CN117070451A - Subculture method of mesenchymal stem cells - Google Patents
Subculture method of mesenchymal stem cells Download PDFInfo
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Abstract
The invention discloses a subculture method of mesenchymal stem cells, and belongs to the technical field of stem cell culture. Firstly, placing a platform in a 37 ℃ cell incubator or a 35-37 ℃ temperature control box, adjusting the level of the platform by using a level gauge with the accuracy of 0.01-0.05mm/m, and placing a cell culture bottle or a cell factory inoculated with cells on the platform; then, cell adherence culture is carried out in a cell culture box or a temperature control box, and subsequent culture is carried out when the cell adherence rate reaches 90%. According to the invention, the influence of the adherent growth condition in the subculture of the mesenchymal stem cells on the cell culture efficiency is considered, so that the cell adherent culture condition is optimized, the cells can be uniformly distributed in an inoculated cell culture bottle or a cell factory, the cell adherent rate is greatly improved, the culture efficiency of the mesenchymal stem cells is further improved, and the technical bottleneck that the culture efficiency cannot be effectively improved by optimizing conditions such as a culture medium in the prior art is broken through.
Description
Technical Field
The invention relates to the technical field of stem cell culture, in particular to a subculture method of mesenchymal stem cells.
Background
Stem cells are cells with proliferation and differentiation potential, capable of producing highly differentiated functional cells, with the potential function of regenerating various tissues and organs and the human body, and are called "universal cells" in the medical community. At present, more than 6000 stem cell clinical tests are underway, and relate to 200 indications and 8 large system diseases, and successful case reports are presented in the fields of multiple systems such as respiratory system, nervous system, endocrine system, motor system, immune system, reproductive system, circulatory system, digestive system and the like. Therefore, how to perform efficient in vitro culture of stem cells under aseptic conditions is of great importance.
At present, in the in-vitro culture process of stem cells, the influence of conditions such as culture medium, culture temperature, culture time, liquid changing frequency and the like on the growth of the cells is generally considered, and the culture conditions are continuously optimized, so that the cell culture efficiency is improved. However, with continuous optimization of culture conditions, technical bottlenecks are encountered, and the degree of improvement in stem cell culture efficiency is limited. Therefore, there is an urgent need for technological breakthroughs to be able to improve stem cell culture efficiency again on the basis of optimized culture conditions.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides the following technical scheme.
The invention provides a subculture method of mesenchymal stem cells, which comprises the following steps:
firstly, placing a platform in a 37 ℃ cell incubator or a 35-37 ℃ temperature control box, adjusting the level of the platform by using a level meter with the accuracy of 0.01-0.05mm/m, and placing a cell culture bottle or a cell factory inoculated with cells on the platform; then, cell adherence culture is carried out in a cell incubator or a temperature control box, and subsequent culture is carried out when the cell adherence rate reaches 90%; the platform comprises a flat plate and 4-6Z-axis displacement tables with scales, wherein the displacement tables are connected with the lower surface of the flat plate, and the flatness of the flat plate is=2× (1+d/1000) mm, wherein d is the diagonal length of a working surface of the flat plate; when in use, the level instrument with the precision of 0.01-0.05mm/m is used for adjusting the Z-axis displacement table with scales to enable the flat plate to be horizontal, and a cell culture bottle or a cell factory inoculated with cells is directly placed on the flat plate.
Preferably, the Z-axis graduated displacement table has the length of 80-120mm, the width of 80-120mm, the moving amount of 10-40mm, the carrying capacity of 5-20kg and the moving regulation and control precision of 1mm.
Preferably, the plate has a size of 100×500mm.
Preferably, the flat plate is provided with 20-100 holes.
Preferably, the slab is a 00-grade granite slab.
Preferably, the temperature control box is prepared by adopting a fireproof heat preservation plate, a radiator and a heating plate are arranged on at least one panel of a box body of the temperature control box, a temperature probe and a temperature controller are further arranged on the temperature control box, and the temperature probe and the heating plate are respectively and electrically connected with the temperature controller.
Preferably, the control precision of the temperature controller is 0.05-0.2 ℃, and the temperature in the temperature control box is controlled to be 35-37 ℃; the heat conductivity coefficient of the fireproof heat-insulating plate is less than or equal to 0.050W/(m.k); the power of the heating plate is 50-200W; the length of the radiator is 50-150mm, the width of the radiator is 50-150mm, and the voltage of the radiator is 12-110V.
