CN118126928A

CN118126928A - Microcarrier culture method of adherent cells

Info

Publication number: CN118126928A
Application number: CN202410135283.4A
Authority: CN
Inventors: 穆光慧; 赖月辉; 陈坚; 张东卓; 江梦雅; 李复坤; 涂玉蓉; 林德锐; 杨傲冰; 齐冬梅; 周晓敏
Original assignee: Guangdong Winsun Biopharmaceuticals Co ltd
Current assignee: Guangdong Winsun Biopharmaceuticals Co ltd
Priority date: 2024-01-31
Filing date: 2024-01-31
Publication date: 2024-06-04

Abstract

The invention belongs to the field of biology, and discloses a microcarrier culture method of adherent cells, which comprises the steps of mixing the adherent cells and microcarriers, and then carrying out flow adherent culture, semi-flow semi-suspension culture or flow suspension culture in a rotary bottle; the rotating speed of the rotating bottle for the flowing adherence culture is lower than that of the rotating bottle for the semi-flowing semi-suspension culture; the rotating speed of the semi-flow semi-suspension culture by adopting the rotating bottle is smaller than that of the semi-flow semi-suspension culture by adopting the rotating bottle. The method utilizes the microcarrier culture method to carry out microcarrier flow adherence culture, semi-suspension semi-flow culture or flow suspension culture, and the culture mode is different from the current general suspension culture and semi-suspension semi-stop culture, thus being an innovative invention on the microcarrier culture process, and the method has the advantages of low shearing force during culture, small cell damage and good culture effect. And the cost of the bottle rotating machine and the bottle rotating machine is lower than that of other modes.

Description

Microcarrier culture method of adherent cells

Technical Field

The invention relates to the field of biology, in particular to a microcarrier culture method of adherent cells.

Background

Cytodex1 microcarriers (GE company in the United states) are microspheres with dextran as the matrix, the surface of which can provide 2D spherical adherent growth space for cells, and 1g of Cytodex1 microcarriers can provide a surface area of about 4400cm ², and because of the special optimization treatment on the surface, the adhesion and the expansion of cells are facilitated, so that Cytodex1 is suitable for almost all adherent cells. The usual culture mode of the microcarrier is constant-speed suspension culture.

At present, in the laboratory stage, the 2D spherical microcarrier culture of adherent cells is usually carried out by using a siliconized cell shake flask and a magnetic rotating flask for constant-speed full suspension culture. The culture principle of the cell shake flask is that the cell shake flask is cultured in a shaking mode of a shaking table, the culture principle of the magnetic rotary flask is that the cell shake flask is cultured in a stirring mode through built-in suspended magnetic beads, the culture cost of the shaking mode is low, the culture effect is poor, the culture effect of the stirring mode is good, and the bottle and a rotating instrument thereof are expensive. In the production stage, bioreactors are used for culturing, the bioreactors mainly comprise mechanical stirring bioreactors and wave bioreactors, the mechanical stirring bioreactors use screw stirring paddles for shearing and stirring to suspend the microcarriers, and the wave bioreactors use disposable reaction bags to enable the microcarriers to incline or suspend left and right through swinging.

Microcarrier culture of adherent cells at home and abroad has been widely applied in the direction of human medicines or veterinary biologicals, but has no great application prospect in the direction of veterinary biologicals in China, and the reason is probably that the comprehensive cost of materials, instruments and equipment is higher, and the microcarrier culture is not suitable for the current veterinary biologicals market in China.

The technical problem that the present case solves is: how to provide a culture mode of adherent cells with lower cost.

Disclosure of Invention

The invention aims to provide a microcarrier culture method of adherent cells, which utilizes the microcarrier culture method to carry out microcarrier flow adherent culture, semi-suspension semi-flow culture or flow suspension culture, and the culture mode is different from the current general suspension culture and semi-suspension semi-stop culture, thus being an innovation invention in microcarrier culture process, having low shearing force during culture, small cell damage and good culture effect. And the cost of the bottle rotating machine and the bottle rotating machine is lower than that of other modes.

