CN114763524A - Subculture method and application of cells - Google Patents

Subculture method and application of cells Download PDF

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CN114763524A
CN114763524A CN202110529912.8A CN202110529912A CN114763524A CN 114763524 A CN114763524 A CN 114763524A CN 202110529912 A CN202110529912 A CN 202110529912A CN 114763524 A CN114763524 A CN 114763524A
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cells
subculturing
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朱灏
吴丹枫
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Dassel Shanghai Life Technology Co ltd
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    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06Animal cells or tissues; Human cells or tissues
    • C12N5/0602Vertebrate cells
    • C12N5/0652Cells of skeletal and connective tissues; Mesenchyme
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    • C12N2501/00Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/15Transforming growth factor beta (TGF-β)

Abstract

The invention belongs to the technical field of cell culture, and particularly relates to a subculture method of cells. The subculture method of the cells can maintain the proliferation capacity when the human umbilical cord mesenchymal stem cells of the generations P1-P25 are cultured; the method comprises the following steps: (1) centrifuging; (2) cleaning; (3) digesting; (4) washing; (5) harvesting and counting; (6) and (4) inoculating the cells. The invention selects specific culture medium and recombinase to ensure that the cells after subculture have good morphology and higher proliferation capacity and survival rate.

Description

Subculture method and application of cells
Technical Field
The invention belongs to the technical field of cell culture, and particularly relates to a subculture method and application of cells.
Background
The mesenchymal stem cell is derived from mesoderm, is an adult stem cell with self-renewal and multidirectional differentiation potential, and mainly has two characteristics of self-renewal and differentiation potential.
Mesenchymal Stem Cells (MSCs) are adult stem cells derived from the mesodermal non-hematopoietic lineage. MSCs were originally isolated from bone marrow, but as research progresses, MSCs were isolated from other tissues of the human body, including fat, muscle, umbilical cord blood, peripheral blood, lung, heart, and the like. The human umbilical cord mesenchymal stem cell is a multifunctional stem cell existing in umbilical cord tissues of newborns, can be differentiated into a plurality of tissue cells, has wide clinical application prospect, and has more and more primary and subculture for the human umbilical cord mesenchymal stem cell. At present, in the process of subculturing umbilical cord mesenchymal stem cells, some pancreatin used for the umbilical cord mesenchymal stem cells is digested, but phenol red recombinase used in some existing pancreatin can cause cells not to adhere to the wall or die after a certain time, the loss of the cells is too large, the multiplication capacity of the cells is reduced, and the phenomena of cell aging and death and the like can occur when the cells are continuously subcultured for multiple times.
Therefore, an effective cell subculture method is urgently needed to be established, and the requirements of the human umbilical cord mesenchymal stem cells on scientific research and application are met.
Disclosure of Invention
In order to solve the above technical problems, the first aspect of the present invention provides a method for subculturing cells, which can maintain the proliferation capacity when culturing human umbilical cord mesenchymal stem cells of generations P1-P25; the method comprises the following steps:
(1) centrifuging: taking a culture bottle with cells, slightly beating the bottle body to enable tissue blocks to fall off, sucking supernatant in the culture bottle by a pipette, centrifuging to obtain centrifugal supernatant for later use, wherein suspended cells are in the culture bottle;
(2) cleaning: cleaning the suspension cells by using a cleaning solution, and then sucking away the cleaning solution;
(3) digestion: adding recombinase into the culture bottle for digestion, stopping digestion after 80% -90% of cells contract and become round, and obtaining cell suspension;
(4) washing: transferring the cell suspension into a centrifuge tube, adding a cleaning solution into a culture bottle, blowing, cleaning, collecting the cleaned cell suspension, and transferring the cell suspension into the centrifuge tube filled with the cell suspension;
(5) and (4) harvesting and counting: centrifuging the centrifuge tubes filled with the cell suspension in the step (4) for the first time, discarding the supernatant, adding a culture medium to resuspend the cell precipitates for the first time, combining the cell precipitates subjected to resuspension of a plurality of centrifuge tubes into 1 tube, adding a cleaning solution to centrifuge, discarding the supernatant, adding the culture medium for the second time, uniformly mixing, and counting the cell number and the survival rate;
(6) cell inoculation: and (3) adding a culture medium into a culture bottle, uniformly mixing, inoculating the cell suspension treated in the step (5), after the operation is finished, labeling the culture bottle, and putting the culture bottle into an incubator at the temperature of 37.0 ℃ and the concentration of CO2 of 5.0%.
