CN115094023A

CN115094023A - MDCK cell microcarrier culture and suspension domestication process

Info

Publication number: CN115094023A
Application number: CN202210779403.5A
Authority: CN
Inventors: 王龙; 姚芸; 王猛
Original assignee: Wuxi Duoning Biotechnology Co ltd
Current assignee: Wuxi Duoning Biotechnology Co ltd
Priority date: 2022-07-04
Filing date: 2022-07-04
Publication date: 2022-09-23

Abstract

The invention discloses a process for culturing and suspending domestication of a MDCK cell microcarrier, which comprises the steps of collecting microcarrier cells prepared by a microcarrier culture method of MDCK cells, and domesticating the collected microcarrier cells by a MDCK cell suspending domestication method; the invention adopts the process of collecting mass cells at one time by culturing the cells by microcarriers and then carrying out suspension domestication, thereby greatly shortening the time for obtaining a large amount of cell suspension by culturing the cells by using a square bottle conventionally; the MDCK cells are continuously subcultured by using the domestication culture medium, the problems of cell agglomeration and wall hanging in the domestication process are solved, and the cells which stop growing or have reduced survival rate are rescued by low-speed centrifugation and addition of fresh healthy cells.

Description

MDCK cell microcarrier culture and suspension domestication process

Technical Field

The invention relates to the technical field of biology, in particular to a process for culturing and suspending domestication of a MDCK cell microcarrier.

Background

MDCK Cells (Madin-Daby Canine Kidney Cells, MDCK) epithelial-like Cells isolated by Madin and Darby in 1958 from the Kidney of healthy male Khaka beagle dogs. The culture is usually grown adherently. Due to the advantages of high virus infection rate, fast proliferation and low liability to mutation of MDCK cells, the cell line has been widely used for amplification and purification of various viruses, such as Adenovirus (Adenovirus), Canine Parvovirus (CPV), Avian Influenza Virus (AIV), Influenza Virus (IV), etc.

MDCK cells are adherently grown cells, have a remarkable adaptability advantage in an adherent state and are insensitive to shearing force and ventilation. Adherent culture by microcarrier is one of the strategies for large-scale culture of MDCK cells, and at present, two solid microcarriers, namely Cytodex1 and Cytodex 3, are used in MDCK cells more. Cytodex1 is based on a cross-linked dextran backbone substituted with positively charged N, N diethylaminoethyl groups, with the charged groups distributed throughout the microcarrier backbone. The denatured collagen layer on Cytodex 3 is easily digested by various proteases such as trypsin and collagenase, and thus, it is possible to separate cells from microcarriers while maintaining the maximum activity, function and integrity of the cells. Different microcarriers affect the growth of the cells, so the selection of different microcarriers is a primary problem for large-scale culture of MDCK cells.

The first report of successful suspension acclimation of the strain is that the Capsule and the like acclimate BHK-21 cells to realize suspension culture and use the suspension culture for producing veterinary vaccines. Then, suspension domesticates suspended CHO, MDCK and other cells. The suspension of adherent cells has been carried out for decades, most of the domestication processes used are methods for gradually reducing serum and gradually increasing the speed for adaptation after the culture medium is replaced, the domestication time of the methods is long (5-10 months), the methods have high failure probability, and even if the domestication is successful, the defects of easy cell agglomeration, low survival rate, long multiplication time and the like easily occur. There is no acclimatization process having a remarkable effect, and it is therefore necessary to provide a culture medium and a culture method having excellent performance.

In 1967, Van Wezel developed microcarriers and enabled large-scale culture of adherent cells in bioreactors. Research on the application of microcarrier culture to suspension acclimatization is only reported, but a large amount of cell suspension can be obtained in a short time by using microcarrier, and the cell density is a key factor for the initial stage of suspension acclimatization.

Disclosure of Invention

The invention provides a process for culturing and suspending domestication of MDCK cell microcarriers, which aims to solve the problems in the prior art.

The scheme of the invention is as follows:

after the microcarrier cells prepared by the microcarrier culture method of the MDCK cells are collected, the collected microcarrier cells are domesticated by the MDCK cell suspension domestication method.

As a preferred technical scheme, the microcarrier culture method of the MDCK cells comprises the following steps:

1) pretreating a microcarrier, namely weighing 2-5 g/L of Cytodex series dry powder microcarrier, soaking the dry powder microcarrier for 4-18h by using PBS (phosphate buffer solution) with the pH of 7.4, washing the dry powder microcarrier twice by using PBS, sterilizing at high pressure, cooling and standing, washing the microcarrier for 2-3 times by using a microcarrier culture medium before use, and then fixing the volume in a microcarrier culture medium of 2% serum;

2) recovering MDCK seed cells, taking out the frozen MDCK cells from liquid nitrogen, rapidly stirring for 2 minutes in a water bath at 37-39 ℃ to recover the cells, and culturing the recovered cells in a T125 square bottle by using a cell culture solution of 10% FBS DMEM until the cells grow to be more than 90% compact on the growth surface of the T125 square bottle;

3) subculturing MDCK seed cells, subculturing when the cell confluence is more than 95%, discarding old culture solution, cleaning for more than 3 times by PBS, adding 5ml of 0.25% pancreatin, digesting for 5-10 minutes at 37 ℃, discarding digestive juice, lightly beating the square bottles up and down, allowing residual digestive juice to permeate into each cell, continuing to soak for 2-5 minutes at 37 ℃ until the bottom of the square bottle T125 is lightly tapped, enabling the cells to uniformly fall off in a flaky manner, adding a proper amount of subculture medium, and performing subculturing according to the following steps of 1: 5-1: inoculating and passaging at a passage ratio of 20;

