetic background, stable karyotype and no exogenous factor pollution, is suitable for large-scale culture, can be produced by a bioreactor, and ensures the homogeneity and safety of the mass production of the vaccine. The Vero cell is a passage cell which is most used for producing vaccines at present, and is widely used for producing vaccines such as encephalitis B vaccines, influenza vaccines, spinal ash vaccines, rotavirus vaccines, yellow fever virus vaccines, hepatitis A vaccines, rabies vaccines for human beings and the like. Therefore, the method has important significance for efficiently preparing the Vero cells and improving the utilization rate of the Vero cells.

However, the current Vero cell preparation procedure used T ₁₅₀ The amplification culture process of the cell culture bottle and the 3L rotary bottle has the problems of complicated operation and fatigue, and the following problems:

1) Compared with the modern high-tech technology, the operation of the existing cell preparation process is relatively backward, and more materials and materials are needed for production;

2) 3L spinner flask cells occupy large area, need to set up the cell culture room alone and cultivate at constant temperature, need transport cell spinner flask culture apparatus to pass in and out the cell culture room many times before and after the operation, need to verify the cell culture room annually at the same time, the verification process is similar to production, very tedious and tired;

3) The existing cell preparation process is manually operated, the opening times are more, and the repeated cross transmission of the A level and the C level exists, so that a lot of operation risks are increased;

4) The existing cell preparation process needs 5 to 6 operators to carry out simultaneously, the production task is heavy, more than 80 bottles of 3L spinner bottles are passaged each time, 80 bottles of 3L spinner bottles of cells are needed for preparing Vero cell suspension, the suspension amount is 6200 to 6500 mL, and the cells are tired when the batch is many;

5) 3L transfer bottles, 10L transfer bottles and the like transferred after the procedures of 3L transfer bottle passage and Vero cell suspension preparation are glass products, and in order to repeatedly wash and sterilize articles, a lot of manpower and material resources are needed to be consumed for cleaning and sterilization, so that the production cost is high;

6) Vero cells are adherent cells, the bottle wall area of a 3L rotary bottle is small, each batch can be transferred into a 40L bioreactor for continuous culture only after more than 80 bottles of passage, and the cell utilization rate is low.

Therefore, the method for preparing the Vero cell suspension has important practical significance for efficiently preparing the Vero cells and improving the utilization rate of the Vero cells.

Disclosure of Invention

In view of this, the present invention provides a method for preparing a suspension of Vero cells.

The invention provides a method for preparing Vero cell suspension by using a WAVE swing bed reactor and a flaky carrier. The WAVE swing bed reactor has an independent control system, the production process is simple and convenient, the number of required passage cells is small, the culture process can be completed without a cell culture chamber, the pollution risk is reduced, and the space and the cost are saved. The WAVE swing bed reactor is matched with a cell culture bag, the cell culture bag contains a flaky carrier, the cell proliferation space is large, and a control system is used for setting the environment which is most suitable for the growth of cells, so that the growth and the proliferation of the cells are more facilitated. The used sheet-shaped carrier can effectively improve the utilization rate of Vero cells, and the prepared cell suspension has high density.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a method for preparing Vero cell suspension, which comprises the following steps:

step 1: obtaining Vero cells after passage;

and 2, step: culturing the Vero cells after passage by a WAVE swing bed;

wherein, the step 2-1: taking the sheet-shaped carrier for pre-incubation to obtain the pre-incubated sheet-shaped carrier;

step 2-2: digesting and resuspending the Vero cells after passage to obtain a first Vero cell suspension;

step 2-3: inoculating the first Vero cell suspension to the pre-incubated sheet-shaped carrier, culturing by a WAVE placing bed, digesting, and re-suspending to prepare a second Vero cell suspension;

and 3, step 3: and taking the second Vero cell suspension for enlarged culture and collecting.

In some embodiments of the invention, the seeding ratio of the passage is 1 to 4 to 1;

the temperature of WAVE swing bed culture is 36-38 ℃, and CO is ₂ The concentration is 5%, the angle is 6-8 ℃, the swing speed is 6-13 rpm, the ventilation flow is 0.06-0.4 SLPM, the PH is 7.1-7.4, and the DO is 55-65%;

the glucose consumption of WAVE swing bed culture comprises: the glucose consumption in the next day is 2 to 4 g, the glucose consumption in the third day is 5 to 10 g, the glucose consumption in the fourth day is 10 to 20 g, and the glucose consumption in the fifth day is 20 to 30 g;

the perfusion amount of the WAVE swing bed culture comprises the following steps: the perfusion volume on the next day is 0.2 to 1.5L, the perfusion volume on the third day is 1.5 to 3.0L, the perfusion volume on the fourth day is 3.0 to 8.0L, and the perfusion volume on the fifth day is 8.0 to 13.0L;

the feed liquor flow rate of WAVE swing bed culture comprises: the liquid inlet flow rate in the next day is 0.1 to 1.0 mL/min; the liquid inlet flow rate is 1.0 to 2.1 mL/min on the third day, the liquid inlet flow rate is 2.1 to 5.6 mL/min on the fourth day, and the liquid inlet flow rate is 5.6 to 9.0 mL/min on the fifth day;

the effluent flow of WAVE swing bed culture comprises: the flow rate of the liquid discharged from the water supply system at the next day is 0.2 to 1.1 mL/min, the flow rate of the liquid discharged from the water supply system at the next day is 1.1 to 2.2 mL/min, the flow rate of the liquid discharged from the water supply system at the next day is 2.2 to 5.8 mL/min, and the flow rate of the liquid discharged from the water supply system at the next day is 5.8 to 9.2 mL/min;

the suspension used for resuspension included: 1% fetal bovine serum and 199 aqueous solution; the 199 aqueous solution includes 199 culture medium, sodium bicarbonate.