Preferably, the platform is placed in a temperature controlled box when the cell factory is greater than 20 layers or cells are cultured on a large scale, otherwise the platform is placed in a cell incubator.
The beneficial effects of the invention are as follows: the invention provides a subculture method of mesenchymal stem cells, which comprises the steps of firstly placing a platform in a 37 ℃ cell incubator or a 35-37 ℃ temperature control box, adjusting the level of the platform by using a level meter with the precision of 0.01-0.05mm/m, and placing a cell culture bottle or a cell factory inoculated with cells on the platform; then, cell adherence culture is carried out in a cell culture box or a temperature control box, and subsequent culture is carried out when the cell adherence rate reaches 90%. By using a platform to place a cell culture bottle or a cell factory and adjusting the horizontal state of the platform to achieve higher levelness, the method optimizes finenessThe conditions in cell adherence culture enable cells to be uniformly distributed in a cell culture bottle or a cell factory, the cell adherence rate is greatly improved, and then the cell culture efficiency is improved. Experiments prove that the method provided by the invention can be used for increasing the yield of cells per T175 bottle from 1X 10 7 The cell/bottle is increased to about 1.4X10 7 About cell/bottle, 10 layers of cell factory yield is changed from 3.1X10 8 Cells/cell up to 5.1X10 8 Cell/cell, factory yield of 20 layers of large-scale cells is increased from 5.6X10 8 Cells/cell up to 9.1X10 8 The method breaks through the technical bottleneck that the culture efficiency cannot be effectively improved by optimizing conditions such as a culture medium in the prior art, and is suitable for large-scale cell factories and large-scale cell culture.
Drawings
FIG. 1 is a schematic flow chart of a method for subculturing mesenchymal stem cells according to the present invention;
FIG. 2 is a schematic view of a platform according to the present invention;
FIG. 3 is a schematic view of a temperature control box according to the present invention;
the meanings of the symbols in the figures are as follows: 1. platform, 2, control by temperature change case, 3, fire prevention heated board, 4, radiator, 5, hot plate, 6, temperature probe, 7, temperature controller, 8, dull and stereotyped, 9, Z axle take scale displacement platform, 10, probe connecting wire, 11, hot plate connecting wire, 12, hole.
Detailed Description
In order to better understand the above technical solutions, the following description will refer to the drawings and specific embodiments.
According to the definition of mesenchymal stem cells by the international cell therapy association (ISCT) in 2006, mesenchymal stem cells have three major characteristics: growing by adherence; cell surfaces express some specific antigens (markers); has the capacity of differentiating into adipocytes, osteoblasts and chondrocytes. Among them, the adherent growth is an important characteristic of the mesenchymal stem cells, and researches show that the adherent growth of the mesenchymal stem cells generally takes 24-48 hours (for example, patent application with the application number of 202210574631.9 and the name of a method for culturing and amplifying the mesenchymal stem cells in vitro in a large scale). It can be seen that adherent growth is the first step in the in vitro culture of mesenchymal stem cells.
In the research of the invention, when the mesenchymal stem cells are cultured by using the existing optimized culture conditions, the condition of cell wall-attached growth can influence the culture efficiency of the cells: firstly, the cell culture bottle or the cell factory is inclined to a certain extent when placed, so that higher levelness cannot be achieved, and the cultured cells are unevenly distributed in the culture bottle or the cell factory, so that the wall attaching efficiency of the cells is affected, the uniformity of the cell confluency is also not high, and the culture efficiency of the mesenchymal stem cells is further affected; secondly, the volume of the cell incubator commonly used in the laboratory at present is generally 180L or slightly more, and the volume of a 20-layer or 40-layer cell factory is relatively larger, so that the operation is troublesome when the cell incubator is put in or taken out. Since the cells are observed under a microscope during the cell attachment growth, the cells are observed under a microscope with a large scale cell factory or a large scale cell culture (10 9 ~10 10 ) During cell attachment growth, it is often carried out at room temperature (18-25 ℃) without being placed in an incubator. And after the adherent growth, placing the culture medium into a cell incubator for culture. Under the condition of room temperature, incomplete cell adhesion can occur, thereby affecting the cell culture efficiency.
