CN115777691A - Method for cryopreserving and recovering placenta tissues and preparing mesenchymal stem cells - Google Patents
Method for cryopreserving and recovering placenta tissues and preparing mesenchymal stem cells Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0668—Mesenchymal stem cells from other natural sources
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
- C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/38—Vitamins
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/70—Undefined extracts
- C12N2500/80—Undefined extracts from animals
- C12N2500/84—Undefined extracts from animals from mammals
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The invention relates to a method for freezing, recovering and preparing mesenchymal stem cells of placenta tissues, which comprises the following steps: cleaning the placenta tissue; cutting placenta lobule from tissue, and pulverizing with pulverizer; preparing a placenta tissue low-temperature protection solution for later use; mixing the pulverized tissue with protective solution, subpackaging into freezing tubes, sequentially refrigerating at 4 deg.C for 0.5 hr, refrigerating at-20 deg.C for 2 hr, freezing at-80 deg.C for 1 day, and freezing in liquid nitrogen; taking out the placenta tissue cryopreservation tube from liquid nitrogen when needed, and unfreezing in a constant-temperature water bath; washing with buffer solution, and digesting placenta tissue by using a double-enzyme method; and filtering and collecting the cells, and culturing and amplifying the mesenchymal stem cells by using a mesenchymal stem cell culture medium. The method can effectively freeze and restore the placenta tissue and prepare the mesenchymal stem cells. The cells obtained from the tissue blocks of the frozen placenta tissue obtained by the cryopreservation method after thawing are directly inoculated in a culture bottle, the adherent survival rate is up to 95 percent, and the cells grow well.
Description
The application is divided case application with the application date of 19.04.2018, the application number of 201711451308.8 and the invention name of a method for cryopreserving and recovering placenta tissues and preparing mesenchymal stem cells.
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for cryopreserving and recovering placenta tissues and preparing mesenchymal stem cells.
Background
Mesenchymal Stem Cells (MSCs) are pluripotent stem cells with the potential of self-renewal and multi-differentiation, can differentiate into cells of various terminal tissues and organs such as nerve cells, cardiac muscle cells, muscle cells and the like, and have wide application prospects in cell replacement therapy, hematopoietic support, gene therapy and the like.
Studies have shown that placental tissue also contains mesenchymal stem cells and can be successfully isolated. The tissue-derived mesenchymal stem cells not only keep the biological characteristics of the mesenchymal stem cells, but also have more primitive isolated stem cells and stronger proliferation and differentiation capacities. The functional activity of immune cells is low, so that the risk of triggering immune response and causing graft-versus-host disease is greatly reduced. The probability of infection and transmission of latent viruses and microorganisms is low. The collecting process is simple, and no harm or injury is caused to the puerpera and the newborn. The above reasons make placental mesenchymal stem cells a hot point for research.
However, the methods and techniques for isolating stem cells from placental tissue are not fully developed, and each treatment of placental tissue and cell culture after isolation requires a certain amount of time and labor. It is therefore relatively more cost effective to cryopreserve placental tissue and resuscitate it for stem cell isolation when needed. Therefore, there is a need in the art for a simple and efficient method for cryopreserving, resuscitating, and preparing mesenchymal stem cells from placenta tissue to meet the needs of the fields of medicine, scientific research, clinical practice, and the like.
Disclosure of Invention
In order to solve the defects of the prior art, the invention provides a method for cryopreserving and recovering placenta tissues and preparing mesenchymal stem cells.
The invention provides a method for cryopreserving and recovering placenta tissues and preparing mesenchymal stem cells, which is characterized by comprising the following steps of:
(1) Washing the placenta tissue by PBS buffer solution to wash out residual blood on the surface of the placenta tissue, wherein no blood clot exists on the surface of the placenta;
(2) Shearing the placenta lobules of the placenta tissue obtained in the step (1) by using a tissue shear, and crushing the placenta lobules into small blocks of tissues by using a crusher;
(3) Adding a precooling protective agent into the small tissue blocks obtained in the step (2) according to the volume ratio of 1:1, uniformly mixing, and subpackaging into a plurality of cryopreservation tubes, wherein the protective agent is a mixed solution of a vitrification solution and a mesenchymal stem cell culture medium;
(4) Freezing the freezing tube obtained in the step (3) at 4 ℃ for 0.5 hour, at-20 ℃ for 2 hours, at-80 ℃ for 1 day, and finally freezing in liquid nitrogen at-196 ℃ for later use;
(5) Taking the tissue block frozen in the step (4) out of liquid nitrogen when needed, unfreezing the tissue block in a constant-temperature water bath, sucking the unfrozen tissue block by a pipette, putting the tissue block into a clean centrifugal tube, adding PBS (phosphate buffer solution) into the centrifugal tube, uniformly mixing the tissue block and the clean centrifugal tube, and centrifuging the mixture to remove a supernatant;
(6) Adding 0.1% type II collagenase and 0.25% pancreatin into the tissue blocks washed in the step (5) successively for digestion, collecting cell solution by filtering with a screen, centrifuging, and removing supernatant;
(7) Resuspending the cell pellet obtained in step (6) with a mesenchymal stem cell culture medium, placing the cell pellet in a culture flask with the mesenchymal stem cell culture medium in advance, and placing the culture flask in CO 2 Culturing in an incubator; and
(8) And (3) carrying out first liquid change 3-4 days after culture, carrying out liquid change 3-4 days later, and carrying out digestion by using trypsin and then carrying out passage after the cell fusion degree reaches more than 70 percent to obtain the placenta mesenchymal stem cells.