In some embodiments of the invention, the temperature of the pre-incubation of the sheet-shaped carrier in the step 2-1 is 37 ℃, the angle is 6-8 ℃, the swinging speed is 10-15 rpm, the pH is 7.2-7.4, and the using amount of the cell growth solution is 500-800 mL or 2500-3000 mL; the cell growth solution comprises: 10% fetal bovine serum and 199 water;

the density of the first Vero cell suspension is 4.83 multiplied by 10 ⁶ cells/mL; the inoculation volume of the first Vero cell suspension is 65 to 75 mL; the culture volume of the inoculated first Vero cell suspension is 1000 mL;

step 2-3, the temperature of WAVE swing bed culture is 37 ℃, and the angle is 6-8 degrees;

the aeration flow rate of the WAVE swing bed culture comprises the following steps: the ventilation flow rate on the first day is 0.06 to 0.1 SLPM, the ventilation flow rate on the second day is 0.1 to 0.2 SLPM, the ventilation flow rate on the third day is 0.2 SLPM, and the ventilation flow rate on the fourth day and the fifth day is 0.2 SLPM;

the swing speed of the WAVE swing bed culture comprises the following steps: the swing speed of the first day is 6 to 7 rpm, the swing speed of the second day is 7 to 9 rpm, the swing speed of the third day is 10 to 12 rpm, and the swing speed of the fourth day and the fifth day is 12 rpm;

the total glucose cultured by the WAVE swing bed is 6.0 g;

the glucose content includes: the glucose content of the second day is 3.5 g, the glucose content of the third day is 3.75 g, the glucose content of the fourth day is 2.7 g, and the glucose content of the fifth day is 2.9 g;

the perfusion amount of glucose comprises: the perfusion volume on the next day is 0.6 to 1.2L, the perfusion volume on the third day is 1.5 to 2.5L, and the perfusion volume on the fourth day and the fifth day is 3.0 to 4.0L;

the density of the second Vero cell suspension is 2.62 multiplied by 10 ⁶ cells/mL; the inoculation volume of the second Vero cell suspension is 600-680 mL, and the culture volume after inoculation is 5000 mL.

In some embodiments of the present invention, the expanded culture in step 3 is WAVE swing bed culture or bioreactor culture;

the temperature of WAVE swing bed culture is 37 ℃, and the angle is 6-8 degrees;

the aeration flow of WAVE swing bed culture comprises the following steps: the ventilation flow rate on the first day is 0.2 to 0.3 SLPM, the ventilation flow rate on the second day and the third day is 0.3 to 0.4 SLPM, and the ventilation flow rate on the fourth day and the fifth day is 0.4 SLPM;

the swing speed of the WAVE swing bed culture comprises the following steps: the swing speed of the first day is 7 to 8 rpm, the swing speed of the second day is 8 to 9 rpm, the swing speed of the third day is 10 to 12 rpm, and the swing speeds of the fourth day and the fifth day are 13 rpm;

the total glucose cultured by the WAVE swing bed is 6.0 g;

the glucose content includes: the glucose content of the second day is 4.3 g, the glucose content of the third day is 2.6 g, the glucose content of the fourth day is 0.9 g, and the glucose content of the fifth day is 1.1 g;

the perfusion amount comprises: the perfusion capacity on the second day is 0.3 to 0.6L, the perfusion capacity on the third day is 2.0 to 3.0L, the perfusion capacity on the fourth day is 5.5 to 6.5L, and the perfusion capacity on the fifth day is 10 to 12L;

the density of the Vero cell suspension prepared by WAVE swing bed culture is 1.71 multiplied by 10 ⁶ cells/mL; the inoculation volume of the Vero cell suspension prepared by WAVE swing bed culture is 5300 to 6200 mL; the culture volume after inoculation is 30L.

The temperature of the bioreactor culture is 36.8 to 37.2 ℃, and CO is added ₂ The concentration is 5%, the rotation speed is 98 to 102 rpm, the pH is 7.2 to 7.3, the DO is 65%, the gas flow is 0.3 to 0.5 SLPM, and the culture is continued for 4 to 5 days; the volume of the bioreactor was 40L.

In some specific embodiments of the invention, the cell density of the inoculated Vero cells in the cell culture solution after the passage is 3.0 to 3.5 cells/mL; the cell density in the cell culture solution after the first Vero cell suspension is inoculated is 3.0 to 3.5 cells/mL; the cell density in the cell culture solution after the second Vero cell suspension is inoculated is 3.0 to 3.5 cells/mL.

As can be seen from the results in FIG. 2, the density of the Vero cell suspension after the sheet-shaped carrier cell culture bag is digested is greatly different from that of the Vero cell suspension after the spherical carrier bag is digested, the cell suspension prepared by the cell culture bag containing the rhombic sheet-shaped carrier has high cell density, and the cell suspension prepared by the cell culture bag containing the Cytodex spherical microcarrier has low cell density. The difference of cell suspensions obtained by digesting and collecting the carrier cell culture bags with the same volume is nearly one time, which indicates that compared with a ball-to-wafer process, the mutual transfer of the sheet carriers is more beneficial to actual production.