Since the influence of cell wall-attached growth on cell culture efficiency in cell culture in vitro is not particularly focused in the prior art, the influence of cell wall-attached growth on cell culture efficiency has not been analyzed, nor has a method for improving cell wall-attached growth to improve cell culture efficiency been more specifically considered. The invention provides a subculture method of mesenchymal stem cells aiming at the cell wall-attaching growth process, wherein the first step is to adjust the levelness of a cell culture bottle or a cell factory, and the second step is to control the temperature in the cell wall-attaching process so as to ensure the effect of cell wall-attaching growth and further improve the culture efficiency of cells.
Specifically, as shown in fig. 1-3, the method for subculturing mesenchymal stem cells provided by the invention comprises the following steps: firstly, placing a platform 1 in a cell incubator or a temperature control box 2, adjusting the level of the platform 1 by using a level meter with the accuracy of 0.01-0.05mm/m, and placing a cell culture bottle or a cell factory inoculated with cells on the platform 1; then, cell adherence culture is carried out in a cell incubator or a temperature control box, and subsequent culture is carried out when the cell adherence rate reaches 90%; the platform 1 comprises a flat plate 8 and 4-6Z-axis graduated displacement tables 9 connected with the lower surface of the flat plate 8, wherein the flatness of the flat plate 8 is=2× (1+d/1000) mm, and d is the diagonal length of the working surface of the flat plate 8; when in use, the flat plate 8 is adjusted to be horizontal by using a level instrument with the precision of 0.01-0.05mm/m through the Z-axis graduated displacement table 9, and a cell culture bottle or a cell factory inoculated with cells is directly placed on the flat plate 8.
In the above method, in order to enable the cell culture flask or the cell factory inoculated with cells to be in a relatively high level state, the cell culture flask or the cell factory inoculated with cells is placed on a flat plate, and the flat plate 8 is adjusted to be horizontal by using a level meter with an accuracy of 0.01 to 0.05mm/m to adjust the Z-axis graduated displacement table 9, thereby enabling the cell culture flask or the cell factory inoculated with cells to be in a horizontal state. Therefore, the cells can be distributed unevenly in a culture bottle or a cell factory, so that the cell wall-attached growth obtains a good effect, the cell confluency has high uniformity, and the mesenchymal stem cells have high culture efficiency. In the embodiment of the invention, a 00-grade granite flat plate with flatness=2× (1+d/1000) mm can be used, so that the flatness of the flat plate and the levelness adjusted by the Z-axis displacement table with scales can be ensured to meet the design requirement, and the flatness of the flat plate can be ensured to meet the requirement of the 00 grade for a long time because the granite is not easy to deform. So as to ensure that cells can carry out adherent growth on a flat plate with high flatness and ensure good adherent growth condition. The invention adopts the culture method of adjusting the level by adopting the platform, can effectively solve the problem that the levelness inside the cell incubator is not high, so that the culture flask or the cell factory is inclined to a certain extent when placed, the cells are unevenly distributed in the culture flask or the cell factory, and the cell wall-attached growth efficiency and the final culture efficiency are further affectedThe problem of the rate. The cell yield per T175 cell culture flask can be increased from 1.0X10 compared with the conventional culture method 7 Left and right are increased to 1.4X10 7 Left and right; the yield improvement effect of the cell factory is more remarkable, and the yield of the 10-layer cell factory can be improved from 3.1 multiplied by 10 8 Lifting to 5.1×10 8 And/or (c).
In addition, in the embodiment of the invention, the cell can be cultured by adhering to the wall by using an incubator or a temperature control box, namely, a cell culture bottle or a cell factory inoculated with the cell is firstly placed in a platform, and then the platform is placed in the incubator or the temperature control box. After the platform is leveled by a level gauge, the wall-attached culture is started under certain conditions, such as a constant temperature state of 35-37 ℃. In particular, when the cell factory is larger than 20 layers or the cells are cultured on a large scale (e.g., 10 9 ~10 10 ) When the platform is placed in the temperature control box, otherwise, the platform is placed in the incubator. Therefore, for cell factories or large-scale cell culture with more than 20 layers, the adherent culture can be carried out under the condition of 35-37 ℃ to obtain better culture effect. Therefore, by adopting the method of the invention, as the temperature control box can provide a constant temperature condition of 35-37 ℃ in a larger space, the cell can be maintained at the temperature of 35-37 ℃ for adherent growth after being inoculated, and the cell can be transferred to a proper cell culture condition for culture after being fully adherent. Thereby ensuring relatively high cell culture efficiency. The temperature control box and the platform are matched for use, so that the problem that the cell culture efficiency is affected due to incomplete adherence in cell culture of a large-size cell factory or a plurality of cell factories can be solved.