4) adapting a culture medium of the MDCK seed cells, directly transferring the subculture cells in the step 3) to a microcarrier culture medium without microcarriers, and adapting to one generation;

5) inoculating a microcarrier: preparing MDCK cell suspension after passage for 5 times into cell suspension with the density of 1000 cells/ml, and adding a microcarrier culture medium containing 2% serum;

6) microcarrier adsorption, namely putting the microcarrier culture medium containing the MDCK cell suspension in the step 5) into a square bottle, putting the square bottle on a side swing shaker, and then putting the square bottle and the side swing shaker together into a carbon dioxide incubator for slow shaking and culturing for 8-12 hours;

7) and (2) microcarrier culture, namely blowing and suspending a microcarrier culture medium containing MDCK cell suspension, transferring the microcarrier culture medium containing the MDCK cell suspension in a square bottle into a 125ml shake flask, wherein the shaking table with the axle distance of 50mm has the rotating speed of 110-130 rpm and the volume fraction of 5-8% of CO ² Continuously culturing for 24 hours at the temperature of 36-38 ℃;

8) carrying out amplification culture on the microcarrier, taking out a 125ml shake flask and standing for 2-4 minutes when microcarrier cells are fully climbed with the carriers and the carriers are sticky, discarding a used microcarrier culture medium, washing the microcarrier containing the cells for 3 times by PBS (phosphate buffer solution), adding 0.25% pancreatin, digesting for 2 hours at the conditions of 110rpm, 5% carbon dioxide concentration and 37 ℃; after digestion and standing, taking the digested supernatant, centrifuging and collecting cells, transferring the digested supernatant cell suspension into a 50ml centrifuge tube, centrifuging at 1000rpm for 5min, and collecting the cells;

mixing the amount of the microcarrier in the step 1) according to a ratio of 1: 10, performing volume expansion culture, then suspending the digested cells in a microcarrier culture medium with 2% serum, then placing the cells into a new shake flask, and culturing the cells under a cell adsorption carrier culture condition for 4-12 hours, wherein the cell adsorption carrier culture condition is that the temperature is 36-38 ℃, the volume fraction is 5-8% of CO2, and the rotation speed is 80-110 rpm; after the culture condition of the cell adsorption carrier is finished, the culture condition of the cell on the carrier is changed, the temperature of the culture condition of the cell on the carrier is 36-38 ℃, and the volume fraction of CO is 5-8% ² The rotating speed is 110-130 rpm, and the time is 24-48 hours; and (5) observing the cell capacity of the carrier by microscopic examination until the cell capacity reaches 80%, starting to harvest the cells, and replacing the culture solution once every two days.

As a preferred technical scheme, the step 1) is to weigh dry powder microcarriers of Cytodex series according to the amount of 3 g/L; the maximum cell harvest amount in the step 8) is concentrated in the 3 rd to 5 th times of washing after digestion; and in the step 8), repeatedly adding sterile PBS to wash the digested microcarrier for multiple times, and centrifugally collecting cells in the supernatant to obtain the cells.

As a preferable technical scheme, the pancreatin digestion condition in the step 3) is that digestion is carried out for two times of incubation, the pancreatin is integrally soaked in cells for the first time, and the cells are separated from each other and connected with the bottom of the bottle; and (3) continuously incubating for 5-10 minutes after the flowing pancreatin is sucked and discarded for the second time, separating the cells from the bottle bottom at the target position, slightly beating the bottle bottom at the moment, separating the cells into pieces, falling and separating, and adding a fresh subculture medium to blow the uniform cells to obtain a uniformly dispersed cell suspension.

As a preferred technical solution, the MDCK cell suspension acclimatization method comprises the following steps:

1) obtaining primary suspension cells, standing and settling the collected microcarrier cells, and absorbing and discarding supernatant; washing for 3-8 times by using PBS buffer solution, adding pancreatin after absorbing and discarding the PBS buffer solution, placing the mixture into a shaking table for digestion in a culture mode, taking out digested cell liquid after digesting for 2 hours, taking cell supernatant after standing for 3 minutes, adding washing solution for 3-8 times to wash the cells dropped from the microcarriers and collect the supernatant, placing the collected supernatant into a centrifuge tube, centrifuging, collecting at room temperature and 1000rpm for 5 minutes, and pouring the collected solution to leave the cells; adding cells into acclimatization culture medium, and adjusting cell density to 20 × 10 ⁵ cells/ml～30×10 ⁵ cells/ml, culturing in a culture mode, sampling every day and observing the change of cell density, cell viability and cell diameter; expanding the acclimated culture volume;

2) treating adherent walls and conglomerations in domestication, wherein cells can have serious conglomeration and wall-hanging phenomena within 2-3 days in a culture process, removing a domestication culture medium by centrifugation, and then treating for 2-3 hours in a culture mode by using pancreatin with the same volume; when the large cells are not digested and aggregated, the large cells are removed by a pipette and discarded, and the rest cells are collected and centrifuged according to the ratio of 10 multiplied by 10 ⁵ cells/ml～20×10 ⁵ One of a cell/ml density passage treatment mode or a liquid change treatment mode;

3) in the domestication, the survival rate is reduced in the culture process, the density is overhigh, and a fresh domestication culture medium is added by adopting passage density; the occurrence rate of cell activity is less than 50% or the cell density is less than 5X 10 ⁵ cells/ml the old acclimation medium was removed by centrifugation at 800rpm for 5 minutes at low speed and fresh, digested healthy cells were added to adjust the cell density to 10X 10 ⁵ cells/ml～20×10 ⁵ continuously culturing cells/ml;

4) stopping cell growth during acclimation, centrifuging at 48 hr intervals, adding fresh digested healthy cells, and adjusting cell density to 10 × 10 ⁵ cells/ml～20×10 ⁵ cells/ml, and continuing culturing;

5) passage of domesticated cells, cell recovery multiplication, and cell density of more than 100 × 10 ⁵ cells/ml, according to 10X 10 ⁵ cells/ml～20×10 ⁵ And (3) carrying out normal cell subculture in a cell/ml density range, wherein a subculture medium is a domestication medium, and repeatedly carrying out subculture according to the method for multiple times until the cell multiplication time is stable, the survival rate is more than 90%, and no mass agglomeration is regarded as the completion of domestication.