The invention provides a method for preparing Vero cell suspension by using a WAVE swing bed reactor and a flaky carrier. The WAVE swing bed reactor has an independent control system, the production process is simple and convenient, the number of required passage cells is small, the culture process can be completed without a cell culture chamber, the pollution risk is reduced, and the space and the cost are saved. The WAVE swing bed reactor is matched with a cell culture bag, contains a flaky carrier, has large cell proliferation space, and is more favorable for the growth and proliferation of cells by setting the most suitable cell growth environment by using a control system. The used sheet-shaped carrier can effectively improve the utilization rate of Vero cells, and the prepared cell suspension has high density.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below.

FIG. 1 shows glucose values during culture of two carrier cell culture bags;

FIG. 2 shows the cell density of cell suspensions prepared from two carrier cell culture bags.

Detailed Description

The invention discloses a method for preparing Vero cell suspension, which can be realized by appropriately improving process parameters by a person skilled in the art with the reference to the content. It is specifically noted that all such substitutions and modifications will be apparent to those skilled in the art and are intended to be included herein. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

Experimental materials:

1. the required liquid proportion is as follows:

199 aqueous solution formulation (taking 10L of formulation as an example): 199 medium 124.9 g, sodium bicarbonate 22.9 g;

cell growth liquid: 10% fetal bovine serum +199 aqueous solution;

suspension of the cell fluid: 1% fetal bovine serum +199 aqueous solution;

cell digestive juice: trypLE ™ Select (1 ×);

other liquids: 7.5% sodium bicarbonate solution, 0.01 mol/L PBS buffer solution, 199 culture medium (manufactured by Tianxin and Biotech Co., ltd.), 199 aqueous solution, fetal bovine serum, etc.

2. Required culture materials: vero cells, cell culture bags (using cell culture bags with sheet carriers of 30 g/L specification, based on culture volume, namely 30 g of sterilized diamond-shaped sheet carriers in 2L of cell culture bags, 150 g of sterilized diamond-shaped sheet carriers in 10L of cell culture bags, purchased from Wuhansaike science and technology Limited), disposable cell culture bottles, and the like

3. Required equipment facilities: WAVE swing bed reactor (purchased from Wuhansaike science and technology Co., ltd.), peristaltic pump, sterile tube connecting machine, inverted biological microscope, and biochemical analyzer.

The production process comprises the following steps:

1. vero cells are passaged according to Vero cell recovery, passage and SOP harvesting, the number of the cells is based on actual needs, and the cells are not allowed to be used for ultrahigh passage (not more than 150 passages).

Specifically, the experimental procedure is as follows:

and (3) carrying out passage on the Vero cells according to a standard operation procedure until the Vero cells are passaged to a required number, when the Vero cells grow into a single layer, the Vero cells are polygonal, are closely and regularly arranged, have clear edges, are orange in culture solution, are clear and transparent, and can be continuously carried out in the next passage or transferred into a WAVE swing bed reactor.

2. The method comprises the steps of culturing Vero cells by using a WAVE swing bed reactor, connecting an air pipeline, an oxygen pipeline, a carbon dioxide pipeline, an alkali supplementing pipeline and a waste liquid discharge pipeline, stabilizing parameters of a WAVE system, sampling cell supernatant by using a special sampling port every day to determine the content of glucose, calculating the consumption of the glucose and the required perfusion amount according to a formula, changing the numerical values of liquid inlet and liquid outlet, and culturing to the required cell number.

Specifically, the experimental procedure is as follows:

1) Pre-incubation of the sheet-shaped carrier cell culture bag: the sheet-shaped carrier cell culture bag needs to be incubated for more than 6 hours at 37 ℃ in advance before use, cell growth liquid (containing 10% fetal calf serum) needs not to cover the carrier, the compensation temperature is set to be 37 ℃, DO and pH calibration parameters of the culture bag are input into a calibration parameter dialog box for calibration after the temperature is stable, the pH is recalibrated after the temperature is stable, and recalibration is not needed if the actual measurement value and the equipment display value are less than or equal to 0.03.

2) According to standard operating proceduresDigesting the cells to prepare Vero cell suspension with required density and quantity, and the specific operation is as follows: t digestion Using TrypLE ™ Select (1 ×) ₁₅₀ Adding 20 mL/bottle of cells in a cell culture bottle, cleaning the cell surface, pouring out, adding 20 mL/bottle, placing the cell surface downwards on an operation table to digest the cells, pouring off a digestive juice when the cell surface is in a ground glass shape, continuing to digest the cells, digesting the cells in each bottle for about 5 minutes, adding a cell suspension (199 aqueous solution containing 1% fetal calf serum) to stop cell digestion when the cell surface has obvious cracks, adding 10 to 20 mL of the cell suspension by using a suction pipe to blow the cell surface, and preparing Vero cell suspension for later use.

3) The dispersed cell suspension is distributed into cell culture bags according to the required inoculation density, the cell culture bags are placed in a swing bed reactor for swing culture, the culture temperature is set to be 36-38 ℃, the angle is 6-8 ℃, a three-gas mode is clicked, and CO is adopted ₂ The concentration is set to be 5%, aeration flow is set, the aeration flow can be adjusted along with cell proliferation, the speed during cell culture can be adjusted according to cell adherence and the cell proliferation speed, the speed during just adherence cannot be too high, otherwise, the cell adherence is not favorable, when the cells enter a proliferation period, the speed can be adjusted at any time, the pH is between 7.1 and 7.4, the DO is between 55 and 65 percent, the WAVE system is provided with more than 3 pump heads, the pump 1 is set to supplement alkali, the pump 2 is set to feed liquid, the pump 3 is set to discharge liquid, a weighing set value is set, meanwhile, a special sampling port is used for sampling cell supernatant every day to measure the glucose content, the glucose consumption and the required perfusion are calculated according to a formula, the numerical values of the feed liquid and the discharge liquid are changed, and the required cell number is cultured.