In the embodiment of the invention, the length and the width of the Z-axis graduated displacement table 9 are respectively 80-120mm, the movement amount is 10-40mm, the carrying capacity is 5-20kg, and the movement regulation and control precision is 1mm.
Wherein, the platform is placed in an incubator or a temperature control box, and the platform is used for cell wall-attached culture after being adjusted to be horizontal by using a level gauge with the precision of 0.01-0.05 mm/m. Therefore, the size (length and width) of the Z-axis graduated displacement table in the platform can be adaptively selected according to the size of the incubator or the temperature control box, so that the displacement table is convenient to take; the moving amount of the displacement table with the scales on the Z axis is required to be ensured to be adjustable to be horizontal, and the height required to be adjusted is not more than 40mm when a table top or a box body of the incubator is placed in a laboratory. The loading capacity is estimated on the basis of the total weight of the cell culture flask or cell factory used and the culture medium therein, and is generally in the range of 5-20kg. The accuracy requirement of mobile regulation is that the level gauge (accuracy requirement of level gauge: 0.01-0.05 mm/m) to be used can be regulated to the level.
In the embodiment of the invention, the size of the flat plate 8 is 100×500mm, the size of the flat plate can be selected according to the size of the incubator or the temperature control box, and the use is convenient. The plate is provided with 20-100 holes 12. The cell culture bottle or the cell factory on the flat plate can be guaranteed to be consistent with the temperature in the temperature control box by a certain number of openings, and the influence of the flat plate on the temperature of the cell culture bottle or the cell factory can be reduced as much as possible.
In the embodiment of the invention, the temperature control box 2 is prepared by adopting a fireproof heat preservation plate 3, a radiator 4 and a heating plate 5 are arranged on at least one panel of the temperature control box 2, the temperature control box 2 is also provided with a temperature probe 6 and a temperature controller 7, and the temperature probe 6 and the heating plate 5 are respectively and electrically connected with the temperature controller 7. During the use, can open the power of hot plate and radiator (such as fan) of control by temperature change case, use temperature probe 6 to detect the temperature in the control by temperature change case to in sending the temperature controller 7 through probe connecting wire 10, temperature controller 7 begins work or stop work according to control program through hot plate connecting wire 11 control hot plate 5, makes the temperature in the box stable in 35~37 ℃.
In this embodiment, the thermal conductivity of the fireproof insulation board 3 is less than or equal to 0.050W/(m·k). The fireproof heat-insulating board has good heat-insulating effect, and can ensure that the temperature in the temperature control box can be controlled within a set smaller range (such as 35-37 ℃). The power of the heating plate 5 is 50-200W, the heating efficiency is high, and the temperature of the temperature control box can be quickly increased to a set temperature (such as 37 ℃). The length and the width of the radiator are respectively 50-150mm, the voltage is 12-110V, the radiating efficiency is high, and the temperature of the temperature control box can be quickly reduced to a set temperature (such as 35 ℃). The control precision of the temperature controller is 0.05-0.2 ℃, the temperature of the temperature control box can be precisely controlled, when the temperature is lower than the set lower temperature limit (such as 35 ℃), the heating plate can be immediately started for heating, and when the temperature is higher than the set upper temperature limit (such as 37 ℃), the radiator can be immediately started for radiating.
The technical scheme provided by the invention is mainly aimed at the condition control of the cell subculture link, and achieves ideal cell subculture efficiency through the condition control during the adherence link which has influence on the cell culture, including the control of levelness, temperature and other conditions during the placement of a cell culture bottle. Specifically, the method of subculturing mesenchymal stem cells is described as follows:
1. mesenchymal stem cells (mesenchymal stem cells, MSCs) which can be used for passage, such as MSCs of the 2 nd generation (P2 generation) -10 th generation (P10 generation) with the culture confluence reaching 85-95 percent, or MSCs after liquid nitrogen freezing preservation and recovery are taken, and are inoculated into a cell culture bottle or a cell factory after cell counting. Wherein the cell culture fluid can be mesenchymal stem cell culture fluid commonly used in laboratory, such as UltraGRO TM Advanced+α -MEM, STEMPRO hMSC SFM, stemCell MesenCult ™, etc.