In a preferred embodiment of the present invention, the local fusion degree of the cells in step (1) is 80% -90%.
As a preferred technical scheme of the invention, the centrifugation in the step (1) is 450-700g for 5 min.
As a preferable technical scheme of the invention, the cleaning solution is 0.9% sodium chloride injection.
As a preferred technical scheme of the invention, the recombinase is a type Select Enzyme (1X), no phenol red.
As a preferred embodiment of the present invention, the method for terminating digestion in step (3) comprises: adding the centrifugal supernatant obtained in the step (1).
As a preferred technical scheme of the invention, the formula of the culture medium is Sf900II serum-free culture medium, FreeStyleTM293 Expression Medium, EliteGroTM-Adv, NEAA, 0.4% histidine solution, growth factor.
As a preferred embodiment of the present invention, the centrifugation in step (5) is performed at 300-450g for 5 min.
As a preferred embodiment of the present invention, the amount of the first addition of the medium in the step (5) is 20 to 50ml/cm2(ii) a The amount of the culture medium added for the second time in the step (5) is 2-4ml/cm2
In a second aspect the invention provides the use of a method of subculturing cells which can be used to culture mesenchymal stem cells.
Has the advantages that:
1. according to the invention, a specific culture medium and a specific recombinase are selected, so that the cells after subculture have good morphology, higher proliferation capacity and higher survival rate;
2. the recombinase mainly comprises the recombinant trypsin, has the advantages that the recombinant trypsin has the same enzymological properties as the animal-derived trypsin but does not contain any animal-derived component, improves the use safety, does not add phenol red into the product, and can avoid the potential influence of the phenol red on the cell culture process;
3. in the step (5) of the invention, a twice centrifugation method is adopted, so that the shape of the cells after subculture is more uniform;
4. the method for subculturing the human umbilical cord mesenchymal stem cells ensures that the cells can maintain the proliferation capacity until the human umbilical cord mesenchymal stem cells are cultured in the P25 generation.
Drawings
FIG. 1 is a cell morphology diagram of the human umbilical cord mesenchymal stem cells of the P23 generation in example 1.
Detailed Description
The disclosure may be understood more readily by reference to the following detailed description of preferred embodiments of the invention and the examples included therein. 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 case of conflict, the present specification, including definitions, will control.
The term "prepared from …" as used herein is synonymous with "comprising". The terms "comprises," "comprising," "includes," "including," "has," "having," "contains," "containing," or any other variation thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.
The conjunction "consisting of …" excludes any unspecified elements, steps or components. If used in a claim, the phrase is intended to claim as closed, meaning that it does not contain materials other than those described, except for the conventional impurities associated therewith. When the phrase "consisting of …" appears in a clause of the subject matter of the claims rather than immediately after the subject matter, it defines only the elements described in the clause; other elements are not excluded from the claims as a whole.
When an amount, concentration, or other value or parameter is expressed as a range, preferred range, or as a range of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when the range "1 to 5" is disclosed, the described range should be interpreted to include the ranges "1 to 4", "1 to 3", "1 to 2 and 4 to 5", "1 to 3 and 5", and the like. When a range of values is described herein, unless otherwise stated, the range is intended to include the endpoints thereof and all integers and fractions within the range.
The singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. "optional" or "any" means that the subsequently described event or events may or may not occur, and that the description includes instances where the event occurs and instances where it does not.
Approximating language, as used herein throughout the specification and claims, is intended to modify a quantity, such that the invention is not limited to the specific quantity, but includes portions that are literally received for modification without substantial change in the basic function to which the invention is related. Accordingly, the use of "about" to modify a numerical value means that the invention is not limited to the precise value. In some instances, the approximating language may correspond to the precision of an instrument for measuring the value. In the present description and claims, range limitations may be combined and/or interchanged, including all sub-ranges contained therein if not otherwise stated.