According to the preferable technical scheme, the culture mode is that the rotating speed of a shaking table with the wheelbase of 50mm is 110-130 rpm, the volume fraction of carbon dioxide is 5-8%, and the temperature is 36-38 ℃.

The preferable technical scheme also comprises a freezing and recovering method for the domesticated cells, wherein the freezing and recovering method for the domesticated cells comprises the following steps:

(1) freezing and storing the domesticated cells according to the domesticated culture medium: DMSM ═ 9: 1 preparing a freezing medium according to a formula of 150X 10 ⁵ cells/ml～200×10 ⁵ Freezing at cell/ml density, freezing by using a program freezing box, transferring into a liquid nitrogen tank for long-term storage after 24 hours, and recovering one cell every half year to 1 year to check cell activity;

(2) and (3) recovering the domesticated cells: quickly thawing MDCK cells taken out from liquid nitrogen by using warm water at 37-39 ℃, adding the MDCK cells into a 10ml acclimation culture medium in a 125ml shake flask, slightly sucking the cells, transferring the cells into a culture medium, recovering the cells for 24-48 hours under the conditions of 110rpm, 5% carbon dioxide concentration and 37 ℃, and carrying out subculture for 3 generations according to acclimation density so as to normally use the cells.

As a preferred technical scheme, the microcarrier culture medium comprises TransVA-01 and 1% -2% of serum, wherein the serum is one of 1% -2% of fetal calf serum or 1% -2% of newborn calf serum; the domestication culture medium comprises a mixed solution of TransVA-01 and 0.025% pancreatin; the subculture medium comprises a mixed solution of TransVA-01 and 0.025 percent of pancreatin.

The invention also discloses application of the MDCK cell microcarrier culture and suspension acclimation process in MDCK cell acclimation culture.

Due to the adoption of the MDCK cell microcarrier culture and suspension domestication process in the technical scheme, after the microcarrier cells prepared by the MDCK cell microcarrier culture method are collected, the collected microcarrier cells are domesticated by the MDCK cell suspension domestication method.

The invention has the advantages that:

the invention adopts the process of collecting mass cells at one time by culturing the cells by microcarrier and then suspending and domesticating the cells, uses a square bottle to revive cell seeds, uses the microcarrier and trypsin to obtain a large amount of cells at one time, and greatly shortens the time of obtaining a large amount of cell suspension by culturing the cells by the square bottle conventionally; the MDCK cells are continuously subcultured by using the domestication culture medium, the problems of cell agglomeration and wall hanging in the domestication process are solved, and the cells which stop growing or have reduced survival rate are rescued by low-speed centrifugation and addition of fresh healthy cells.

In the invention, pancreatin digestion and co-incubation are carried out for 2 times, the pancreatin is integrally soaked in cells for the first time, and the connection between the cells and the bottle bottom is separated; after the flowing pancreatin is sucked and discarded for the second time, the incubation is continued for a few minutes, the aim is mainly to separate the cells from the bottle bottom, at the moment, the bottle bottom is lightly tapped, the cells can be flaked, separated and added with a fresh culture medium to blow the uniform cells, and the uniformly dispersed cell suspension can be obtained.

The advantages of the method of the invention and the traditional method are as follows:

1. MDCK belongs to cells which are difficult to digest, the traditional digestion method has the problems of incomplete digestion and repeated cleaning, and if the pancreatin is not completely digested, pancreatin can be directly supplemented without cleaning.

2. The traditional digestion method has the problem of over digestion, the digestion is divided into 2 stages, the excessive digestion of excessive pancreatin can be prevented by discarding pancreatin in the first stage, and the purpose of continuous digestion can be achieved by a small amount of residual pancreatin in the second stage.

3. Traditional pancreatin digestion has the problem of uneven digestion, the bottom of the bottle is tapped to enable liquid to permeate each cell and be evenly distributed, the phenomenon of local unevenness is avoided, meanwhile tapping is helpful for the cells to fall off, and compared with the operation of blowing and suspending the cells by a suction pipe or a gun head, the operation is warmer and softer and is not easy to contaminate bacteria.

The process fundamentally solves the problems of long production period, complex operation, large workload, easy pollution, bottle-to-bottle difference and the like when square bottle culture is adopted to obtain a large amount of cell suspension, and reduces the production cost for obtaining a large amount of single cell suspension while the existing adherent cell culture process is technically upgraded.

The process also solves the problems of cell agglomeration, wall hanging, growth stopping, activity rate reduction and the like commonly occurring in the suspension domestication of the MDCK cells, greatly improves the success rate of the suspension domestication of the adherent cells, and is far superior to the domestication period (from half a year to one year) of the traditional process because the period of the front domestication and the back domestication is only 1 month.

Therefore, the process research invention has very practical application prospect in practical production.