2. Shaking uniformly after inoculation, and then placing a cell culture flask or a cell factory in a cell culture box or a temperature control box for adherent culture. Firstly, placing a platform in a cell culture box or a temperature control box, using a level meter with the accuracy of 0.01-0.05mm/m to adjust the level of the platform, and then placing a cell culture bottle or a cell factory on the platform to ensure the levelness of the cell culture bottle or the cell factory so as to ensure that cells in the cell culture bottle or the cell factory are uniformly distributed.
3. In the process of cell wall-attaching culture in a cell culture box or a temperature control box, the temperature can be controlled at 35-37 ℃ to ensure that the cell wall-attaching rate in a cell culture bottle or a cell factory reaches 90 percent, and then the subsequent culture process is carried out. Thus, the flatness of a platform for placing a cell culture bottle or a cell factory in a cell culture box or a temperature control box and the temperature in the cell culture box or the temperature control box are controlled, so that the cell adherence rate can reach 90% or more, and the subsequent cell culture can be ensured to have higher efficiency.
4. In the subsequent culturing process after the cell adherence culturing, the adherence cells in the temperature control box can be transferred into the incubator (if the adherence process is carried out in the incubator, the culturing in the incubator is continued), and the following culturing conditions are adopted: 37+ -1deg.C, 5+ -0.5% CO 2 And saturated humidity, culturing for 3-4 days, and observing the cell confluency.
5. After meeting the requirements of cell confluence, adding a digestive enzyme solution in proportion (the digestive enzyme solution is obtained by uniformly mixing digestive enzyme and 0.9% sodium chloride injection solution according to the volume ratio of 1:6), wherein the digestive enzyme can be pancreatin including but not limited to animal derived pancreatin (Gibco 0.25% Trypsin-EDTA) and non-animal derived recombinase (GibcoTrypLE) TM Express)). Digestion is carried out for 10 to 15 minutes at 37+/-1 ℃, and digestion is stopped after cell shedding and rounding. Digestive terminating fluids include, but are not limited to, equal volumes of fetal bovine serum, equal volumes of supernatant, or double volumes of supernatant.
6. After digestion, the cells were resuspended by centrifugation and then examined for cell number and cell viability.
The method provided by the invention is used for subculturing mesenchymal stem cells, and experiments prove that the method has the following beneficial effects: in the case of cell wall-attached culture in incubator, the yield per T175 bottle can be adjusted from 1X 10 after level adjustment by using a level gauge with accuracy of 0.01-0.05mm/m, compared with the existing culture method 7 Left and right are increased to 1.4X10 7 About, the production of 10 layers of cells can be changed from 3.1X10 8 Lifting to 5.1×10 8 And/or (c). For larger cell factories or large scale cell cultures (e.g., 10 9 ~10 10 ) When the cell is subjected to wall-attached culture in the temperature control box, the temperature condition of 35-37 ℃ can be kept after the cell is inoculated, and the cell is transferred to the culture box for subsequent cell culture after being completely attached, so that the relatively high cell culture efficiency is ensured. The method can produce 20 layers of large-scale cells with factory yield of 5.6X10 8 Lifting to 9.1×10 8 And/or (c).
Experimental example 1 subculturing of umbilical cord mesenchymal stem cells (UC-MSCs) T175 cell culture flask.
Collecting P4 generation UC-MSCs cells with confluence degree reaching 85% -95%, counting the cells, and respectively adding 1.6X10 6 Cells/flask were inoculated into 6T 175 cell culture flasks. The culture solution is UltraGRO TM Advanced+α -MEM. After shaking, 3T 175 cell culture flasks were placed in cell incubator A, and another 3T 175 cell culture flasks were placed in cell incubator B (the culture conditions of both incubators were 37.+ -. 1 ℃, 5.+ -. 0.5% CO.) 2 And saturated humidity). Before culturing, the platform was placed in cell incubator A and leveled using a level gauge with an accuracy of 0.01-0.05 mm/m. Cell incubator B is a conventional cell incubator with a partition plate (the partition plate has a low levelness and a certain degree of inclination). After 3 days of culture, the cell confluency of each flask was observed, the number of cells was examined after digestion, centrifugation and resuspension, and the cell viability was calculated according to formula (1), and the results are shown in Table 1.
Cell viability (%) =number of viable cells/total number of cells×100% (1).