In order to achieve the above purpose, the present invention provides a microcarrier culture method for adherent cells, wherein the adherent cells and microcarriers are mixed and then subjected to flow adherent culture, semi-flow semi-suspension culture or flow suspension culture in a roller bottle;

The rotating speed of the rotating bottle for the flowing adherence culture is lower than that of the rotating bottle for the semi-flowing semi-suspension culture; the rotating speed of the semi-flow semi-suspension culture by adopting the rotating bottle is lower than that of the semi-flow semi-suspension culture by adopting the rotating bottle;

The rotary bottle comprises a bottle body;

the inner wall of the bottle body is provided with a plurality of raised strips raised along the inner wall, and two adjacent raised strips form a groove extending along the axial direction of the bottle body;

In the direction vertical to the axis of the bottle body, the section of the inner wall of the bottle body is unfolded to form a continuous undulating curve.

In the microcarrier culture method, the number of grooves is 10-40, and the depth of the grooves is 5-15% of the radius of the bottle body.

In the microcarrier culture method, the number of grooves is 15-30, and the depth of the grooves is 8-12% of the radius of the bottle body.

In the microcarrier culture method, the raised strips are formed by inwards sinking the walls of the bottle body, and/or the surfaces of the raised strips are cambered surfaces; and/or the bottom surface of the groove is a plane or an arc surface.

In the microcarrier culture method, the cross section of the raised line is semicircular or semi-elliptic.

In the microcarrier culture method, the bottle body comprises a bottle body, a bottle neck connected with the bottle body and a bottle mouth connected with the bottle neck; the groove extends to the bottleneck position, and the groove extends to the bottleneck position close to the bottleneck.

In the microcarrier culture method, the bottle body is made of glass.

In the microcarrier culture method, the adherent cells and the microcarriers are uniformly mixed to a roller bottle, culture solution is added, and the roller bottle is placed on a roller bottle machine for culture.

In the microcarrier culture method, the concentration of the microcarrier in the solution after the nutrient solution is added in the step2 is 1.25 g/L-3.75 g/L.

In the microcarrier culture method, the adherent cells are one of Vero cells, ST cells and PK-15 cells.

Because the rotating speed requirements of the bottles with different specifications are different, taking the bottle body 1 disclosed by the invention as an example, the rotating speed of the bottle body for carrying out flow adherence culture is 60-90 r/h; the rotating speed of the semi-flow and semi-suspension culture by adopting a rotating bottle is 90-270 r/h; the rotating speed of the rotary bottle for the flow suspension culture is 270-800 r/h.

The rotational speed should be flexibly adjusted by those skilled in the art according to the actual bottle specifications to present the desired flow pattern.

In the microcarrier culture method, the volume of the solution in the roller bottle is 8-12% of the volume of the roller bottle.

The beneficial effects of the invention are as follows:

The microcarrier culture method is used for microcarrier flow adherence culture, semi-suspension semi-flow culture or flow suspension culture, the culture mode is different from the current general suspension culture and semi-suspension semi-stop culture, the microcarrier culture method is an innovative invention on the microcarrier culture process, the shearing force is low during culture, the damage to cells is small, and the culture effect is good. And the cost of the bottle rotating machine and the bottle rotating machine is lower than that of other modes.

Drawings

Fig. 1A is a perspective view of a bottle 1 of a first part of the present invention;

fig. 1B is a front view of a bottle 1 of the first part of the present invention;

fig. 2 is a cross-sectional view perpendicular to the axis of the bottle body 1 of the first part of the present invention;

Fig. 3 is a schematic cross-sectional view of the inner wall of the bottle 1 of the first part of the present invention;

fig. 4 is a schematic cross-sectional view of the inner wall of the bottle body 2 of the first part of the present invention;

fig. 5 is a schematic cross-sectional view of the inner wall of the bottle body 3 of the first part of the present invention;

fig. 6 is a schematic cross-sectional view of the inner wall of the bottle body 4 of the first part of the present invention;

FIG. 7 is a schematic cross-sectional view of the inner wall of the bottle 5 of the first part of the present invention;

FIG. 8 is a schematic diagram of cell culture in a straight-walled roller bottle of the first section of the present invention;

FIG. 9 is a schematic diagram of the cultivation of the bottle body 1 of the second part of the present invention at different rotational speeds;

FIG. 10 is a state diagram of the third part of the first day of Vero cells of the invention under flow suspension culture conditions;

FIG. 11 is a state diagram of the third part of the Vero cells of the invention on the next day under flow suspension culture conditions;

FIG. 12 is a state diagram of the third part of the Vero cells of the invention on the third day under the conditions of flow suspension culture.