Drawings

FIG. 1 shows a state diagram of cells in DMEM medium containing 10% FBS;

FIG. 2 is a diagram showing the state of cell suspension obtained by two trypsinization processes;

FIG. 3 shows a diagram of MDCK adherent cells after completion of microcarrier adsorption in a square flask;

FIG. 4 shows a diagram of MDCK adherent cells after microcarrier expansion in shake flasks;

FIG. 5 is a graph showing a comparison of the state of first-generation cells at the time of starting suspension acclimation of MDCK adherent cells and the state after completion of suspension acclimation;

fig. 6 shows cell density, cell viability and doubling time profiles for cell passages during MDCK cell suspension acclimation;

fig. 7 shows cell density, cell viability and doubling time curves of the passages of the cells recovered after the MDCK suspension cells successfully subjected to suspension acclimation are cryopreserved.

Detailed Description

In order to make up for the above deficiencies, the present invention provides a process for culturing and suspending acclimatization of an MDCK cell microcarrier to solve the above problems in the background art.

After microcarrier cells prepared by a microcarrier culture method of MDCK cells are collected, the collected microcarrier cells are domesticated by an MDCK cell suspension domestication method.

2)2) recovering MDCK seed cells, taking out the frozen MDCK cells from liquid nitrogen, rapidly stirring for 2 minutes in a water bath at 37-39 ℃ to recover the cells, and culturing the cells in a T125 square bottle by using a cell culture solution of 10% FBS DMEM after recovery until the cells grow to be more than 90% compact on a growth surface of the T125 square bottle;

7) and (3) performing microcarrier culture, namely blowing and suspending the microcarrier culture medium containing the MDCK cell suspension, transferring the microcarrier culture medium containing the MDCK cell suspension in a square bottle into a 125ml shake flask, wherein the shaking table with the axle distance of 50mm has the rotating speed of 110-130 rpm and the volume fraction of 5-8% CO ² Continuously culturing for 24 hours at the temperature of 36-38 ℃;

8) carrying out amplification culture on the microcarrier, taking out a 125ml shake flask and standing for 2-4 minutes when microcarrier cells are fully climbed over the carrier and the carrier is sticky, discarding the used microcarrier culture medium, washing the microcarrier containing the cells for 3 times by PBS (phosphate buffer solution), adding 0.25% pancreatin, digesting for 2 hours under the conditions of 110rpm, 5% carbon dioxide concentration and 37 ℃; after digestion and standing, taking the digested supernatant, centrifuging and collecting cells, transferring the digested supernatant cell suspension into a 50ml centrifuge tube, centrifuging at 1000rpm for 5min, and collecting the cells;

mixing the amount of the microcarrier in the step 1) according to a ratio of 1: 10, then re-suspending the digested cells in a microcarrier culture medium with 2% serum, then placing the cells into a new shake flask, and culturing the cells under a cell adsorption carrier culture condition of 36-38 ℃ for 4-12 hours, wherein the volume fraction of the cells is 5-8% of CO2, and the rotation speed is 80-110 rpm; after the culture condition of the cell adsorption carrier is finished, the culture condition of the cell on the carrier is changed, the temperature of the culture condition of the cell on the carrier is 36-38 ℃, and the volume fraction of CO is 5-8% ² The rotating speed is 110-130 rpm, and the time is 24-48 hours; and (5) observing the cell capacity of the carrier by microscopic examination until the cell capacity reaches 80%, starting to harvest the cells, and replacing the culture solution once every two days.

Weighing Cytodex series dry powder microcarrier with the amount of 3g/L in the step 1); the maximum cell harvest amount in the step 8) is concentrated in the 3 rd to 5 th times of washing after digestion; and in the step 8), repeatedly adding sterile PBS to wash the digested microcarrier for multiple times, and centrifugally collecting cells in the supernatant to obtain the cells.

The pancreatin digestion condition in the step 3) is that digestion is carried out for two times of incubation, the pancreatin is integrally soaked in cells for the first time, and the connection between the cells and the bottle bottom is separated; after the flowing pancreatin is sucked and discarded for the second time, the incubation is continued for 5-10 minutes, the cells are separated from the bottle bottom at the target position, at the moment, the bottle bottom is slightly tapped, the cells can be flaked, separated, and then a fresh subculture medium is added to blow the cells evenly to obtain a uniformly dispersed cell suspension.

The MDCK cell suspension domestication method comprises the following steps:

1) obtaining primary suspension cells, standing and settling the collected microcarrier cells, and absorbing and discarding supernatant; washing for 3-8 times by PBS buffer solution to wash more than 80% of cells, removing the PBS buffer solution by suction, adding pancreatin, putting the cells into a shaking table for digestion in a culture mode, taking out digested cell liquid after digesting for 2 hours, taking cell supernatant after standing for 3 minutes, adding washing solution for 3-8 times to wash the cells fallen off from the microcarrier and collecting the supernatant, putting the collected supernatant into a centrifuge tube, centrifuging, collecting at room temperature and 1000rpm for 5 minutes, pouring the collected solution and leaving the cells; adding cells into acclimatization culture medium, and adjusting cell density to 20 × 10 ⁵ cells/ml～30×10 ⁵ cells/ml, culturing in a culture mode, sampling every day and observing the change of cell density, cell viability and cell diameter; expanding the acclimated culture volume;

3) in the domestication, the survival rate is reduced in the culture process, the density is overhigh, and a fresh domestication culture medium is added by adopting passage density; the occurrence rate of cell viability is less than 50 percent or the cell density is denseDegree less than 5X 10 ⁵ In the case of cells/ml, old acclimation medium was removed by centrifugation at low speed 800rpm for 5 minutes and fresh digested healthy cells were added to adjust the cell density to 10X 10 ⁵ cells/ml～20×10 ⁵ cells/ml continue to be cultured;

5) passage of domesticated cells, cell recovery multiplication, and cell density of more than 100 × 10 ⁵ cells/ml, according to 10X 10 ⁵ cells/ml～20×10 ⁵ And (3) carrying out normal cell subculture in a cell/ml density range, wherein a subculture medium is a domestication medium, and repeatedly carrying out subculture according to the method for multiple times until the cell multiplication time is stable, the survival rate is over 90 percent, and no mass agglomeration is regarded as domestication completion.