According to one embodiment of the invention, the placental leaflets are 5cm x 5cm in size; the small tissue block is 3mm multiplied by 3mm in size.
According to one embodiment of the invention, the protective agent is formed by mixing a vitrification solution and a mesenchymal stem cell culture medium according to a volume ratio of 1:9.
According to one embodiment of the invention, the vitrification solution contains glycerol 27-32% by volume, ethylene glycol 12-16% by volume, dimethyl sulfoxide 12-16% by volume, and sucrose 0.3-0.5M by mass.
According to one embodiment of the invention, the vitrification solution contains 30% by volume of glycerol, 15% of ethylene glycol, 15% of dimethyl sulfoxide and 0.4M by mass of sucrose.
According to one embodiment of the invention, the digestion time in step (6) is 20-30 minutes.
According to one embodiment of the present invention, the mesenchymal stem cell culture medium contains: 12-18 parts of FBS, 0.8-1 part of L-glutamine, 0.02-0.05 part of gentamicin and 80-90 parts of DMEM-F12.
According to one embodiment of the present invention, the mesenchymal stem cell culture medium contains: 15 parts by weight of FBS, 1 part by weight of L-glutamine, 0.04 part by weight of gentamicin and 85 parts by weight of DMEM-F12.
According to one embodiment of the present invention, the method for cryopreserving, resuscitating, and preparing mesenchymal stem cells of the placenta tissue of the present invention further comprises the steps of:
(9) Detecting at least one of the following items aiming at the placenta mesenchymal stem cells obtained in the step (8): cell viability, cell contamination, genetic disease, HLA-ABC/DR match; or
(10) And (4) freezing and storing the placenta mesenchymal stem cells obtained in the step (8) after passage in liquid nitrogen for later use.
In another aspect of the present invention, a method for establishing a placental stem cell database is provided, the method comprising the steps of the method according to the first aspect of the present invention, and the steps of: and (3) cryopreserving the cells after passage in liquid nitrogen, recording related fetal information, identifying biological characteristics and multidirectional differentiation potential of the cells, performing molecular genetic diagnosis on the cells, storing all related data of the cells, establishing a database of the placental stem cells, and associating the database with the cryopreserved cells.
The method is simple to operate, convenient and practical, can effectively protect the cryopreserved placenta tissues, avoids the loss of the survival rate of cells in the recovery process, can obtain a large amount of mesenchymal stem cells, and has good differentiation performance and good cell differentiation capacity. The invention establishes a placental stem cell bank by utilizing a large amount of mesenchymal stem cells with higher purity separated from placenta so as to store the stem cells with great application prospect. The method is simple and easy to implement, and because the placenta is the same as cord blood, the cell components are more immature, the source is wide, and the method is convenient and easy to obtain, the method has wide prospect in the clinical application of stem cells. The results of the examples show that: the cells of the tissue block of the thawed frozen placenta tissue obtained by the cryopreservation method are directly inoculated in a culture bottle, the adherent survival rate is up to 95 percent, and the cells grow well.
Drawings
FIG. 1 shows the growth status of P1 generation cells after thawing recovery according to one embodiment of the present invention.
FIG. 2 shows the growth status of P1 generation cells after thawing recovery, according to one embodiment of the present invention.
Detailed Description
The present application will be described in further detail below with reference to the accompanying drawings by way of specific embodiments.