Detailed Description

The following describes the technical scheme of the invention in further detail, but does not limit the invention in any way.

Performance verification of first part vials in microcarrier culture

Referring to fig. 1-2, the embodiment provides 4 kinds of specifications of bottles and one kind of specification of comparison bottles, wherein the different specifications of bottles are obtained by casting high borosilicate glass materials through a mold;

the concrete structure comprises: comprises a bottle body; the inner wall of the bottle body is provided with a plurality of raised strips 1 raised along the inner wall, and two adjacent raised strips 1 form a groove 2 extending along the axial direction of the bottle body; in the direction vertical to the axis of the bottle body, the section of the inner wall of the bottle body is unfolded to form a continuous undulating curve.

The bottle body specifically comprises a bottle body, a bottle neck 3 connected with the bottle body and a bottle opening 4 connected with the bottle neck.

The diameter of the bottle bottom of each specification bottle body is about 11.5cm, the height of the bottle body is about 26cm, and the volume is about 2L; the main differences are the number, specification, etc. of grooves, and the specific differences are shown in table 1 below;

TABLE 1 bottle Specification Table

Numbering device	Number of raised strips	Number of grooves	Diameter of convex strip is mm	Drawings
					Bottle body 1	16	16	10	3
Bottle body 2	40	40	5.5	4
					Bottle body 3	10	10	16.5	5
Bottle body 4	15	15	13.2	6

Comparison bottle body

Substantially identical to the bottle 1, except for the following dimensional specifications, with reference to fig. 7:

The number of the raised strips is 4, the diameter of the raised strips is 10mm, and the number is named as bottle body 5.

The microcarrier cell culture is carried out by adopting the bottle body 1, the bottle body 5 and the cell shake flask and the disposable reaction flask with the built-in impeller, and the specific steps are as follows:

1) Pretreatment of Cytodex1 (GE Co., USA) microcarriers:

Pretreatment of Cytodex1 microcarriers: weighing 10gCytodex pieces of microcarrier, soaking in 400ml of PBS buffer solution with pH of 7.2 for 3 hours at room temperature, naturally settling, discarding supernatant, adding 200ml of PBS again, naturally settling after resuspension, discarding supernatant, adding 200ml of PBS again, sterilizing at 121 ℃ under high pressure for 30 minutes by steam, standing and cooling, absorbing PBS, adding 200ml of DMEM culture solution containing 10% new born calf serum again, and storing in a refrigerator at 4 ℃ for later use. The volume of the microcarrier precipitate after hydration was about 20ml/g microcarrier.

2) Passage of Vero cells to bottle 1

Four bottles of 80% -95% confluent layer or thin monolayer Vero cells (T175 cell bottle) are taken, cell culture solution is discarded, PBS is used for washing once, 5ml of pancreatin is added for digestion, after digestion is finished, digestion solution is discarded, 5ml of DMEM culture solution containing 10% new born calf serum is taken, after being blown out and resuspended, 100ul of cell suspension is taken and added with 100ul of 0.1% trypan blue staining solution, 20ul of cell suspension is absorbed on a cell counting plate after uniform mixing, cell counting is carried out on a Countstar cell counter, 2x10 ⁷ cells are respectively taken after counting and added into a siliconized sterile bottle body 1, 10ml of microcarriers processed in step 1 are respectively poured into the centrifuge tube, finally 200ml of culture solution is added, shaking and mixing are carried out, and then the mixture is transferred to a cell culture bottle rotating machine for culture, one is rotated and cultured at 300r/h and 37 ℃, and the other is rotated and cultured at 600r/h and 37 ℃. Samples were taken daily to observe the growth of cells on microcarriers and to adjust the pH around neutrality to avoid peracid or overbase.