3. And (4) digesting the passage Vero cells by using a WAVE swing bed reactor to prepare a Vero cell suspension for enlarged culture (continuously using the WAVE swing bed reactor for enlarged culture or transferring the Vero cell suspension into a bioreactor for enlarged culture).

Specifically, the experimental procedure is as follows:

1) Cell culture bag cell digestion: connecting the cell culture bag with each pipeline, firstly discharging supernatant, washing the surface of the carrier with 0.01 mol/L PBS buffer solution, then discharging, then adding digestive juice for digestion, discharging the digestive juice when a small amount of cells are separated out, continuing digestion at room temperature (18-26 ℃) until a large amount of cells are separated out visible to the naked eye, ending digestion, timely adding cell suspension liquid into the cell culture bag for cell suspension, repeatedly adding the cell suspension liquid for multiple times until the liquid in the flaky carrier cell culture bag visible to the naked eye is basically clear, supplementing fetal calf serum to 10% concentration, uniformly mixing, sampling and counting the cells;

2) Calculating the inoculation number required by cell passage according to the cell density and the cell suspension number, and adding the inoculated cell suspension number according to the density of 3.0 to 3.5 cells/mL of the final volume. And timely transferring the cells into a cell culture bag of a WAVE swing bed reactor to continue culturing, and repeating the culturing steps according to the method.

4. Transferring into a bioreactor for culture: after the cells in the cell culture bag after the culture are digested, suspended and counted according to the method, the cells are transferred into a bioreactor to be cultured according to the number of the cells required by the bioreactor, and the culture method of the bioreactor can be executed according to the production standard of the invention.

Specifically, the experimental procedure is as follows:

inoculating cells into a bioreactor for continuous culture, inoculating a Vero cell suspension according to the final density of 3.0-3.5 cells/mL, setting control parameters, setting the rotating speed at the set value of 98-102 rpm, the temperature of 36.8-37.2 ℃ and 5% CO ₂ And (3) culturing for 4 to 5 days at a pH of 7.2 to 7.3 and a DO of 65 percent and a gas flow rate of 0.3 to 0.5 SLPM.

And (4) analyzing results:

1) The WAVE swing bed reactor is used for culturing cells, only disposable cell culture bottles are needed to be used for culturing the cells, 3L spinner bottles are not needed to be used for culturing the cells, the required amount of the cells is small, and the operation is convenient;

2) The WAVE swing bed reactor is provided with an independent control system, can control the culture temperature, the culture angle, the culture speed per hour, the pH value, the DO value, the aeration flow, the weighing setting, the compensation temperature and the like, can display the set numerical value, the accumulated value, the trend chart and the like in real time, and is convenient for personnel to observe and trace;

3) The WAVE swing bed reactor is matched with a cell culture bag, the cell culture bag contains a flaky carrier, the cell proliferation space is large, a control system is used for setting the environment which is most suitable for the growth of cells, the growth and the proliferation of the cells are more facilitated, the cell growth state is good, and the proliferation is fast;

4) The WAVE swing bed reactor is used for digestion and passage, the original pure manual operation is replaced by equipment operation, the pollution risk is reduced, the passage number is small, and the density of the prepared Vero cell suspension is high.

Based on the above developments, the protection scope of the present invention includes but is not limited to:

the specific method and the flow provided by the invention, namely the specific cell type, the cell generation, the passage proportion, the passage mode, the concentration of the digestive juice, the type of the digestive juice, the model specification of the WAVE swing bed reactor, the setting parameters of the WAVE swing bed reactor and the like can be adjusted according to the actual production requirement;

the invention relates to a matching system of a WAVE swing bed reactor, which comprises a culture volume of a cell culture bag, and the area and the number of flaky carriers in the culture bag, and can be adjusted according to the production requirement; meanwhile, the cells are transferred to the bioreactor for continuous culture after being cultured by only one cell culture bag, or transferred to the bioreactor for continuous culture after being cultured by two or more cell culture bags, so that the requirements are not met, and the adjustment can be carried out according to the production requirements;

the cells used in the invention are Vero cells, and other cells can be used for culture according to production requirements; meanwhile, the cell generation does not make requirements, but can not exceed the ultrahigh generation set in the cell stability investigation;

through the increase of the volume of the cell culture bags and the increase of the number of the flaky carriers, 1 small-volume cell culture bag can correspond to 1 or more cell culture bags with larger volume, and 1 cell culture bag can also correspond to 1 or more bioreactors which can be adjusted according to actual production requirements; meanwhile, the number of cell generation days can be adjusted according to actual needs, the method is not limited to the existing production level (the standard of the invention is 4 to 6 days), and a WAVE swing bed reactor can be used for preparing high-concentration cell suspension, so that the cell density and the cell number can be adjusted according to the actual production needs, and the method is not limited to the existing production level (the standard of 1 40L bioreactor is that the amount of Vero cell suspension is 6000 to 6500 mL, and the cell density is 3.0 to 3.5 cells/mL).

The raw materials and reagents used in the method for preparing the Vero cell suspension provided by the invention can be purchased from the market.