Example 1
(1) The placenta tissue after normal parturition of a healthy lying-in woman is taken, and immediately placed in 0.9% physiological saline at 4 ℃ after being isolated. Stripping the decidua and the placenta with hemostatic forceps and tissue scissors, and washing with PBS buffer solution to wash the residual blood on the surface of placenta tissue and make the surface of placenta tissue have no blood clot.
(2) Shearing the placenta tissue obtained in the step (1) into placenta lobules with the size of 5cm multiplied by 5cm by using a tissue shear, and crushing the placenta lobules into tissue blocks with the size of about 3mm multiplied by 3mm by using a crusher.
(3) And (3) adding the tissue blocks obtained in the step (2) into a precooling protective agent according to the volume ratio of 1:1, uniformly mixing, and subpackaging into a plurality of freezing tubes.
(4) Freezing the freezing tube obtained in the step (3) at 4 ℃ for 0.5 hour, at-20 ℃ for 2 hours, at-80 ℃ for 1 day, and finally freezing in liquid nitrogen at-196 ℃ for later use;
(5) Taking the tissue block frozen in the step (4) out of liquid nitrogen when needed, unfreezing the tissue block in a constant-temperature water bath, sucking the unfrozen tissue block by a pipette, putting the tissue block into a clean centrifugal tube, adding PBS (phosphate buffer solution) into the centrifugal tube, uniformly mixing the tissue block and the PBS, centrifuging the tissue block at 2500rpm for 5 minutes, and removing a supernatant; the washing was repeated twice.
(6) And (3) adding 0.1% type II collagenase and 0.25% pancreatin into the tissue mass washed in the step (5) to digest the tissue mass for 30 minutes, filtering and collecting the digested cell solution by using a 200-mesh screen, collecting the filtrate, centrifuging the cell solution for 10 minutes at 1200rmp, and removing the supernatant.
(7) Resuspending the cell pellet obtained in step (6) using a mesenchymal stem cell culture medium, placing the pellet in a culture flask previously filled with a mesenchymal stem cell culture medium, and placing the pellet in 5% CO 2 Culturing at 37 deg.C in incubator.
(8) The first liquid change is carried out 3-4 days after the culture, the liquid change is carried out 3-4 days later, when the cell fusion degree reaches more than 70%, 0.25% trypsin is used for digesting for 2 minutes at 37 ℃, 4-5 times of volume of culture medium is added to stop the digestion reaction, then the centrifugation is carried out for 10 minutes at 1200rpm, and the supernatant is discarded. And (4) resuspending the precipitate by using a culture medium, and carrying out passage at a ratio of 1:3 to obtain the placenta mesenchymal stem cells.
Fig. 1 and 2 show the growth state of P1 generation cells. The result shows that the survival rate of the thawed tissue blocks directly inoculated in the culture bottle reaches up to 95 percent and the cells grow well.
The formula of the used reagent is as follows:
vitrification solution: 30 percent of glycerol, 15 percent of glycol, 15 percent of dimethyl sulfoxide and 0.4M of cane sugar by mass fraction.
Mesenchymal stem cell culture medium: 15 parts by weight of FBS, 1 part by weight of L-glutamine, 0.04 part by weight of gentamicin and 85 parts by weight of DMEM-F12.
A protective agent: the vitrification solution and the mesenchymal stem cell culture medium are mixed according to the volume ratio of 1:9.
Example 2
The placenta mesenchymal stem cells obtained after the passage of the embodiment 1 are tested for the cell activity and the cell contamination.
The results show that the cell activity is good and no cell contamination occurs.
Example 3
With respect to the placental mesenchymal stem cells obtained after the passage in example 1, with reference to the methods of step (4) and step (5) in example 1, placental mesenchymal stem cells were cryopreserved in liquid nitrogen for use.
The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. It will be apparent to those skilled in the art from this disclosure that many more simple derivations or substitutions can be made without departing from the spirit of the disclosure.