The culture mode is that the rotating speed of a shaking table with the wheelbase of 50mm is 110-130 rpm, the volume fraction of carbon dioxide is 5-8%, and the temperature is 36-38 ℃.

The method also comprises a freezing and thawing method for the domesticated cells, and the freezing and thawing method for the domesticated cells comprises the following steps:

(1) and (3) freezing and storing the domesticated cells according to the domesticated culture medium: DMSM ═ 9: 1 preparing a freezing medium according to a formula of 150X 10 ⁵ cells/ml～200×10 ⁵ Performing cryopreservation at a cell/ml density, performing cryopreservation by using a program cryopreservation box, transferring into a liquid nitrogen tank for long-term preservation after 24 hours, and recovering one cell every half year to 1 year to check cell viability;

(2) and (3) recovering the domesticated cells: rapidly thawing MDCK cells taken out from liquid nitrogen by using warm water at 37-39 ℃, adding the MDCK cells into a 10ml acclimation culture medium 125ml shake flask, slightly sucking the cells, transferring the cells into a culture medium, recovering the cells for 24-48 hours under the conditions of 110rpm, 5% carbon dioxide concentration and 37 ℃, and carrying out subculture for 3 generations according to acclimation density so as to normally use the MDCK cells.

The microcarrier culture medium comprises TransVA-01 and 1% -2% serum, wherein the serum is one of 1% -2% fetal calf serum or 1% -2% newborn calf serum; the domestication culture medium comprises a mixed solution of TransVA-01 and 0.025% pancreatin; the subculture medium comprises a mixed solution of TransVA-01 and 0.025 percent of pancreatin.

In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

In the present invention, the original concentration of pancreatin is 0.25% by mass of pancreatin, and the phosphate buffer is PBS with pH 7.4, unless otherwise stated, wherein the culture methods are all conventional in cell culture and related fields.

Example 1: microcarrier culture of MDCK cells

1. Main reagents and consumables:

firstly, microcarrier: cytodex1 type, GE, 3.1X 10 per gram ⁶ And (3) a carrier.

(ii) MDCK cells: provided by the Tongzhi biology of Guangzhou, duoning group.

③ TransVA-01 Medium: contains glutamine, prepared according to the specification, Shanghai duoning biological corporation.

DMEM medium: dry powder, formulated as per the instructions, GBICO.

0.25% trypsin (liquid): GBICO.

Sixthly, fetal bovine serum and newborn bovine serum: shanghai Shuang wet BioLimited.

The T125 cell culture square bottle: CORNING is provided.

Eighthly, shaking the bottle: shanghai duoning bionics GmbH.

2. Main equipment and model:

name(s)	Company(s)	Type number
			Micro electronic scale	Sartorius	BSA2202S
Carbon dioxide incubator	WIGGENS	WCI-180
			Carbon dioxide shaking table	Shanghai duoning (medicine for treating Shanghai disease)	DNSI0101
Super clean bench	Suzhou Jingan	SW-CJ-2FD
			Cell counter	CountStar	IC1000
Microscope	Cai Kang	XDS-200C

3. The process method comprises the following steps:

weighing 3g of dry powder microcarrier, soaking the carrier for 4-18h by using PBS buffer solution with the pH value of 7.4, washing the carrier for two times by using PBS, sterilizing the carrier under high pressure, cooling and standing the carrier.

② before using, the microcarrier is cleaned for 2-3 times by TransVA-01 and then is fixed in 2% serum of TransVA-01.

Thirdly, standing and culturing the recovered MDCK cells in a T125 square flask until the cells grow to more than 90% of the growth surface of the square flask for digestion and passage.

Fourthly, the old culture solution is discarded, the old culture solution is washed for more than 3 times by PBS, 5ml of 0.25 percent pancreatin is added, the mixture is digested for 10 minutes at 37 ℃, then the digestion solution is discarded, and the square bottle is tapped up and down, left and right to ensure that the residual digestion solution permeates into each cell.

Fifthly, continuing to digest for 2-5 minutes at 37 ℃ until the bottom of the square bottle is tapped, enabling the cells to be flaked uniformly, adding 20ml of DMEM, mixing uniformly, discarding 19ml of cell suspension, replenishing the culture medium to 20ml again, and adding 10% serum.

After passage for 3 times, the passage culture medium is replaced by a micro carrier culture medium (without micro carrier) of the MDCK cells and passage is carried out for 1 time.

Seventhly, when the cell is subcultured for the 5 th time, adding the subcultured cell suspension and 20ml of microcarrier culture medium (containing microcarriers) into a square bottle.

Placing the square bottle on a side swing shaking bed, and placing the square bottle and the side swing shaking bed together into a carbon dioxide incubator to carry out slow shaking culture for 12 hours.

Ninthly, transferring the microcarrier culture medium into a 125ml shake flask after blowing and suspending, and setting shaking table parameters according to culture conditions for culture.

And (c) when the microcarrier cells are fully climbed with the carrier and the carrier is sticky, carrying out carrier amplification culture, taking out a shake flask and standing for 2-4 minutes, discarding the old culture medium, washing the carrier containing the cells for 3 times by PBS, adding pancreatin with the volume of 20ml, and digesting for 2 hours at the conditions of 110rpm, 5% carbon dioxide concentration and 37 ℃.

11 the cell suspension of the digested supernatant was transferred to a 50ml centrifuge tube and centrifuged at 1000rpm for 5min to collect the cells.

12 according to 1: 10 times (i.e. 200ml), the digested cells were resuspended in 200ml of carrier medium and the culture conditions were changed to adsorption conditions after 12 hours of culture using a 1L shake flask.