Results: the flow suspension culture was performed by using the cell culture method recommended by the official authority of Cytodex1 (the microcarrier amount was 1.25g/L, the cell seeding amount was 20 cells/microcarrier, the cell seeding amount of other microcarriers in this example was about 20 cells/microcarrier), the cell count of T175 cell bottles with nearly 80% confluence layer was 2.6x10 ⁶ cells/ml, vero cells in both sets of bottles could grow good cell monolayers on microcarriers within 3 days, and the growth rate and cell pellet uniformity were not significantly different, and all could grow good cell monolayers within about 3 days, indicating that the shear force damage of the arc column (convex bar) of the present invention to cells and microcarriers was not great.

In the suspension culture mode, the rotation speed of the microcarrier for culturing the adherent cells by using a shaking mode such as a cell culture shake flask is generally 90-110 r/min; the rotation speed of culturing the adherent cells by using a stirring mode such as a swing arm shake flask or a magnetic shake flask and the like for the microcarrier is generally 60-80 r/min; the bottle body 1 can achieve the purpose of suspension culture by using the rolling shaft rotating speed of 300r/h, and from the culture result, the cell and the microcarrier can be subjected to flowing suspension culture at a low rotating speed when being cultured along with the rotation of the wavy circular arc column after being uniformly mixed due to the large transverse area and the low height of the culture solution, so that a good uniform culture effect can be obtained without mechanical high-rotating-speed stirring.

3) Vero cells are passaged to cell shake flasks, disposable reaction flasks with built-in impellers and flask body 5

Taking 80% -95% confluent layer or thin monolayer Vero cells (T175 cell bottle), discarding cell culture solution, washing with PBS once, adding 5ml pancreatin to digest, discarding digestion solution after digestion, taking 5ml DMEM culture solution containing 10% new born calf serum per bottle, blowing and resuspension, collecting together, taking 100ul cell suspension, adding 100ul of 0.1% trypan blue staining solution, mixing uniformly, sucking 20ul onto a cell counting plate, performing cell counting on Countstar cell counter, respectively taking 5.4x10 ⁶ cells after counting, adding into siliconized cell shake flask and built-in impeller disposable reaction flask, pouring 2.5ml microcarriers processed in step 1 into cell shake flask and built-in impeller disposable reaction flask, finally respectively supplementing 50ml culture solution, shaking and mixing uniformly, placing cell shake flask for culturing, shaking at 90r/min, and 37 ℃ and 5% carbon dioxide; placing the built-in impeller disposable reaction bottle on a cell culture magnetic stirrer for culture, wherein the speed is 70r/min, the temperature is 37 ℃, and the carbon dioxide is 5%; adding 2x10 ⁷ cells into a siliconized sterilized bottle body 5, pouring 10ml microcarriers into a centrifuge tube, pouring into a bottle, supplementing 200ml culture solution, placing into a cell culture bottle rotating machine for culture, and rotating at 800r/h and 37 ℃; samples were taken daily to observe the growth of cells on microcarriers and to adjust the pH around neutrality to avoid peracid or overbase.

Results: the microcarrier is better in suspension culture by using a disposable reaction bottle with an internal impeller, the growth speed and the ball pasting uniformity of the microcarrier are not different from the effect of the bottle body 1 in flow suspension culture, the microcarrier and the bottle body grow into a good monolayer within 3 days, cells on each microcarrier grow uniformly, no obvious empty balls exist, and the cell uniformity is better. However, the culture effect of using the cell shake flask is poor, the cell shake flask is difficult to culture into a good ball-attached monolayer, the growth is easy to stop in the later period of culture, a large number of dead cells float in the culture solution, the stirring suspension or flowing suspension effect is difficult to culture even if repeated for several times, and a great amount of time is required for technological fumbling by using a shaking suspension mode to culture good cells possibly. The microcarrier of the bottle body 5 is in a state that a part of microcarrier is suspended and a part of microcarrier is subjected to flow adherence culture under the rotation speed of 800r/h, and the structure of a single convex strip cannot support the whole culture system to carry out flow suspension culture, so that cells on the part of microcarrier grow uniformly, cells on the part of microcarrier grow less, hemispherical cells or empty spheres exist, and the microcarrier with more good cell monolayers can be observed in 4 days as a whole.