The invention is further illustrated by the following examples:

example 1 preparation of Vero cell suspension with WAVE pendulum bed reactor and sheet Carrier

1. Passage of disposable cell culture bottle

The Vero cells are subcultured according to the standard operating procedure (table 1) until the Vero cells are required to be subcultured, when the Vero cells grow into a monolayer and are polygonal, the Vero cells are closely and regularly arranged, the edge of the Vero cells is clear, the culture solution is orange, the Vero cells are clear and transparent, and the Vero cells can be subcultured next time or transferred into a WAVE swing bed reactor for continuous operation.

TABLE 1 recovery and passage of Disposable cell culture flasks

Name of procedure	Description of the procedures
		Cell resuscitation	Reviving 2 Vero cells
Cell-changing liquid	Changing the liquid after 16 to 24 hours
		Cell passage

		1, 4 to 1
Preparation of Vero cell suspension			After digesting the cells, preparing a Vero cell suspension by using the cell suspension for later use.

2. Transferring to WAVE swing bed reactor for culture

Now, taking a 2L sheet-shaped carrier cell culture bag as an example (1L:

1) The sheet carrier cell culture bags were pre-incubated (table 2): the sheet-shaped carrier cell culture bag needs to be incubated for more than 6 hours at 37 ℃ in advance before use, cell growth liquid (containing 10% fetal calf serum) needs not to cover the carrier, the compensation temperature is set to 37 ℃, DO and pH calibration parameters of the culture bag are input into a calibration parameter dialog box for calibration after the temperature is stabilized, the pH is recalibrated after the temperature is stabilized, and recalibration is not needed if the actual measurement value and the equipment display value are less than or equal to 0.03.

TABLE 2 Pre-incubation of sheet Carrier cell culture bags (2L cell culture bag for example)

Item	Specific parameter ranges
		Time of pre-incubation	More than 6 hours
Pre incubation temperature	37 deg.C (set value)
		Angle of pre-incubation	6~8°
Pre-incubation swing speed	10~15 rpm
		pH value setting	7.2~7.4
Amount of cell growth liquid used	500 to 800 mL (without overload body)

2) Digesting the cells according to a standard operating procedure to prepare a Vero cell suspension with required density and quantity, which comprises the following specific operations: t digestion Using TrypLE ™ Select (1 ×) ₁₅₀ Adding 20 mL/bottle of cells in a cell culture bottle, cleaning the cell surface, pouring out, adding 20 mL/bottle, placing the cell surface downwards on an operation table to digest the cells, pouring off a digestive juice when the cell surface is in a ground glass shape, continuing to digest the cells, digesting the cells in each bottle for about 5 minutes, adding a cell suspension (199 aqueous solution containing 1% fetal calf serum) to stop cell digestion when the cell surface has obvious cracks, adding 10 to 20 mL of the cell suspension by using a suction pipe to blow the cell surface, and preparing Vero cell suspension for later use.

3) The dispersed cell suspension is distributed into cell culture bags according to the required inoculation density, the cell culture bags are placed in a swing bed reactor for swing culture, the culture temperature is set to be 36 to 38 ℃, the angle is 6 to 8 ℃, a three-gas mode is clicked, and CO is adopted ₂ The concentration is set to be 5%, the aeration flow is set, the aeration flow can be adjusted along with cell proliferation, the speed during cell culture can be adjusted according to the cell adherence and the cell proliferation speed, the speed cannot be too high when the cell is just adhered to the wall, otherwise, the cell adherence is not facilitated, the cell can be adjusted at any time when the cell enters the proliferation period, the pH is 7.1-7.4, the DO is 55-65%, the WAVE system is provided with more than 3 pump heads, the pump 1 is set to supplement alkali, the pump 2 is set to feed liquid, the pump 3 is set to discharge liquid, a weighing set value is set, meanwhile, a special sampling port is used for sampling cell supernatant liquid every day to measure the glucose content, the glucose consumption and the required perfusion amount (table 5) are calculated according to a formula, the numerical values of the feed liquid and the discharge liquid are changed, and the required cell number is cultured.

The glucose detection method and formula are as follows:

(1) the ultraviolet spectrophotometer is started, and the product is used after preheating for at least 30 minutes.

(2) And starting the constant-temperature water tank, setting the temperature to 37 ℃, and using after the temperature is stable.

(3) The working solution R and the sample (culture solution) were sampled into test tubes by a pipette in the proportions shown in Table 3 and mixed. After mixing, the test tubes were placed on a test tube rack.

TABLE 3 proportion of the solutions used in the glucose assay

	Blank tube	Calibration tube	Sample tube
				Sample(s)	-	-	0.02 mL
Calibration article	-	0.02 mL	-
				Water for injection	0.02 mL	-	-
R (working fluid)	3 mL	3 mL	3 mL

(4) The test tube on the test tube rack is placed in a water bath at 37 ℃ for 10 minutes in a constant temperature water tank.

(5) Adjusting the wavelength of the ultraviolet spectrophotometer to 505nm, adjusting the wavelength to zero by using a blank tube, and performing machine measurement on the sample in the test tube after water bath and the calibrator to respectively read the absorbance of the calibrator and the absorbance of the sample.