13, after culturing for 48 hours, observing that the carrying capacity of the carrier cells exceeds 80 percent by microscopic examination, starting to harvest the cells, and if the cell amount is insufficient, continuing to culture, wherein the carrier culture medium needs to be replaced once in 48 hours after long-time culture.

Note that the carrier medium used in the above embodiment was 2% NBS TransVA-01.

Further, the generation ratio in the embodiment may be 1: 5-1: 20, the passage ratio being related to the subsequent amplification time.

Further, the time for adsorbing the carrier by the cells may be any time between 4 and 12 hours, the 12-hour adsorption time is used in the above examples, and the culture mode used is the adsorption condition mode.

FIG. 1 shows the adherent state of MDCK cells cultured in the original 10% FBS DMEM medium, and the cells are tightly attached to the bottom of the flask in an epithelial manner. After addition of microcarrier adsorption culture, fig. 3 shows the state of MDCK adherent cells after 12 hours of adsorption in the flask, cells can still grow adsorbed to the bottom of the flask, and most of the cells have grown adsorbed to the microcarriers. After the trypsinization expansion, fig. 4 shows the state of MDCK adherent cells in the shake flask after the adsorption conditions and culture condition patterns, the cells can be uniformly dispersed on the surface of each vector and stably grow.

Example 2: microcarrier cultured MDCK cell harvesting and suspension domestication

1. Main reagents and consumables:

(ii) MDCK cells: vector MDCK cells in example 1.

② TransVA-01 culture medium: contains glutamine, prepared according to the specification, Shanghai duoning biological corporation.

③ 0.25% trypsin (liquid): GIBCO.

Fourthly, shaking the flask: shanghai duoning biology.

2. Main equipment and model:

name(s)	Company(s)	Type number
			Carbon dioxide shaking table	Shanghai duoning (medicine for treating Shanghai disease)	DNSI0101
Super clean bench	Suzhou Jingan	SW-CJ-2FD
			Cell counter	CountStar	IC1000
Centrifugal machine	Sigma	BBI-8570892

3. The process method comprises the following steps:

firstly, taking 200ml of cultured microcarrier cells, standing and settling for 10 minutes, and sucking and removing supernatant.

And washing the PBS for 5 times, if adding 300ml of PBS, shaking clockwise for 10 times, shaking anticlockwise for 10 times, standing for 10 minutes, sucking and discarding, and adding the PBS washed in the next round.

③ after the PBS cleaning solution is removed, 200ml of pancreatin is added, and the mixture is put into a culture condition mode for digestion for 2 hours.

Fourthly, the cell fluid is taken out, kept stand for 3 minutes and the cell supernatant is taken.

And fifthly, adding PBS or culture medium repeatedly for a plurality of times to wash the cells dropped from the carrier and collecting the supernatant.

Sixthly, putting the collected supernatant into a 50ml centrifuge tube, collecting at room temperature and 1000rpm for 5 minutes, and pouring the collected liquid to leave the cells.

Seventhly, adding an acclimatization culture medium to adjust the cell density to an interval of 20 × 105 cells/ml-30 × 105 cells/ml.

Culturing in a culture condition mode, sampling every day and observing the change of cell density, cell viability and cell diameter.

Ninthly, when the cell density exceeds 100 × 105cells/ml, carrying out subculture according to the density range of 10 × 105cells/ml to 20 × 105cells/ml, wherein the subculture medium is an acclimatization medium.

Repeatedly carrying out passage according to the method for many times until the cell doubling time is stable, the survival rate is more than 90 percent, and no mass agglomeration is considered to be the acclimation completion.

It should be noted that the standing time for taking the cell supernatant is not too long, otherwise the cells will settle and lose much, and experimental tests show that the settling time of the harvested cell supernatant with 3 minutes of settling is preferred.

Further, the number of repetitions in the examples was 3 to 8, and at least 3 times of the repeated repetitions were performed, and the cell mass was concentrated in the supernatant obtained by 3 to 5 times of washing.

Furthermore, when cell wall build-up and agglomeration occurred in the above experiment, the cell wall build-up and agglomeration were continued by centrifugation, followed by digestion with the same amount of trypsin for 1 hour, followed by centrifugation to adjust the cell density.

Further, when the cell viability is lower than 50% or the cell density is lower than 5 × 105cells/ml in the above experiment, the old culture medium and dead cells can be removed by centrifugation at low speed 800rpm for 5 minutes, and then fresh cell suspension is added again to adjust the cell density to the acclimation density range, and the culture is continued.

Furthermore, when cell growth stagnation occurs in the experiment, centrifugation is carried out at intervals of 48 hours, a fresh acclimation culture medium is added to adjust the cell density to be within the acclimation density range, and the culture is continued.

It should be noted that the "acclimatization medium" described in the above experiment is a mixed solution of transVA-01 and 0.025% pancreatin, and the "culture conditions" are that the rotation speed of a shaker with a wheelbase of 50mm is 110-130 rpm, the volume fraction of carbon dioxide is 5-8%, and the temperature is 36-38 ℃.

The results shown in fig. 6 show that the cells in the late stage of MDCK cell suspension acclimation can be stably passaged and multiplied, the survival rate is maintained above 90%, and the acclimation is successful. And a comparison graph of the first generation cell state when the MDCK adherent cells start suspension acclimation and the state after suspension acclimation is completed, which is shown in fig. 5, shows that the cell aggregation activity rate is decreased during early acclimation, and the cells are dispersed into a single cell suspension state after acclimation is successful and grow with a good activity rate all the time.

