CN112458047B - Method for separating placenta mesenchymal stem cells and serum-free culture medium used by same - Google Patents

Method for separating placenta mesenchymal stem cells and serum-free culture medium used by same Download PDF

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CN112458047B
CN112458047B CN202011381195.0A CN202011381195A CN112458047B CN 112458047 B CN112458047 B CN 112458047B CN 202011381195 A CN202011381195 A CN 202011381195A CN 112458047 B CN112458047 B CN 112458047B
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CN112458047A (en
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王正
刘冰
肖海蓉
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BOYALIFE Inc
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Abstract

The invention relates to a method for separating placenta mesenchymal stem cells and a serum-free culture medium used by the method. Specifically, the method for separating and culturing the primary placental mesenchymal stem cells comprises the following steps: processing placenta samples(ii) a Cleaning with D-Hanks liquid, shearing placenta chorion, shearing, cleaning, filtering to remove residual blood cells in tissue, adding collagenase II for oscillating digestion, adding D-Hanks liquid for dilution, centrifuging, collecting the bottom cell precipitate, adding primary complete culture medium for resuspension, sampling and counting, and inoculating to a culture flask according to the specified cell amount; placing CO 2 And (3) culturing in an incubator, carrying out half-exchange for 4D and complete-exchange for 7D until the cell fusion degree reaches more than 80%, removing the old culture medium, cleaning the cells with D-hanks liquid, adding recombinant pancreatin solution to digest the cells, enabling the cells to fall off, adding D-hanks liquid to dilute, centrifuging, and carrying out heavy suspension on cell precipitates with a primary complete culture medium to obtain the primary placenta mesenchymal stem cells. The method of the present invention exhibits excellent technical effects as described in the specification.