In addition, the indefinite articles "a" and "an" preceding an element or component of the invention are not intended to limit the number requirement (i.e., the number of occurrences) of the element or component. Thus, "a" or "an" should be read to include one or at least one, and the singular form of an element or component also includes the plural unless the stated number clearly indicates that the singular form is intended.
In order to solve the above-mentioned problems, the present invention provides, in a first aspect, a method for subculturing cells which can maintain the proliferation potency even when P1-P25 generation cells are cultured; the method comprises the following steps:
(1) centrifuging: taking a culture bottle with cells, slightly beating the bottle body to enable tissue blocks to fall off, sucking supernatant in the culture bottle by a pipette, centrifuging to obtain centrifugal supernatant for later use, wherein suspended cells are in the culture bottle;
(2) cleaning: washing the suspended cells with a washing solution, and then removing the washing solution by suction;
(3) digestion: adding recombinase into the culture bottle for digestion, stopping digestion after 80% -90% of cells contract and become round, and obtaining cell suspension;
(4) washing: transferring the cell suspension into a centrifugal tube, adding a cleaning solution into a culture bottle, blowing, cleaning, collecting the cleaned cell suspension, and transferring the cell suspension into the centrifugal tube filled with the cell suspension;
(5) and (4) harvesting and counting: centrifuging the centrifuge tubes filled with the cell suspension in the step (4) for the first time, discarding the supernatant, adding a culture medium for resuspending the cell precipitates for the first time, merging the cell precipitates resuspended in the centrifuge tubes into 1 tube, adding a cleaning solution for centrifuging, discarding the supernatant, adding the culture medium for the second time, mixing uniformly, and counting the cell number and the survival rate;
(6) cell inoculation: and (3) adding a culture medium into a culture bottle, uniformly mixing, inoculating the cell suspension treated in the step (5), after the operation is finished, labeling the culture bottle, and putting the culture bottle into an incubator at the temperature of 37.0 ℃ and the concentration of CO2 of 5.0%.
The applicant finds that the number of the cells after subculture is increased by twice centrifugal washing after twice centrifugation in the system, probably because twice centrifugal washing of the system can better separate the cells from the recombinase in the solution and the supernatant after centrifugation, so that the cell can be conveniently re-inoculated for subculture, and the growth capacity of the cells is better during subculture.
In one embodiment, the continuous subculture may be performed according to the subculture method of the above-described cells: when the generation of the cells is from P0 generation to P1 generation, the culture bottle filled with the cells in the step (1) refers to a culture bottle which is subjected to primary culture cells; when the cells are Pi-1 passage, the flask containing the cells in step (1) is referred to as the flask containing the Pi cells (i is 1 or more, and i may be 1, 2, 3, 4, 5, etc.).
In one embodiment, the cell is a human umbilical cord mesenchymal stem cell.
In one embodiment, the degree of local fusion of the cells in step (1) is 80% to 90%.
The degree of fusion refers to the area occupied by the cells after the cells are attached to the wall and fully expanded, which is the surface area of the flask.
In one embodiment, the step of primary culturing the cells comprises removing residual blood from umbilical cord tissue, peeling off blood vessels and umbilical cord adventitia, collecting Wharton's jelly, shearing Wharton's jelly to obtain minced tissue, inoculating the tissue into a culture flask, culturing in a medium, placing at 37 deg.C and 5% CO, and culturing2Culturing in an incubator, and performing fluid infusion operation after 4-6 days to finally obtain primary culture cells with over 50 percent of crawled cells around the tissue blocks and 80-90 percent of local fusion degree.
In one embodiment, cell morphology is observed under light microscopy every 24h after passage, and depending on cell growth status, further cell passage or cryopreservation and other uses are performed depending on cell growth status.
In one embodiment, the centrifugation in step (1) is performed at 450-700g for 5 min; further preferably, the centrifugation in the step (1) is 600g centrifugation for 5 min.