TABLE 1
| The method of the invention | Existing methods | |
| Cell yield | 1.4×10 7 Bottle(s) | 1.0×10 7 Bottle(s) |
| Confluence degree | 94.7% | 85.6% |
| Cell viability | 97.3% | 91.7% |
As can be seen from Table 1, the cell culture vessel with a platform having a high levelness in the method of the present invention can produce a cell yield of 1.0X10 cells per T175 bottle, relative to the cell culture vessel with a partition plate in the prior art method 7 Left and right are increased to 1.4X10 7 Left and right.
Experimental example 2 subculturing of umbilical cord mesenchymal stem cells (UC-MSCs) 10-layer cell factories.
Collecting P4 generation UC-MSCs cells with confluence degree reaching 85% -95%, counting the cells, and respectively adding 5.7X10 s 7 Cells/cell factories were seeded into 2 10-layer cell factories. The culture solution is UltraGRO TM Advanced+α -MEM. After shaking up, 1 cell factory was placed in cell incubator A, and another 1 cell factory was placed in cell incubator B (the culture conditions of both incubators were 37.+ -. 1 ℃, 5.+ -. 0.5% CO 2 And saturated humidity). Before culturing, the platform was placed in cell incubator A and leveled using a level gauge with an accuracy of 0.01-0.05 mm/m. Cell incubator B is a conventional cell incubator with a partition plate (the partition plate has a low levelness and a certain degree of inclination). After culturing for 3 to 4 days, the cell confluence of each cell factory was observed, the cell number was detected after digestion, centrifugation and resuspension, and the cell viability was calculated according to formula (1), and the results are shown in Table 2.
As can be seen from Table 2, the cell incubator with a platform having a high levelness in the method of the present invention can produce a 10-layer cell factory of 3.1X10-th yield, relative to the cell incubator with a partition plate in the conventional method 8 Lifting to 5.1×10 8 And/or (c).
TABLE 2
| The method of the invention | Existing methods | |
| Cell yield | 5.1×10 8 Number/number | 3.1×10 8 Number/number |
| Confluence degree | 94.1% | 86.5% |
| Cell viability | 96.5% | 91.1% |
Experimental example 3 temperature condition control in the adherent culture was performed on the cell factory.
The platform was placed in a cell incubator and adjusted to horizontal using a level gauge with an accuracy of 0.01-0.05 mm/m. Collecting P4 generation UC-MSCs cells with confluence degree reaching 85% -95%, counting the cells, and then adding the cells with the concentration of 5.7X10 7 Inoculating cells into 10-layer cell factory with UltraGRO culture solution TM Advanced+α -MEM. And placing the 10-layer cell factory after inoculation on a platform which is regulated to be horizontal, controlling the temperature of 35-37 ℃ at time points of 2 hours, 4 hours and 6 hours respectively, observing the cell adherence growth condition, and calculating the cell adherence rate according to a formula (2), wherein the result is shown in a table 3.
Cell attachment rate (%) =number of attached cells/number of inoculated cells×100% (2).
TABLE 3 Table 3
| The time is 35-37 DEG C | 2hr | 4hr | 6hr |
| Wall sticking rate | 91.3% | 95.2% | 94.4% |
As can be seen from Table 3, the temperature conditions were controlled (35 to 37 ℃ C.) so as to promote cell adhesion to 90% or more. The cell attachment efficiency (< 80%) was significantly improved over 2-6 hours at room temperature.
Experimental example 4 cell culture in larger-scale cell factories.
Collecting P4 generation UC-MSCs cells with confluence degree reaching 85% -95%, counting the cells, and respectively according to 1.14X10 8 Inoculating density of cell factory, culture solution volume of 4L/cell factory to 2 20-layer cell factories, culture solution being UltraGRO TM Advanced+α -MEM. After shaking, 1 cell factory was placed in a temperature-controlled box for cell wall-attaching culture, and the other 1 cell factory was placed in a cell incubator for cell wall-attaching culture. Before the adherent culture, a platform is placed in a temperature control box, and a level gauge with the precision of 0.01-0.05mm/m is used for adjusting the level. The cell incubator is a conventional incubator with a partition plate (the partition plate has low levelness and a certain inclination)Cell incubator.