Calculating the formula:

glucose (g/L) = (sample absorbance ÷ calibrator absorbance) × 5.55 × 0.18

Glucose consumption during perfusion culture is referenced in table 4:

TABLE 4 glucose consumption, perfusion, liquid inlet and outlet flows during perfusion culture

Glucose consumption g/day	Perfusion volume L/day	Inlet flow rate mL/min	The effluent flow is mL/min
				2~4	0.2~1.5	0.1~1.0	0.2~1.1
5~10	1.5~3.0	1.0~2.1	1.1~2.2
				10~20	3.0~8.0	2.1~5.6	2.2~5.8
20~30	8.0~13.0	5.6~9.0	5.8~9.2

TABLE 5 inoculation of Disposable WAVE System (2L cell culture bag as an example)

Item	Specific parameter ranges
		Density before inoculation	4.83×10 ⁶ cells/mL (actual measured value is standard)
Volume of inoculation	65~75 mL
		Culture volume	1000 mL (culture volume)
Temperature of culture	37 ℃ (setting)
		Cultivation angle	6~8°
Day one ventilation flow/swing speed	0.06~0.1 SLPM/6~7 rpm
		Second day ventilation flow/swing velocity	0.1~0.2 SLPM/7~9 rpm
Ventilation flow/swing speed on day three	0.2 (maximum value) SLPM/10 to 12 rpm
		Ventilation flow/swing speed on day four	0.2 (maximum) SLPM/12 (maximum) rpm
Fifth day ventilation flow/swing velocity	0.2 (maximum) SLPM/12 (maximum) rpm
		Glucose total sugar	6.0 (actual measured value is accurate)
Glucose content/perfusion on day	3.5 L/(0.6 to 1.2L) (actual measurement value is standard)
		Third day glucose content/perfusion volume	3.75 L/(1.5 to 2.5L) (actual measurement value is standard)
Glucose content/perfusion on day four	2.7 L/(3.0 to 4.0L) (actual measurement values: L:. RTM.) (L/(3.0 to 4.0L)Standard)
		Glucose content/perfusion on day five	2.9 L/(3.0 to 4.0L) (actual measurement value is standard)

3, digesting and passaging by using WAVE swing bed reactor

1) Cell culture bag cell digestion: connecting a cell culture bag with each pipeline, firstly discharging supernatant, cleaning the surface of a carrier with 0.01 mol/L PBS buffer solution, then discharging, then adding digestive juice for digestion, discharging the digestive juice when a small amount of cells are separated out, continuing digestion at room temperature (18 to 26 ℃), ending digestion until a large amount of cells are separated out visible to the naked eye, timely adding cell suspension into the cell culture bag for cell suspension, repeatedly adding the cell suspension for multiple times until the liquid in the flaky carrier cell culture bag is basically clarified, adding fetal calf serum to the concentration of 10%, uniformly mixing, preparing Vero cell suspension, sampling and counting the cells.

2) The inoculation number required for cell passage is calculated according to the cell density and the cell suspension number, and the number of the inoculated cell suspensions is added according to the density of 3.0 to 3.5 cells/mL of the final volume (Table 6). And timely transferring the cells into a 10L cell culture bag of a WAVE swing bed reactor for continuous culture.

TABLE 6 preparation of Vero cell suspension (2L cell culture bag as an example)

Item	Specific parameter ranges
		Volume of cell suspension	800~1000 mL
Cell suspension number	2 to 3 times
		Cell density	2.62×10 ⁶ cells/mL (actual measured value is standard)
Volume of inoculation	600~680 mL
		Is inoculated to	10 L-shaped sheet carrier cell culture bag

Example 2 preparation of Vero cell suspension with WAVE pendulum bed reactor and flaky Carrier

1. Transferring to WAVE swing bed reactor for culture

Now, taking a 10L sheet-shaped carrier cell culture bag as an example (10L:

1) Sheet-like carrier cell culture bags were pre-incubated (table 7): the sheet-shaped carrier cell culture bag needs to be incubated for more than 6 hours at 37 ℃ in advance before use, cell growth liquid (containing 10% fetal calf serum) needs not to cover the carrier, the compensation temperature is set to 37 ℃, DO and pH calibration parameters of the culture bag are input into a calibration parameter dialog box for calibration after the temperature is stabilized, the pH is recalibrated after the temperature is stabilized, and recalibration is not needed if the actual measurement value and the equipment display value are less than or equal to 0.03.

TABLE 7 Pre-incubation of sheet Carrier cell culture bags (10L cell culture bags are exemplified)

Item	Specific parameter ranges
		Time of pre-incubation	Over 6 hours
Pre incubation temperature	37 ℃ (setting)
		Pre-incubation angle setting	6~8°
Pre-incubation swing speed	10~15 rpm
		pH value setting	7.2~7.4
Amount of cell growth liquid used	2500 to 3000 mL (no overload body)

2) Adding Vero cell suspension obtained after cell digestion of the 2L sheet-shaped carrier cell culture bag in example 1 into a 10L cell culture bag according to the density of 3.0-3.5 cells/mL of the final volume, placing the cell culture bag in a swing bed reactor for swing culture, setting the culture temperature to be 36-38 ℃ and the angle to be 6-8 ℃, and clicking a three-gas mode to obtain CO ₂ The concentration is set to be 5 percent, the aeration flow is set, the aeration flow can be adjusted along with cell proliferation, the speed can be adjusted according to the cell adherence and the cell proliferation speed during cell culture, the speed cannot be too high when the cell is adhered to the wall, otherwise, the cell adherence is not facilitated, the cell can be adjusted at any time when the cell enters the proliferation period, the pH is between 7.1 and 7.4, the DO is between 55 and 65 percent, the WAVE system is provided with more than 3 pump heads, the pump 1 is set to supplement alkali, the pump 2 is set to feed liquid, the pump 3 is set to discharge liquid, a weighing set value is set, and meanwhile, a special sampling port is used for sampling cell supernatant every dayMeasuring glucose content, calculating glucose consumption and required perfusion amount according to formula (Table 8), changing the values of feed liquid and effluent liquid, and culturing to required cell number.