In one embodiment, the cleaning solution is 0.9% sodium chloride injection.
In one embodiment, the amount of washing solution added in step (2) and step (4) is 5ml/75cm2
In one embodiment, the recombinase is Tryple Select Enzyme (1X), no phenol red.
In one embodiment, the Tryple Select Enzyme (1X), no phenyl red is GIBCO brand.
In the present invention, the use of Tryple Select Enzyme (1X), no phenoxy red, allows for good cell yield and cell survival of the subcultured cells. Probably because the main component of the Tryple Select Enzyme (1X) and the no phenol red is recombinant trypsin, the trypsinic property of the product is the same as that of animal-derived trypsin, but the product does not contain any animal-derived component, the use safety is improved, and in addition, the product is not added with phenol red, which is different from phenol red recombinase, and the potential influence of the phenol red on the cell culture process can be avoided.
In one embodiment, the method for terminating digestion in step (3) is: adding the centrifugal supernatant obtained in the step (1).
In one embodiment, the ratio of the volume of centrifugation supernatant to the volume of recombinase added in step (1) (0.8-1.2): 1; further preferably, the volume of the centrifuged supernatant in step (1) added to 1: 1.
in the present invention, the centrifugation supernatant of step (1) is selected to stop digestion, and in the present invention, the addition amount of the centrifugation supernatant of step (1) is controlled, and when it is added too much or too little, the survival rate of the cells after passaging or the number of the cells is affected.
In one embodiment, the formulation of the medium is Sf900II serum free medium, FreeStyleTM293 Expression Medium, EliteGroTM-Adv, NEAA, 0.4% histidine solution, growth factor.
In one embodiment, the FreeStyleTM293 Expression Medium volume of 5-10% of the culture Medium volume; further preferably, the FreeStyleTMThe volume of 293 Expression Medium was 8% of the volume of the Medium.
In one embodiment, the volume of the EliteGroTM-Adv is 3-6% of the volume of the culture medium; further preferably, the volume of the EliteGroTM-Adv is 4% of the volume of the culture medium.
In one embodiment, the volume of the 0.4% histidine solution is 0.4-0.7% of the volume of the culture medium; further preferably, the volume of the 0.4% histidine solution is 0.5% of the volume of the culture medium.
In one embodiment, the growth factor: culture medium 10-30 ng: 1 mL; further preferably, the growth factor: medium 22 ng: 1 mL.
In one embodiment, the growth factor is selected from at least one of bFGF, PDGF, EGF, TGF β, IGF-1; further preferably, the growth factor is EGF, TGF β; wherein the weight ratio of EGF to TGF beta is (1.8-2.5): 1; further preferably, the weight ratio of EGF to TGF β is 2.3: 1.
in one embodiment, the Sf900II serum-free medium is GIBCO brand.
In one embodiment, the FreeStyleTM293 Expression Medium is the GIBCO brand.
In one embodiment, the EliteGroTM-Adv is purchased from Biotechnology, Inc. of Beijing Dataceae.
In one embodiment, the NEAA is purchased from Wuhan Punuo race Life technologies, Inc.
In one embodiment, the 0.4% histidine solution is purchased from Ku Ladbo technologies, Inc. of Beijing.
In one embodiment, the EGF is purchased from Wuhan Poncirus Life technologies, Inc.
In one embodiment, the TGF β is purchased from Wuhan Pond Life technologies, Inc.
The culture medium of the invention is used for subculture, and the cultured cells have good proliferation capacity. And the applicant unexpectedly found that adding EliteGroTM-Adv to the culture medium increased the proliferation of subcultured cells, probably because of the polypeptide additive (GlutaMAX) contained in EliteGroTM-AdvTM-I), which has better stability in cooperation with a certain amount of EliteGroTM-Adv and 0.4% histidine solution, thereby providing nutrition for the cells well and enabling the cells to proliferate, and the EliteGroTM-Adv, NEAA and the growth factors cooperate with each other, so that the use concentration of the Sf900II serum-free medium is reduced, and the proliferation capacity is better provided for the cells.