In a temperature control box, the temperature is controlled at 35-37 ℃ for adherence culture, and after 20 layers of cells in a cell factory are ensured to be adhered (the adherence rate is more than 90%), the cells are transferred into a cell culture box with proper volume for subsequent culture process.
In the cell incubator, cell attachment culture and subsequent culture were performed according to the conventional method (no leveling of the partition plates in the incubator).
The subsequent culture conditions for 2 20-layer cell factories were: 37+ -1deg.C, 5+ -0.5% CO 2 Saturated humidity. After culturing for 3 to 4 days, the cell confluence of each cell factory was observed, the cell number was detected after digestion, centrifugation and resuspension, the cell attachment rate was calculated according to formula (2), the cell activation rate was calculated according to formula (1), and the results are shown in Table 4.
TABLE 4 Table 4
| The method of the invention | Existing methods | |
| Cell attachment rate | 96.3% | - |
| Confluence degree | More than 95% | Non-uniformity, local greater than 95% and local less than 50% |
| Cell viability | 95.2% | 91.1% |
| Cell number | 9.1×10 8 Number/number | 5.6×10 8 Number/number |
As can be seen from Table 4, according to the method of the present invention, the cell attachment rate was 96.3% by adjusting the levelness of the stage and controlling the temperature during the cell attachment culture stage (when the conventional method was used for culturing, it was not possible to observe the cell attachment rate due to the inconvenience of taking and placing 20 layers of cells in the incubator). The cell culture efficiency is also significantly improved (for example, the cell number is significantly improved, and the cell culture is 5.6X10 by the existing method 8 The cell/cell increase was 9.1X10 8 Cell/cell, while achievable cell confluence and cell viability are also improved).
Experimental research shows that the method provided by the invention is suitable for large-scale cell culture (such as 10 9 ~10 10 Individual cells are cultured), the cell attachment rate and the cell culture efficiency can be improved. Examples are not listed here.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention. It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (8)
1. A method for subculturing mesenchymal stem cells, comprising:
firstly, placing a platform in a 37 ℃ cell incubator or a 35-37 ℃ temperature control box, adjusting the level of the platform by using a level meter with the accuracy of 0.01-0.05mm/m, and placing a cell culture bottle or a cell factory inoculated with cells on the platform; then, cell adherence culture is carried out in a cell incubator or a temperature control box, and subsequent culture is carried out when the cell adherence rate reaches 90%; the platform comprises a flat plate and 4-6Z-axis displacement tables with scales, wherein the displacement tables are connected with the lower surface of the flat plate, and the flatness of the flat plate is=2× (1+d/1000) mm, wherein d is the diagonal length of a working surface of the flat plate; when in use, the level instrument with the precision of 0.01-0.05mm/m is used for adjusting the Z-axis displacement table with scales to enable the flat plate to be horizontal, and a cell culture bottle or a cell factory inoculated with cells is directly placed on the flat plate.
2. The method for subculturing mesenchymal stem cells of claim 1, wherein the Z-axis graduated displacement table has a length of 80-120mm, a width of 80-120mm, a movement amount of 10-40mm, a load capacity of 5-20kg and a movement regulation accuracy of 1mm.
3. The method for subculturing mesenchymal stem cells of claim 1, wherein the plate has a size of 100 x 500mm.
4. The method for subculturing mesenchymal stem cells of claim 1, wherein the plate is provided with 20 to 100 holes.
5. The method of claim 1, wherein the plate is a 00-grade granite plate.
6. The method for subculturing mesenchymal stem cells of claim 1, wherein the temperature control box is prepared by using a fireproof insulation board, a radiator and a heating plate are arranged on at least one panel of a box body of the temperature control box, the temperature control box is further provided with a temperature probe and a temperature controller, and the temperature probe and the heating plate are respectively and electrically connected with the temperature controller.
7. The method for subculturing mesenchymal stem cells of claim 6, wherein the control accuracy of the temperature controller is 0.05-0.2 ℃, and the temperature in the temperature control box is controlled to be 35-37 ℃; the heat conductivity coefficient of the fireproof heat-insulating plate is less than or equal to 0.050W/(m.k); the power of the heating plate is 50-200W; the length of the radiator is 50-150mm, the width of the radiator is 50-150mm, and the voltage of the radiator is 12-110V.
8. The method of claim 1, wherein the platform is placed in a temperature-controlled box when the cell factory is larger than 20 layers or cells are cultured on a large scale, and otherwise the platform is placed in a cell incubator.
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