The glucose measurement method and formula, and the glucose consumption during perfusion culture were the same as in example 1.

TABLE 8 inoculation of Disposable WAVE System (10L cell culture bag as an example)

Item	Specific parameter ranges
		Density before inoculation	2.62×10 ⁶ cells/mL (actual measured value is standard)
Volume of inoculation	600~680 mL
		Culture volume	5000 mL (culture volume)
Temperature of cultivation	37 deg.C (set value)
		Cultivation angle	6~8°
Day one ventilation flow/swing speed	0.2~0.3 SLPM/7~8 rpm
		Second day ventilation flow/swing velocity	0.3~0.4 SLPM/8~9 rpm
Third day ventilation flow/swing speed	0.3~0.4 SLPM/10~12 rpm
		Ventilation flow/swing speed on day four	0.4 (maximum) SLPM/13 (maximum) rpm
Fifth day ventilation flow/swing velocity	0.4 (maximum) SLPM/13 (maximum) rpm
		Glucose Total sugar	6.0 (actual measured value is accurate)
Glucose content/perfusion on day	4.3 L/(0.3 to 0.6L) (actual measurement value is standard)
		Third day glucose content/perfusion volume	2.6 L/(2.0 to 3.0L) (actual measurement value is standard)
Glucose content/perfusion on day four	0.9 L/(5.5 to 6.5L) (actual measurement value is standard)
		Glucose content/perfusion on day five	1.1 L/(10 to 12L) (actual measurement value is standard)

And 3, digesting and passaging by using a WAVE swing bed reactor:

1) Cell culture bag cell digestion: connecting a cell culture bag with each pipeline, firstly discharging supernatant, cleaning the surface of a carrier with 0.01 mol/L PBS buffer solution, then discharging, then adding digestive juice for digestion, discharging the digestive juice when a small amount of cells are separated out, continuing digestion at room temperature (18 to 26 ℃) until a large amount of cells are separated out, finishing digestion, adding cell suspension into the cell culture bag in time for cell suspension, repeatedly adding the cell suspension for multiple times until the liquid in the flaky carrier cell culture bag is basically clear, adding fetal calf serum to the concentration of 10%, uniformly mixing, preparing Vero cell suspension, sampling and counting cells.

2) The number of inoculations required for cell passaging was calculated from the cell density and the number of cell suspensions, and the number of inoculated cell suspensions was added at a density of 3.0 to 3.5 cells/mL in final volume (Table 9). And timely transferring the culture medium into a bioreactor for continuous culture, and repeating the culture steps according to the method.

TABLE 9 preparation of Vero cell suspension (10L cell culture bag as an example)

Item	Specific parameter ranges
		Volume of cell suspension	5000-10000 mL
Cell suspension number	2 to 3 times
		Cell density	1.71×10 ⁶ cells/mL (actual measured value is standard)
Volume of inoculation	5300~6200 mL
		Inoculating into a bioreactor	Inoculating the mixture into a 40L bioreactor according to the final concentration of 3.0 to 3.5 cells/mL.

4. Transferring into a bioreactor for culture: after the cells in the cell culture bag after the culture are digested, suspended and counted according to the method, the cells are transferred into a bioreactor to be cultured according to the number of the cells required by the bioreactor, and the culture method of the bioreactor can be executed according to the production standard of the invention. The culture method of the bioreactor is specifically described as follows: inoculating cells into a bioreactor for continuous culture, inoculating Vero cell suspension according to the final density of 3.0-3.5 cells/mL, setting the culture volume of 30L, setting the control parameters, setting the rotation speed at 98-102 rpm, the temperature at 36.8-37.2 ℃ and 5% CO ₂ And (3) culturing for 4 to 5 days at a pH of 7.2 to 7.3 and a DO of 65 percent and a gas flow rate of 0.3 to 0.5 SLPM.

Comparative example

Sheet carrier cell culture bag (containing diamond sheet carrier): wuhansaike science and technology Limited;

disposable spherical carrier bag (containing Cytodex spherical microcarrier): GE medical treatment in China.

A cell culture bag is used, inoculation is carried out according to the final density of 3.0-3.5 cells/mL from a 10L cell culture bag with a spherical microcarrier of 30 g/L, the usage amount and related control parameters are basically consistent with those in the table 8 with the summary of the experimental results of the disposable WAVE system, the cell growth state is good, but after Vero cell suspension is prepared, namely in the table 9, a liquid outlet of the spherical microcarrier cell culture bag is above a cut-off film, vero cells are large and are under the carrier cell culture bag when a large amount of Vero cells exist, the Vero cells are not easy to pass through the cut-off film when the cell concentration is high, meanwhile, a large amount of cells are attached to the surface of the spherical microcarrier again, waste is serious, and the cell density of the carrier cell culture bag with the same volume (compared with a sheet-shaped carrier cell culture bag with a liquid outlet at the lower end) cannot reach the cell number of a 40L bioreactor to be inoculated.