In one embodiment, the centrifugation in step (5) is 300-450g for 5 min; further preferably, the centrifugation in step (5) is 400g for 5 min.
In one embodiment, the amount of the washing solution added in the step (5) is 10 to 15ml/75cm2(ii) a Further preferably, the amount of the washing solution added in the step (5) is 12ml/75cm2
In one embodiment, the amount of the first addition of the medium in step (5) is 20 to 50ml/75cm2(ii) a Further preferably, the first addition in step (5)The amount of the culture medium added is 30ml/75cm2
In one embodiment, the amount of the second addition of the medium in step (5) is 2 to 4ml/75cm2(ii) a Further preferably, the amount of the medium added in the second addition in the step (5) is 3ml/75cm2
In one embodiment, the cells are seeded at a density of 6000 to 8000 cells/cm in step (6)2(ii) a Further preferably, the seeding density of the cells in step (6) is 7000 cells/cm2
In a second aspect, the invention provides the use of a method of subculturing cells, which method may be used to culture mesenchymal stem cells.
The present invention will be specifically described below by way of examples. It should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and that the insubstantial modifications and adaptations of the present invention by those skilled in the art based on the above disclosure are still within the scope of the present invention.
Examples
Example 1
Taking primary cells which are finally prepared and have more than 50 percent of cells crawled out of the periphery of the tissue block and the local fusion degree of 85 percent, namely P0 generation human umbilical cord mesenchymal stem cells, and carrying out continuous subculture till 30 generations, wherein the subculture method comprises the following steps: (1) centrifuging: taking a culture bottle with cells, lightly tapping the bottle body to enable tissue blocks to fall off, sucking supernatant in the culture bottle by a pipette, centrifuging to obtain centrifugal supernatant for later use, wherein suspended cells are in the culture bottle; (2) cleaning: washing the suspended cells with a washing solution, and then removing the washing solution by suction; (3) digestion: adding recombinase into the culture bottle for digestion, stopping digestion after 80% -90% of cells contract and become round, and obtaining cell suspension; (4) washing: transferring the cell suspension into a centrifugal tube, adding a cleaning solution into a culture bottle, blowing, cleaning, collecting the cleaned cell suspension, and transferring the cell suspension into the centrifugal tube filled with the cell suspension; (5) and (4) harvesting and counting: centrifuging the centrifuge tubes filled with the cell suspension in the step (4), discarding the supernatant, adding a culture medium for resuspending cell precipitates for the first time, combining the cell precipitates subjected to resuspension in a plurality of centrifuge tubes into 1 tube, adding a cleaning solution for centrifuging, discarding the supernatant, adding the culture medium for the second time, uniformly mixing, and counting the number of cells and the survival rate; (6) cell inoculation: adding a culture medium into a culture bottle, uniformly mixing, inoculating the cell suspension treated in the step (5), after the operation is finished, sticking a label on the culture bottle, and putting the culture bottle into an incubator at the temperature of 37.0 ℃ and the concentration of CO2 of 5.0%;
wherein, when Pi is substituted to Pi +1 is substituted for the human umbilical cord mesenchymal stem cells, the culture flask filled with the cells in the step (1) refers to a culture flask filled with Pi cells (i is an integer of 1-29);
the local fusion degree of the cells in the step (1) is 80-90%; the centrifugation in the step (1) is 600g for 5 min; the cleaning solution is 0.9% sodium chloride injection; the adding amount of the cleaning solution in the step (2) and the step (4) is 5ml/75cm2(ii) a The recombinase is a Tryple Select Enzyme (1X) and no phenol red; the method for terminating digestion in the step (3) comprises the following steps: adding the centrifugal supernatant obtained in the step (1); adding the volume of the centrifugation supernatant in the step (1) and 1: 1; the formula of the culture medium is Sf900II serum-free culture medium and FreeStyleTM293 Expression Medium, EliteGroTM-Adv, NEAA, 0.4% histidine solution, growth factor; the FreeStyleTMThe volume of 293 Expression Medium was 8% of the volume of the Medium; the volume of the EliteGroTM-Adv is 4% of the volume of the culture medium; the volume of the 0.4% histidine solution is 0.5% of the volume of the culture medium; the growth factor is as follows: medium 22 ng: 1 mL; the growth factor is EGF and TGF beta; the weight ratio of the EGF to the TGF beta is 2.3: 1; centrifuging at 400g in the step (5) for 5 min; the adding amount of the cleaning solution in the step (5) is 12ml/75cm2(ii) a The amount of the first addition of the medium in the step (5) was 30ml/75cm2(ii) a The amount of the culture medium added for the second time in the step (5) is 3ml/75cm2(ii) a The seeding density of the cells in the step (6) is 7000 cells/cm2
The Tryple Select Enzyme (1X) and no phenol red are GIBCO brands; the Sf900II serum-free culture medium is a GIBCO brand; the FreeStyleTM293 ExpressThe ion Medium is the GIBCO brand; the EliteGroTM-Adv is purchased from Beijing Dataceae, Biotechnology, Inc.; the NEAA was purchased from Wuhan Punuo race Life technologies, Inc.; the 0.4% histidine solution was purchased from Ku Laibobo technologies, Beijing; the EGF is purchased from Wuhan Punuoise Life technologies, Inc.; the TGF beta was purchased from Wuhan Punuo race Life technologies, Inc.