In addition, the part adopts borosilicate straight-wall roller bottles to culture microcarriers, and the culture method comprises the following steps:

taking two bottles of 80% -95% confluent layer or thin monolayer Vero cells (T175 cell bottles), discarding cell culture solution, washing once with PBS, adding 5ml pancreatin for digestion, discarding digestion solution after digestion, taking 5ml DMEM culture solution containing 10% new born calf serum for blowing-off and resuspension for each bottle, collecting together, taking 100ul of cell suspension, adding 100ul of 0.1% trypan blue staining solution, uniformly mixing, absorbing 20ul onto a cell counting plate, performing cell counting on a Countstar cell counter, respectively taking 2x10 ⁷ cells after counting, adding the 2L borosilicate straight wall rotary bottles after the counting, pouring 10ml microcarriers processed in step 1 into the rotary bottles, finally respectively supplementing to 200ml culture solution, shaking and uniformly mixing, culturing on a cell culture rotary bottle machine, culturing at 800r/h and 37 ℃ and culturing at 300r/h and 37 ℃. Taking samples every day to observe the growth of cells on the microcarrier, and regulating the pH value to be about neutral, so as to avoid peracid or alkali.

Results: the microcarrier was not well suspended in a conventional straight-walled roller bottle, and it was observed that most microcarriers were grown under a flow-by-wall at 300r/h and most microcarriers were suspended in a limited range at 800r/h (see FIG. 8 for culture at two rotational speeds). The rotational speed of 800r/h is close to the highest rotational speed of a commercial cell culture bottle rotating machine, and in theory, the use of a very high rotational speed can also enable the straight wall bottle rotating to carry out full suspension culture, but the cost is high.

Meanwhile, the part also carries out digestion passage of Vero cells when the bottle body 1 carries out microcarrier flow suspension culture, and the operation is specifically as follows:

Taking bottle bodies 1 of Vero cells after being cultured on microcarriers for 72 hours, slowly tilting and pouring out culture solution after standing for 1 minute, then adding 30ml of 0.25% pancreatin, slowly tilting and pouring out pancreatin after rotating for a few minutes by hand, then adding 50ml of 0.25% pancreatin, slowly tilting and pouring out part of pancreatin after rotating for 2 minutes at 300r/h, continuously rotating and digesting for 2 minutes, and then forcefully whipping a few bottles for most cells to fall off, adding DMEM nutrient solution to terminate digestion, and distributing microcarriers and cell suspension according to the requirement of experiments at the moment.

Results: by using the bottle body 1, the microcarrier is precipitated at the bottleneck when being slowly inclined due to the arrangement of the curved bottleneck, so that the excellent separation effect of the microcarrier and the liquid is achieved, the operation during digestion is very convenient, and if the microcarrier is digested for passage, the digestion time is only required to be searched or the quantity and concentration of the digestive juice are optimized. In addition, operations such as discarding liquid, harvesting microcarriers, harvesting culture liquid, washing microcarriers during recovery and the like are very convenient. These advantages are also a disadvantage of other microcarrier culture vessels in digestion and passage or in collection of isolated microcarriers and supernatant.

In addition, the part also verifies the functions of other bottle bodies

The verification method is adopted to verify the microcarrier culture of the Vero cells, and the microcarrier culture mode is applicable to the bottle bodies 2-4;

Through experiments, the recommended rotation speed range can be referred to in table 2;

TABLE 2 adaptive rotational speed and culture Effect for different flasks

Culturing microcarrier of second part of Vero cells in bottle 1 at different rotation speeds

1) Pretreatment of Cytodex1 (GE Co., USA) microcarriers:

Pretreatment of Cytodex1 microcarriers: weighing 10gCytodex pieces of microcarrier, soaking in 400ml of PBS buffer solution with pH of 7.2 for 3 hours at room temperature, naturally settling, discarding supernatant, adding 200ml of PBS again, naturally settling after resuspension, discarding supernatant, adding 200ml of PBS again, sterilizing at 121 ℃ under high pressure for 30 minutes by steam, standing and cooling, absorbing PBS, adding 200ml of DMEM culture solution containing 10% new born calf serum again, and storing in a refrigerator at 4 ℃ for later use. The volume of the microcarrier after hydration was about 20ml/g microcarrier.