Effects of the invention

FIGS. 1 and 2 are graphs showing the comparison between the culture conditions and the digestion and inoculation conditions of cell culture bags of two different carriers, i.e., spherical microcarriers and pellet-like carriers, of 10L. And (4) analyzing results: FIG. 1 is a graph showing the trend of glucose values during the culture of two carrier cell culture bags, with the abscissa of the time of the day of culture, i.e., after 1 day, 2 days, 3 days and 4 days of culture, sampled through a sampling port, and the glucose content was measured, and this data is the average of three tests. The white area is Vero cells cultured by a sheet-shaped carrier cell culture bag made of Setaria, and the black area is Vero cells cultured by a GE disposable spherical carrier bag. The difference of the two culture processes is small, the cell growth is good, and the production requirements can be met.

FIG. 2 is a graph showing the trend of cell density of cell suspensions prepared from two carrier cell culture bags, and the abscissa shows the number of detection times, i.e., three experimental studies are performed. The white area is the Vero cell suspension density after digestion of the Sertaceae flaky carrier cell culture bag, and the black area is the Vero cell suspension density after digestion of the GE disposable spherical carrier bag. The result difference is larger, the difference of the cell suspension after digestion and collection of the carrier cell culture bag with the same volume is about one time, which indicates that compared with the ball-rotating-sheet process, the mutual transfer of the sheet-shaped carriers is more beneficial to the actual production.

The result of comparing two different carrier cell culture bags shows that the culture mode of the bioreactor used by the invention is also a sheet carrier, cells need to be digested from the sheet carrier and then added with cell suspension, the cells are transferred into the bioreactor to be cultured after being prepared into cell suspension, the carrier in the cell culture bag is a rhombic sheet carrier, the area is large, the outflow of the cell suspension cannot be influenced when liquid is transferred out through a peristaltic pump, and the liquid outlet is arranged below the carrier cell culture bag, so that the outflow under the action of the gravity of the cells is facilitated, and the cell density of the prepared cell suspension is high. And the spherical microcarrier is small, the liquid outlet end cannot be arranged below, so that the pipeline is blocked, the liquid outlet can be arranged on the carrier cell culture bag, and meanwhile, in order to prevent the pipeline from being blocked, an interception membrane is added, but the interception membrane can also block the passage of part of Vero cells, so that waste is caused, and the cell density of the prepared cell suspension is low. If the carrier cell culture bag is used, the carrier cell culture bag with larger culture volume is selected for carrying out the culture, but the larger the carrier cell culture bag is, the more troublesome the operation is, and the materials are wasted, so the spherical microcarrier of the GE cell culture bag is not suitable for the production process of the ball rotor, and the test comparison analysis shows that the sheet-shaped carrier cell culture bag is more suitable for culturing the Vero cells.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.

Claims

1. A method of preparing a Vero cell suspension, comprising the steps of:

step 1: obtaining Vero cells after passage;

step 2: culturing the Vero cells after passage by a WAVE swing bed;

and step 3: and taking the second Vero cell suspension for enlarged culture and collecting.

2. The method according to claim 1, wherein the seed differentiation rate of the passage is 1 to 4 to 1.

3. The method of claim 2, wherein the temperature of the WAVE swing bed culture is 36 to 38 ℃, and the CO is ₂ Concentration of 5%, angle6-8 degrees, the swing speed is 6-13 rpm, the ventilation flow is 0.06-0.4 SLPM, the PH is 7.1-7.4, and the DO is 55-65%.

4. The method of claim 3, wherein the resuspension is performed using a cell suspension comprising: 1% fetal bovine serum and 199 aqueous solution.

5. The method as claimed in claim 4, wherein the pre-incubation temperature of the sheet-like carrier in the step 2-1 is 37 ℃, the angle is 6-8 ℃, the oscillation speed is 10-15 rpm, and the pH is 7.2-7.4.

6. The method of claim 5 wherein the first Vero cell suspension has a density of 4.83 x 10 ⁶ cells/mL。

7. The method as claimed in claim 6, wherein the temperature of WAVE swing bed culture in the step 2-3 is 37 ℃ and the angle is 6 to 8 ℃;

the swing speed of the WAVE swing bed culture comprises the following steps: the swing speed on the first day is 6 to 7 rpm, the swing speed on the second day is 7 to 9 rpm, the swing speed on the third day is 10 to 12 rpm, and the swing speed on the fourth day and the fifth day is 12 rpm.

8. The method of claim 7 wherein the second Vero cell suspension has a density of 2.62 x 10 ⁶ cells/mL。

9. The method of claim 8, wherein the expanded culture in step 3 is WAVE swing bed culture or bioreactor culture;

the swing speed of the WAVE swing bed culture comprises the following steps: the swing speed on the first day is 7 to 8 rpm, the swing speed on the second day is 8 to 9 rpm, the swing speed on the third day is 10 to 12 rpm, and the swing speeds on the fourth day and the fifth day are 13 rpm;

the density of the Vero cell suspension prepared by WAVE swing bed culture is 1.71 multiplied by 10 ⁶ cells/mL；

The temperature for culturing the bioreactor is 36.8 to 37.2 ℃, and CO is added into the bioreactor ₂ The culture medium is cultured for 4 to 5 days at a concentration of 5%, a rotation speed of 98 to 102 rpm, a pH of 7.2 to 7.3, a DO of 65% and a gas flow of 0.3 to 0.5 SLPM.

10. The method according to claim 9, wherein the cell density in the cell culture solution after the inoculation of the passaged Vero cells is 3.0 to 3.5 cells/mL;

the cell density in the cell culture solution after the first Vero cell suspension is inoculated is 3.0 to 3.5 cells/mL;

the cell density in the cell culture solution after the second Vero cell suspension is inoculated is 3.0 to 3.5 cells/mL.