The morphology of the human umbilical cord mesenchymal stem cells from generation P1 to generation P25 is relatively uniform, and the morphology of the human umbilical cord mesenchymal stem cells from generation P26 to generation P30 is changed. From fig. 1, it can be seen that the P23 generation human umbilical cord mesenchymal stem cells also have good morphology.
Example 2
Taking primary cells which are finally prepared and have more than 50 percent of cells crawled out of the periphery of the tissue block and the local fusion degree of 85 percent, namely P0 generation human umbilical cord mesenchymal stem cells, and carrying out continuous subculture till 30 generations, wherein the subculture method comprises the following steps: (1) centrifuging: taking a culture bottle with cells, lightly tapping the bottle body to enable tissue blocks to fall off, sucking supernatant in the culture bottle by a pipette, centrifuging to obtain centrifugal supernatant for later use, wherein suspended cells are in the culture bottle; (2) cleaning: washing the suspended cells with a washing solution, and then removing the washing solution by suction; (3) digestion: adding recombinase into the culture bottle for digestion, stopping digestion after 80% -90% of cells contract and become round, and obtaining cell suspension; (4) washing: transferring the cell suspension into a centrifuge tube, adding a cleaning solution into a culture bottle, blowing, cleaning, collecting the cleaned cell suspension, and transferring the cell suspension into the centrifuge tube filled with the cell suspension; (5) and (4) harvesting and counting: centrifuging the centrifuge tubes filled with the cell suspension in the step (4), discarding the supernatant, adding culture medium to suspend cell precipitates, merging the cell precipitates suspended by a plurality of centrifuge tubes into 1 tube, and counting the number of cells and the survival rate after uniformly mixing; (6) cell inoculation: adding a culture medium into a culture bottle, uniformly mixing, inoculating the cell suspension treated in the step (5), after the operation is finished, sticking a label on the culture bottle, and putting the culture bottle into an incubator at the temperature of 37.0 ℃ and the concentration of CO2 of 5.0%;
wherein, when Pi is replaced to Pi +1 generation of human umbilical cord mesenchymal stem cells, the culture bottle filled with cells in the step (1) refers to a culture bottle filled with Pi cells (i is an integer of 1-29);
the local fusion degree of the cells in the step (1) is 80-90%; the centrifugation in the step (1) is 600g for 5 min; the cleaning solution is 0.9% sodium chloride injection; the adding amount of the cleaning solution in the step (2) and the step (4) is 5ml/75cm2(ii) a The recombinase is a Tryple Select Enzyme (1X) and no phenol red; the method for terminating digestion in the step (3) comprises the following steps: adding the centrifugal supernatant obtained in the step (1); adding the volume of the centrifugation supernatant in the step (1) and 1: 1; the formula of the culture medium is Sf900II serum-free culture medium and FreeStyleTM293 Expression Medium, EliteGroTM-Adv, NEAA, 0.4% histidine solution, growth factor; the FreeStyleTMThe volume of 293 Expression Medium was 8% of the volume of the Medium; the volume of the EliteGroTM-Adv is 4% of the volume of the culture medium; the volume of the 0.4% histidine solution is 0.5% of the volume of the culture medium; the growth factor is as follows: medium 22 ng: 1 mL; the growth factor is EGF and TGF beta; the weight ratio of the EGF to the TGF beta is 2.3: 1; centrifuging at 400g for 5min in the step (5); the adding amount of the cleaning solution in the step (5) is 12ml/75cm2(ii) a The amount of the medium added in step (5) was 33ml/75cm2(ii) a The seeding density of the cells in the step (6) is 7000 cells/cm2