Claims (10)
1. A method of cryopreserving placental tissue, comprising the steps of:
(1) Washing the placenta tissue by PBS buffer solution to wash out residual blood on the surface of the placenta tissue, wherein no blood clot exists on the surface of the placenta;
(2) Shearing the placenta lobules of the placenta tissue obtained in the step (1) by using a tissue shear, and crushing the placenta lobules into small blocks of tissues by using a crusher;
(3) Adding a precooling protective agent into the small tissue blocks obtained in the step (2) according to the volume ratio of 1:1, uniformly mixing, and subpackaging into a plurality of cryopreservation tubes, wherein the protective agent is a mixed solution of a vitrification solution and a mesenchymal stem cell culture medium;
the protective agent is formed by mixing a vitrification solution and a mesenchymal stem cell culture medium according to the volume ratio of 1:9;
the vitrification solution contains 27-32% of glycerol, 12-16% of glycol, 12-16% of dimethyl sulfoxide and 0.3-0.5M of cane sugar in percentage by volume;
the mesenchymal stem cell culture medium contains: 12-18 parts of FBS, 0.8-1 part of L-glutamine, 0.02-0.05 part of gentamicin and 80-90 parts of DMEM-F12;
(4) And (4) sequentially refrigerating the cryopreserved tubes obtained in the step (3) at 4 ℃ for 0.5 hour, refrigerating at 20 ℃ for 2 hours, freezing at 80 ℃ for 1 day, and finally freezing in liquid nitrogen at 196 ℃ below zero for later use to obtain the cryopreserved placenta tissues.
2. The method according to claim 1, wherein the vitrification solution contains 30% by volume of glycerol, 15% of ethylene glycol, 15% of dimethyl sulfoxide and 0.4M by mass of sucrose.
3. The method of claim 1, wherein the mesenchymal stem cell culture medium comprises: 15 parts by weight of FBS, 1 part by weight of L-glutamine, 0.04 part by weight of gentamicin and 85 parts by weight of DMEM-F12.
4. The method of claim 1, wherein said placental leaflets are 5cm x 5cm in size; the small tissue block is 3mm multiplied by 3mm in size.
5. Use of the method of any one of claims 1 to 4 for cryopreservation of placental tissue.
6. A method for resuscitating and preparing mesenchymal stem cells, comprising the steps of:
I. removing the frozen tissue mass obtained by the method of any one of claims 1 to 4 from liquid nitrogen, thawing in a thermostatic water bath, sucking the thawed tissue mass with a pipette, placing into a clean centrifuge tube, adding PBS buffer solution, mixing, centrifuging, and removing the supernatant;
II. Adding 0.1% type II collagenase and 0.25% pancreatin to the tissue mass after washing in step I, digesting, collecting the cell solution by filtration using a sieve, then centrifuging, and removing the supernatant;
III, resuspending the cell sediment obtained in the step II by using a mesenchymal stem cell culture medium, putting the cell sediment into a culture bottle which is provided with the mesenchymal stem cell culture medium in advance, and putting the culture bottle in CO 2 Culturing in an incubator; and
IV, carrying out first liquid change 3-4 days after culture, changing liquid once 3-4 days later, digesting by using trypsin after the cell fusion degree reaches more than 70%, and then carrying out passage to obtain the placenta mesenchymal stem cells.
7. The method of claim 6, wherein the digestion of step II is for a period of 20 to 30 minutes.
8. The method according to claim 6, wherein in step IV, 0.25% trypsin is used for digestion at 37 ℃ for 2 minutes, 4-5 times volume of the culture medium is added to stop the digestion reaction, then the mixture is centrifuged at 1200rpm for 10 minutes, and the supernatant is discarded; resuspending the sediment in a culture medium, and carrying out passage at the ratio of 1:3 to obtain the placenta mesenchymal stem cells; after thawing, the tissue block is directly inoculated in a culture bottle, the adherent survival rate is up to 95 percent, and the cell growth is good.
9. The method of claim 6, further comprising the step of:
detecting at least one of the following items aiming at the placenta mesenchymal stem cells obtained in the step IV: cell viability, cell contamination, genetic disease, HLA-ABC/DR match; or freezing the placenta mesenchymal stem cells obtained in the step IV after passage in liquid nitrogen for later use.
10. A method of establishing a placental stem cell database, comprising the steps of the method of any one of claims 5-9, and the steps of: and (3) cryopreserving the cells after passage in liquid nitrogen, recording related fetal information, identifying biological characteristics and multidirectional differentiation potential of the cells, performing molecular genetic diagnosis on the cells, storing all related data of the cells, establishing a database of the placental stem cells, and associating the database with the cryopreserved cells.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202211453756.2A CN115777691A (en) | 2018-04-19 | 2018-04-19 | Method for cryopreserving and recovering placenta tissues and preparing mesenchymal stem cells |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202211453756.2A CN115777691A (en) | 2018-04-19 | 2018-04-19 | Method for cryopreserving and recovering placenta tissues and preparing mesenchymal stem cells |
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| CN102660497A (en) * | 2012-05-21 | 2012-09-12 | 博雅干细胞科技有限公司 | Method for freezing and reviving umbilical cord tissues and for separating and increasing stem cells |
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