2) Passage of Vero cells to bottle 1

Taking 6 bottles of Vero cells (T175 cell bottles) with 80% confluence layers, discarding cell culture solutions, respectively washing with PBS once, respectively adding 5ml of pancreatin for digestion, respectively taking 10ml of DMEM culture solution containing 10% of new born calf serum after digestion, blowing off and re-suspending uniformly, taking 100ul of cell suspension, adding 100ul of trypan blue dye solution with 0.1% of cell suspension, uniformly mixing, absorbing 20ul of cell suspension onto a cell counting plate, performing cell counting on a Countstar cell counter, respectively taking 2x10 ⁷ cells after counting, adding the 2x10 cells into three sterile bottle bodies 1 with well siliconized surfaces in the example 2, respectively pouring 10ml of microcarriers processed in the step 1 into the bottle bodies 1, finally supplementing 200ml of culture solution, shaking and uniformly mixing, transferring to a cell culture bottle rotating machine for culture, and performing rotary culture at the temperature of 37 ℃; one set at 120r/h, rotation culture at 37 ℃; one set at 240r/h, and the culture was rotated at 37 ℃. Sampling every day to observe the growth condition of cells on the microcarrier, and regulating the pH value to be about neutral, so as to avoid peracid or alkali.

Results: the same microcarrier addition density and cell inoculation density were used to culture microcarriers of cells using flask 1 at low and medium speeds, and they were all capable of growing a good monolayer of cells, but at different growth rates and growth conditions. FIG. 9 shows a culture schematic diagram and a flow suspension culture schematic diagram under different rotation speeds;

The best culture effect is semi-flow semi-suspension culture close to flow suspension culture, and the comparison result of the culture effect of the disposable reaction flask with built-in impeller and the cell shake flask in section 3) of the first part and the culture effect of the first part is shown in Table 3.

TABLE 3 cultivation results under different cultivation conditions

Culturing Vero cells in bottle body 1 and disposable reaction bottle with built-in impeller at different microcarrier concentrations

1) Pretreatment of Cytodex1 (GE Co., USA) microcarriers:

2) Vero cells are passaged to bottle body 1 and disposable reaction bottle with built-in impeller

8 Bottles of Vero cells (T175 cell bottles) with 80% confluence layers are taken, cell culture solutions are discarded, PBS is used for washing once respectively, 5ml of pancreatin is added respectively for digestion, 10ml of DMEM culture solution containing 10% of new born calf serum is taken respectively for blowing off and re-suspending evenly after digestion, 100ul of trypan blue staining solution with the concentration of 0.1% is taken and added into 100ul of cell suspension, 20ul of the cell suspension is absorbed onto a cell counting plate after evenly mixing, and cell counting is carried out on a Countstar cell counter. Counting, adding 4.3x10 ⁷ cells into a siliconized sterile bottle body 1 in the embodiment 2, pouring 20ml of microcarriers treated in the step 1 into a rotary bottle by a centrifuge tube, adding 200ml of culture solution, shaking, mixing uniformly, transferring to a cell culture rotary bottle machine for culturing, and rotating at the temperature of 480r/h and 37 ℃; adding 2.15X10 ⁷ cells into a siliconized sterile bottle body 1, pouring 10ml of microcarriers treated in the step 1 into a centrifuge tube, pouring the microcarriers into the bottle body 1, supplementing 200ml of culture solution, shaking, mixing uniformly, transferring to a cell culture bottle rotating machine for culturing, and rotating at the temperature of 480r/h and 37 ℃. Sampling every day to observe the condition, regulating the pH value to be about neutral, avoiding peracid or alkali, and measuring the glucose content.

1.075X10 ⁷ cells and 5ml microcarrier suspension are taken and added into a disposable reaction bottle with an internal impeller, and are added into 50ml culture solution; 5.37X10 ⁶ cells and 2.5ml microcarrier suspension are taken and added into a disposable reaction bottle with an internal impeller, added into 50ml culture solution, and placed on a cell culture magnetic stirrer for culture at 70r/min and 37 ℃. Sampling every day to observe the condition, regulating the pH value to be about neutral, avoiding peracid or alkali, and measuring the glucose content.