The Tryple Select Enzyme (1X) and no phenol red are GIBCO brands; the Sf900II serum-free culture medium is a GIBCO brand; the FreeStyleTM293 Expression Medium is the GIBCO brand; the EliteGroTM-Adv is purchased from Beijing Dake as Biotechnology, Inc.; the NEAA was purchased from Wuhan Punuo race Life technologies, Inc.; the 0.4% histidine solution was purchased from Ku Laibobo technologies, Beijing; the EGF is purchased from Wuhan Punuoise Life technologies, Inc.; the TGF beta was purchased from Wuhan Punuo race Life technologies, Inc.
The morphology of the human umbilical cord mesenchymal stem cells from generation P1 to generation P15 is relatively uniform through observation by using an optical microscope, and the morphology of the human umbilical cord mesenchymal stem cells from generation P16 to generation P30 is changed.
Example 3
Taking primary cells which have more than 50% of crawled cells around the finally prepared tissue blocks and have the local fusion degree of 85%, namely P0 generation human umbilical cord mesenchymal stem cells, and carrying out continuous subculture till 30 generations, wherein the subculture method is the same as that of example 1, except that EliteGroTM-Adv is not used.
Example 4
Taking the primary cells which have more than 50% of crawled cells around the finally prepared tissue blocks and have the local fusion degree of 85%, namely the P0 generation human umbilical cord mesenchymal stem cells, and carrying out continuous subculture till 30 generations, wherein the subculture method is the same as that in example 1, except that the volume of the EliteGroTM-Adv is 1% of the volume of the culture medium.
Example 5
And (3) taking the primary cells which are finally prepared and have more than 50 percent of creeping cells around the tissue blocks and 85 percent of local fusion degree, namely the P0 generation human umbilical cord mesenchymal stem cells, and carrying out continuous subculture till 30 generations, wherein the subculture method is the same as that in example 1 except that 0.4 percent histidine acid solution is not used.
Example 6
Taking the primary cells which are finally prepared and have more than 50 percent of crawled cells around the tissue block and 85 percent of local fusion degree, namely the human umbilical cord mesenchymal stem cells of P0 generation, and carrying out continuous subculture till 30 generations, wherein the subculture method is the same as the embodiment 1, except that the volume of the centrifugal supernatant obtained in the step (1) and the volume of the recombinase are added to be 1: 1.5.
example 7
Taking the primary cells which are finally prepared and have more than 50 percent of crawled cells around the tissue block and 85 percent of local fusion degree, namely the human umbilical cord mesenchymal stem cells of P0 generation, and carrying out continuous subculture till 30 generations, wherein the subculture method is the same as example 1, except that the volume of the centrifugal supernatant obtained in the step (1) and the volume of the recombinase are added in the ratio of 1.5: 1.
performance testing
Recording the cell number and the survival rate of the cells in the step (5) when the human umbilical cord mesenchymal stem cells of the P0 generation are cultured to the cells of the P1 generation, wherein the survival rate and the cell number are the cells of the P0 generation; recording the cell number and the survival rate of the cells in the step (5) when the human umbilical cord mesenchymal stem cells of the P1 generation are cultured to the cells of the P2 generation, wherein the survival rate and the cell number are the cells of the P1 generation; the cell number and the survival rate in the step (5) are recorded when the human umbilical cord mesenchymal stem cells of the P2 generation are cultured to the cells of the P3 generation, and the cell number and the survival rate are the cells of the P2 generation. The results are shown in table 1:
TABLE 1
Figure BDA0003067025980000111
According to the test results, the invented subculture method of the cells can be used for culturing the human umbilical cord mesenchymal stem cells to the P25 generation, and has good cell morphology; the cells cultured by the subculture method of the cells have good proliferation capacity and survival rate.