Example 3 trypsinization of MDCK cells

Firstly, a square bottle digestion process: taking a bottle of T125 MDCK cells (cultured by 10% FBS DMEM), removing old culture solution, washing 3-5 times by PBS (20 ml of washing volume each time), removing washing solution, adding 5ml of pancreatin, digesting for 10 minutes at 37 ℃, removing digestive juice, slightly beating the square bottle up and down, allowing residual digestive juice to permeate into each cell, continuing to digest for 3 minutes at 37 ℃ until the bottom of the square bottle is slightly tapped, uniformly dropping the cells, adding a proper amount of fresh culture medium, and blowing and suspending to prepare cell suspension for later use.

② a carrier digestion process: taking a bottle of 125ml shake flask microcarrier MDCK cells, and standing for 10 minutes; adding PBS for washing for 5 times (30 ml washing volume each time, standing for 10 minutes and removing by suction), removing the washing liquid, adding 20ml pancreatin, and digesting for 2 hours under the culture condition; standing for 3 min after digestion is completed, taking the digested supernatant, and repeating for multiple times (3-8 times, wherein each time of washing is performed by clockwise rotation for 10 times and anticlockwise rotation for 10 times); the digested supernatant was collected in a 50ml centrifuge tube at 1000rpm for 5 minutes at room temperature, the supernatant was discarded, and fresh culture medium was added and blown into a cell suspension for use.

③ suspension digestion process: : taking a bottle of 125ml MDCK cells cultured in a shake flask in a suspension manner, collecting the cells under the centrifugal condition of 1000rpm at room temperature for 5 minutes, adding 20ml of pancreatin, removing the pancreatin under the centrifugal condition after the cells are digested for 1 hour under the culture condition, adding a fresh culture medium, and blowing and suspending the cells to prepare a cell suspension for later use.

FIG. 2 shows that the cell suspension obtained after two times of pancreatin digestion has good state and better cell dispersion.

Example 4 Carrier adsorption of MDCK cells

Firstly, a square bottle adsorption process: and adding the cell suspension after passage and 20ml of microcarrier culture medium (containing microcarrier) into a T125 square bottle, placing the square bottle on a side shaking table, and placing the square bottle and the side shaking table together into a carbon dioxide incubator to culture for 4-12 hours by slow shaking.

Optimized, and the adsorption culture is optimal for 12 hours.

The shaking bottle adsorption process comprises the following steps: culturing the cell suspension after passage and a new microcarrier culture medium under an adsorption condition mode, and then switching to a culture condition mode for culturing, wherein the adsorption condition mode is that the rotating speed of a shaking table with the axle base of 50mm is 80-100 rpm, the volume fraction of carbon dioxide is 5-8%, and the temperature is 36-38 ℃.

Example 5cell cryopreservation

Preparing a freezing storage culture medium: 9ml of TransVA-01+1ml of pancreatin.

Preparing a freezing solution: 9ml of the frozen medium +1ml of DMSO.

Collecting cells: the cells to be centrifuged were calculated according to the freezing density of 200X 105cells/ml, centrifuged at 1000rpm for 5 minutes at room temperature, and the supernatant was discarded.

And fourthly, resuspending the cells by using the freezing medium, and transferring the cells into a freezing tube with the volume of 1.5ml per tube.

Fifthly, the freezing tube is placed into a program freezing box after being turned upside down and mixed evenly, and is directly placed into a freezing box for freezing overnight at the temperature of minus 80 ℃.

Sixthly, after 24 hours, the cells are transferred into liquid nitrogen for long-term storage.

Further, the frozen seeds were revived every half year to check viability to determine the optimal shelf life of the cell line.

Example 6 cell recovery

Firstly, preheating a water bath tank until the water temperature is adjusted to 37-39 ℃.

② the cells are taken out of the liquid nitrogen.

And thirdly, rapidly transferring the cell to warm water, and stirring and melting the cell for 2 minutes until the cell is completely melted.

And fourthly, taking out all the frozen cells and adding the frozen cells into 10ml of fresh culture medium.

Fifthly, centrifuging at room temperature of 800rpm for 5 minutes, discarding supernatant, re-suspending cells by using 10-20ml of fresh culture medium, and adjusting the cell density to be within the range of 10 × 105 cells/ml-20 × 105 cells/ml.

Sixthly, putting the mixture into a shaking table with the axle distance of 50mm, setting the parameter rotating speed of the shaking table to be 110-130 rpm, the volume fraction of carbon dioxide to be 5-8 percent, and the temperature to be 36-38 ℃, and culturing for 24 hours for counting.

Seventhly, after the cell density is doubled, the cells are passaged with the density of 30 multiplied by 105cells/ml to 40 multiplied by 105cells/ml, and the passaging density is 5 multiplied by 105 cells/ml.

After 5 continuous passages, the cells can be subjected to experiments such as growth or virus inoculation.

The culture medium used for the recovered cell culture was TransVA-01 containing 1% by volume of trypsin.

The results in fig. 7 show that the cells recovered from the frozen MDCK suspension cells after successful suspension acclimation can recover normal multiplication after 3 rd to 4 th generations after recovery, the cell survival rate is always maintained above 95%, and the acclimated cell strain can be subjected to cell subculture normally and stably, so that the MDCK suspension cell strain is successfully established.

The foregoing shows and describes the general principles, principal features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are given by way of illustration of the principles of the present invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, and such changes and modifications are within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

1. A process for culturing and suspending domestication of MDCK cells by microcarriers is characterized in that: after the microcarrier cells prepared by the microcarrier culture method of the MDCK cells are collected, the collected microcarrier cells are acclimated by an MDCK cell suspension acclimation method.