The glucose content determination method comprises the following steps:

The method comprises the steps of using an SBA-40E biosensing analyzer to measure glucose content of a culture medium, standing a culture bottle for 1 minute, absorbing a small amount of supernatant, taking 40ul of supernatant, diluting 5 times with PBS, pressing a switch key by the biosensing analyzer in a long-term standby state, accurately absorbing 25ul of glucose standard solution by using a microinjector when a 'sample' lamp flashes, rapidly injecting the glucose standard solution into a reaction tank, continuously using the microinjector to accurately absorb 25ul of glucose standard solution when the 'sample' lamp flashes next time after reaction, repeatedly injecting the glucose standard solution by the method until the 'sample' lamp is always on, accurately absorbing 25ul of culture solution for 5 times of diluent for measurement after the 'sample' lamp is always on, repeatedly measuring three times each sample, taking an average value by the three times, and obtaining the glucose concentration of the culture solution, wherein the glucose concentration unit is mg/L.

Results: the two concentrations of 1.25g/L and 2.5g/L are used for carrying out the flowing suspension culture and the stirring suspension culture respectively, the glucose content is sampled, observed and measured every day, the culture observation results are not obviously different, the effect diagram of the flowing suspension culture in the embodiment is shown in fig. 10-12, the glucose consumption rate is not obviously different, the effect of the flowing suspension culture mode is not obviously different from that of the stirring culture mode, but the culture container and the culture instrument equipment of the flowing suspension culture are better in cost consideration.

Note that: the DMEM used in all examples of the invention contains 1.5g/L sodium bicarbonate, and the concentration is found out through experiments, and the bottle body 1 is used for culturing vero cells with different microcarrier concentrations by culturing without placing a carbon dioxide incubator, and only needs to adjust the pH value once in 48 hours or 24 hours.

Specific culture results are shown in Table 4;

TABLE 4 calculated values after measurement of glucose in culture at different concentrations in different modes

In conclusion, when the adherent cells are cultured in microcarriers, the flow suspension culture in the flow culture method can replace other suspension culture methods, and the culture effect is excellent. In addition, the new flow adherence culture and semi-flow semi-suspension culture modes can be selected according to the characteristics of different cells or different test requirements.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims

1. A microcarrier culture method of adherent cells is characterized in that the adherent cells and microcarriers are mixed and then subjected to flow adherent culture, semi-flow semi-suspension culture or flow suspension culture in a rotary bottle;

The rotary bottle comprises a bottle body;

2. The microcarrier culture method of claim 1, wherein the number of grooves is 10-40, and the depth of the grooves is 5% -15% of the radius of the bottle body.

3. The microcarrier culture method of claim 2, wherein the number of grooves is 15-30, and the depth of the grooves is 8% -12% of the radius of the bottle body.

4. The microcarrier culture method of claim 1, wherein the raised strips are formed by inward recessing of the walls of the bottle body and/or the raised strips have cambered surfaces; and/or the bottom surface of the groove is a plane or an arc surface.

5. The microcarrier culture method of claim 4, wherein the cross section of the ridge is semicircular or semi-elliptical.

6. The microcarrier culture method of claim 1, wherein the bottle body comprises a bottle body, a bottle neck connected with the bottle body, and a bottle mouth connected to the bottle neck; the groove extends to the bottleneck position, and the groove extends to the bottleneck position close to the bottleneck.

7. The microcarrier culture method of claim 1, wherein the bottle is made of glass.

8. The method according to any one of claims 1 to 7, wherein the adherent cells and the microcarriers are mixed uniformly to a roller bottle, the culture solution is supplemented, and the roller bottle is placed on a roller bottle machine for culturing.

9. The method according to claim 8, wherein the concentration of the microcarrier in the solution after the nutrient solution is added in step 2 is 1.25g/L to 3.75g/L.

10. The microcarrier culture method of any one of claims 1-9, wherein the adherent cells are Vero cells.

11. A roller bottle according to any one of claims 1 to 7, wherein the volume of solution in the roller bottle corresponds to 8 to 12% of the roller bottle volume.