The foregoing examples are merely illustrative and serve to explain some of the features of the method of the present invention. The appended claims are intended to claim as broad a scope as is contemplated, and the examples presented herein are merely illustrative of selected implementations in accordance with all possible combinations of examples. Accordingly, it is applicants' intention that the appended claims are not to be limited by the choice of examples illustrating features of the invention. Also, where numerical ranges are used in the claims, subranges therein are included, and variations in these ranges are also to be construed as possible being covered by the appended claims.

Claims (10)

1. A subculturing method of cells is characterized in that the subculturing method of the cells can maintain the proliferation capacity when the subculturing method of the cells can culture P1-P25 generation cells; the method comprises the following steps:
(1) centrifuging: taking a culture bottle with cells, lightly tapping the bottle body to enable tissue blocks to fall off, sucking supernatant in the culture bottle by a pipette, centrifuging to obtain centrifugal supernatant for later use, wherein suspended cells are in the culture bottle;
(2) cleaning: washing the suspended cells with a washing solution, and then removing the washing solution by suction;
(3) digestion: adding recombinase into the culture bottle for digestion, stopping digestion after 80% -90% of cells contract and become round, and obtaining cell suspension;
(4) washing: transferring the cell suspension into a centrifuge tube, adding a cleaning solution into a culture bottle, blowing, cleaning, collecting the cleaned cell suspension, and transferring the cell suspension into the centrifuge tube filled with the cell suspension;
(5) and (4) harvesting and counting: centrifuging the centrifuge tubes filled with the cell suspension in the step (4) for the first time, discarding the supernatant, adding a culture medium for resuspending the cell precipitates for the first time, merging the cell precipitates resuspended in the centrifuge tubes into 1 tube, adding a cleaning solution for centrifuging, discarding the supernatant, adding the culture medium for the second time, mixing uniformly, and counting the cell number and the survival rate;
(6) cell inoculation: and (4) adding a culture medium into a culture bottle, uniformly mixing, inoculating the cell suspension treated in the step (5), after the operation is finished, labeling the culture bottle, and putting the culture bottle into an incubator at 37.0 ℃ and 5.0% of CO 2.
2. A method for subculturing cells according to claim 1, wherein the local fusion degree of the cells in the step (1) is 80% to 90%.
3. A method for subculturing cells according to claim 1, wherein the centrifugation in step (1) is performed at 450-700g for 5 min.
4. A method for subculturing cells according to claim 1, wherein the washing solution is 0.9% sodium chloride injection.
5. A method for subculturing cells according to claim 1, wherein the recombinant Enzyme is Tryple Select Enzyme (1X), no phenol red.
6. A method for subculturing cells according to claim 1, wherein the digestion is terminated in step (3) by: adding the centrifugal supernatant obtained in the step (1).
7. A method for subculturing cells according to claim 1, wherein the formula of the medium is Sf900II serum-free medium, FreeStyleTM293 Expression Medium, EliteGroTM-Adv, NEAA, 0.4% histidine solution, growth factor.
8. A method for subculturing cells according to claim 1, wherein the centrifugation in step (5) is performed at 300-450g for 5 min.
9. A method for subculturing cells according to claim 1, wherein the amount of the medium added for the first time in the step (5) is 20 to 50ml/cm2(ii) a The amount of the culture medium added for the second time in the step (5) is 2-4ml/cm2
10. Use of a method of subculturing a cell according to any of claims 1-9, wherein the method of subculturing is used for culturing mesenchymal stem cells.
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Publication number Priority date Publication date Assignee Title
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115340976A (en) * 2022-10-18 2022-11-15 长春卓谊生物股份有限公司 Application of non-animal-derived recombinase in digestion of Vero cells

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