2. The process for microcarrier culture and suspension acclimatization of MDCK cells according to claim 1, wherein the method for culturing the microcarrier of MDCK cells comprises the following steps:

1) pretreating a microcarrier, namely weighing 2-5 g/L of Cytodex series dry powder microcarrier, soaking the dry powder microcarrier for 4-18h by using PBS buffer solution with the pH of 7.4, washing the dry powder microcarrier twice by using PBS, sterilizing at high pressure, cooling and standing, washing the microcarrier for 2-3 times by using a microcarrier culture medium before use, and then fixing the volume in a microcarrier culture medium with 2% serum;

2) recovering MDCK seed cells, taking out the frozen MDCK cells from liquid nitrogen, rapidly stirring for 2 minutes in a water bath at 37-39 ℃ to recover the cells, and culturing the recovered cells in a T125 square bottle by using a cell culture solution of 10% FBSDMEM until the cells grow to be more than 90% of the growth surface of the T125 square bottle;

5) inoculating a microcarrier: preparing the MDCK cell suspension after passage for 5 times into cell suspension with the density of 1000 cells/ml, and adding a microcarrier culture medium with 2% serum;

3. The process for culturing and suspending domestication of MDCK cell microcarriers as claimed in claim 2, wherein: weighing Cytodex series dry powder microcarrier with the amount of 3g/L in the step 1); the maximum cell harvest amount in the step 8) is concentrated in the 3 rd to 5 th times of washing after digestion; and in the step 8), repeatedly adding sterile PBS to wash the digested microcarrier for multiple times, and centrifugally collecting cells in the supernatant to obtain the cells.

4. The process for culturing and suspending domestication of MDCK cell microcarriers as claimed in claim 2, wherein: the pancreatin digestion condition in the step 3) is that digestion is carried out for two times of incubation, the pancreatin is integrally soaked in cells for the first time, and the connection between the cells and the bottle bottom is separated; and (3) continuously incubating for 5-10 minutes after the flowing pancreatin is sucked and discarded for the second time, separating the cells from the bottle bottom at the target position, slightly beating the bottle bottom at the moment, separating the cells into pieces, falling and separating, and adding a fresh subculture medium to blow the uniform cells to obtain a uniformly dispersed cell suspension.

5. The process for culturing and suspension acclimatization of MDCK cells by microcarriers according to claim 1, wherein the method for suspension acclimatization of MDCK cells comprises the following steps:

1) obtaining primary suspension cells, standing and settling the collected microcarrier cells, and sucking and removing supernatant; washing for 3-8 times by using PBS buffer solution, adding pancreatin after absorbing and removing the PBS buffer solution, placing the mixture into a shaker for digestion in a culture mode, taking out digested cell fluid after digesting for 2 hours, taking cell supernatant after standing for 3 minutes, adding washing solution for 3-8 times to wash cells dropped from microcarriers and collect supernatant, putting the collected supernatant into a centrifuge tube, centrifuging, collecting at room temperature and 1000rpm for 5 minutes, and pouring the collected solution to leave the cells; adding cells into acclimatization culture medium, and adjusting cell density to 20 × 10 ⁵ cells/ml～30×10 ⁵ cells/ml, culturing in a culture mode, sampling every day and observing the change of cell density, cell viability and cell diameter; expanding the domesticated culture volume;

2) treating adherent walls and conglomerations in domestication, wherein cells can have serious conglomeration and wall-hanging phenomena within 2-3 days in a culture process, removing a domestication culture medium by centrifugation, and then treating for 2-3 hours in a culture mode by using pancreatin with the same volume; when the large cells are not digested and aggregated, the large cells are removed by a pipette and discarded, and the rest cells are collected and centrifuged according to the ratio of 10 multiplied by 10 ⁵ cells/ml～20×10 ⁵ One of cell/ml density passage treatment mode or liquid change treatment modeThe method is as follows;

3) in the domestication, the survival rate is reduced in the culture process, the density is too high, and a fresh domestication culture medium is added by adopting passage density; the occurrence rate of cell activity is less than 50% or the cell density is less than 5X 10 ⁵ cells/ml the old acclimation medium was removed by centrifugation at 800rpm for 5 minutes at low speed and fresh, digested healthy cells were added to adjust the cell density to 10X 10 ⁵ cells/ml～20×10 ⁵ continuously culturing cells/ml;

6. The process for culturing and suspending acclimatization of the MDCK cell microcarriers as claimed in claim 5, wherein: the culture mode is that the rotation speed of a shaking table with the axle distance of 50mm is 110-130 rpm, the volume fraction of carbon dioxide is 5-8%, and the temperature is 36-38 ℃.

7. The MDCK cell microcarrier culture and suspension acclimatization process according to claim 5, further comprising a cryopreservation and recovery method for the acclimatized cells, wherein the cryopreservation and recovery method for the acclimatized cells comprises the following steps:

(1) freezing and storing the domesticated cells according to the domesticated culture medium: DMSM ═ 9: 1 preparing a freezing medium according to a formula of 150X 10 ⁵ cells/ml～200×10 ⁵ Freezing and storing at cell/ml density, freezing and storing in a programmed freezing and storing box, and transferring into a liquid nitrogen tank after 24 hoursPreserving for a long time, and recovering one cell every half to 1 year to check the cell viability;

8. The process for culturing and suspension acclimatization of MDCK cell microcarriers according to claim 2 or 5, wherein the process comprises the following steps: the microcarrier culture medium comprises TransVA-01 and 1% -2% serum, wherein the serum is one of 1% -2% fetal calf serum or 1% -2% newborn calf serum; the domestication culture medium comprises a mixed solution of TransVA-01 and 0.025% pancreatin; the subculture medium comprises a transVA-01 and 0.025% pancreatin mixed solution.

9. Use of the MDCK cell microcarrier culture and suspension acclimatization process of claim 1 for acclimatizing MDCK cells.