Description

Method for separating placenta mesenchymal stem cells and serum-free culture medium used by same
Technical Field
The invention belongs to the technical field of biology, and relates to a method for separating adipose-derived mesenchymal stem cells from placenta. The invention also relates to a culture medium and a related test solution used in the culture of the placenta mesenchymal stem cells. When the method is used for separating and culturing the placenta mesenchymal stem cells, the excellent technical effect can be shown. In particular, the present invention relates to methods for isolating mesenchymal stem cells from placenta.
Background
Mesenchymal Stem Cells (MSCs), such as human mesenchymal stem cells, were first isolated from bone marrow and a class of tissue stem cells derived from the mesoderm, which have the potential for multipotent differentiation and the ability to self-renew, have the ability to differentiate into various adult cells, such as osteoblasts, chondrocytes, adipocytes, endothelial cells, nerve cells, muscle cells, hepatocytes, etc., under specific conditions in vivo and in vitro (Cap AI. mesenchymal stem cells. J Orthop Res.1991,9:641-650.Pittenger MF, Mackay AM, Beck, et al. multilineage patent of epithelial man stem cells. science.1999; 284:143 Across 147). The latest research shows that the mesenchymal stem cells have the functions of immunoregulation and hematopoiesis support, and are easy to introduce and express exogenous genes. Therefore, the mesenchymal stem cells are not only seed cells in the construction of tissue engineering bone, cartilage and cardiac muscle and important carrier cells in gene therapy, but also have wide application prospect in hematopoietic stem cell transplantation and organ transplantation because the mesenchymal stem cells promote hematopoietic reconstruction and inhibit graft-versus-host reaction. Mesenchymal stem cells have the characteristic of adherent growth in vitro, and by utilizing the characteristic, the mesenchymal stem cells are successfully separated and cultured from various tissues such as liver, kidney, pancreas, muscle, cartilage, skin, peripheral blood and the like.
Stem cells are progenitors of human cells, and all cells in our body are derived from stem cells. When cells in the body age, die or damage denatures, stem cells grow and transform out of the cells that can replace them. As seed cells, the compound is mainly used for treating various refractory diseases of tissue cells and organ injuries which cannot be naturally repaired by an organism clinically; as immunoregulatory cells, for the treatment of immune rejection and autoimmune diseases. Human mesenchymal stem cells are important members of a stem cell family, are derived from mesoderm in early development and belong to pluripotent stem cells, and are discovered in bone marrow initially, so that the human mesenchymal stem cells are increasingly concerned because of the characteristics of multidirectional differentiation potential, hematopoietic support, stem cell implantation promotion, immune regulation, self-replication and the like. Initial clinical studies were conducted in 1995 by Lazarus et al, who collected autologous MSCs of patients with hematological tumors in remission, cultured for 4-7 weeks in vitro for expansion, and then injected intravenously into patients, who were divided into 3 groups, administered different doses of MSCs, respectively, and no toxic side effects were observed after injection, suggesting that MSCs are safe and reliable for transplantation therapy. Then, clinical reports of autologous MSCs are gradually increased, and the disease types comprise hematopoietic reconstruction after radiotherapy and chemotherapy, graft-versus-host disease (GVHD), heart system diseases and the like, and clinical intravenous infusion is proved to be safe and reliable in the reports.
The isolation culture and subculture process of mesenchymal stem cells are key steps related to the use safety of stem cells as therapeutic drugs. The composition of the culture process, in particular of the culture medium, is a major influencing parameter. In the methods described in the prior documents, when mesenchymal stem cells are isolated and subcultured, the culture medium mostly needs to be supplemented with serum, such as fetal bovine serum, and particularly, 10% fetal bovine serum is usually required to be supplemented, and for example, the mesenchymal stem cells are isolated and subcultured usually by using MSC complete medium (which is DMEM-F12 medium containing 10% fetal bovine serum).
On the one hand, however, the cost of fetal bovine serum is rather high, which is disadvantageous for the culture of mesenchymal stem cells; on the other hand, the presence of fetal bovine serum as an exogenous material of animal origin in stem cells poses a potential risk to the safety of clinical use of the cells. Therefore, serum-free isolation and subculture of mesenchymal stem cells are of interest.
The existing methods for establishing placental stem cell banks by isolating stem cells from the placenta have many disadvantages, such as insufficient purity, complicated process, and/or low number, and thus show that these methods are not satisfactory. For example, CN101270349A (chinese patent application No. 200810061267.6) discloses a method for separating and in vitro amplification culturing placenta mesenchymal stem cells, which comprises collecting placenta maternal periostracum tissue cells, performing adherent amplification of primary cells, then performing a positive and negative immune sorting combination method to purify placenta mesenchymal stem cells to obtain CD 34-CD 105+ cells, and performing passage amplification culturing in a serum-free in vitro culture system. The method disclosed by the invention is believed to achieve a good sorting effect only by a small amount of primary cells (1 × 105 cells) at a time, and further improve the purification rate of the placenta mesenchymal stem cells. The culture system not only has obvious amplification advantages for the placenta mesenchymal stem cells, but also has multi-differentiation potential for the amplified cells. Can be applied to purifying the placenta mesenchymal stem cells and in vitro amplification culture. CN101693884A (chinese patent application No. 200910117522.9) discloses a method for separating and extracting stem cells from placenta, umbilical cord or adipose tissue, which comprises the following process steps: mixing placenta, umbilical cord or adipose tissue and a cell maintenance solution according to a weight ratio of 2.5-4: 1, putting the mixture into a tissue crushing barrel, crushing, adding collagenase, uniformly mixing, incubating at about 37 ℃, filtering, adding a precipitator, standing, sucking supernatant, centrifuging, removing supernatant, adding concentrated cells to a sodium diatrizoate-ficoll 400# liquid, centrifuging again, collecting a middle 10-15ml cell layer, cleaning with the cell maintenance solution, counting collected cells, and detecting that the cell survival rate is more than or equal to 95%, so that the placenta, umbilical cord or adipose tissue can be used clinically. The invention is believed to not only realize the separation and extraction of all stem cells from placenta, umbilical cord or adipose tissue, but also realize the industrialized production, so that doctors can conveniently, safely and normatively obtain adult stem cells clinically, use the adult stem cells to treat diseases of patients like medication, solve the bottleneck that the adult stem cells are difficult to obtain clinically, and popularize the cell treatment technology. CN102146359A (chinese patent application No. 201110005964.1) discloses a method for extracting original mesenchymal stem cells from placenta and serum-free expansion. The invention utilizes the fetal placenta tissue of early abortion to extract more original human placenta mesenchymal stem cells and establishes a system for separating and in-vitro amplification culture of the placenta mesenchymal stem cells. The invention is believed to make the genetic information and phenotype of the stem cell more stable, the characteristics of the stem cell more original, and the invention can effectively expand the cell, improve the product quality, obtain stable and uniform placenta mesenchymal stem cell, solve the problem that the stem cell treatment needs a large amount of stem cells, and can be used for treating various diseases. In addition, CN102676451A (chinese patent application No. 201210044648X) discloses a method for isolating mesenchymal stem cells from placenta, the method comprising the steps of: (a) taking placenta lobules, and fully washing with PBS buffer solution to remove residual blood in the placenta; (b) cutting placenta lobule into pieces, adding PBS buffer solution containing tissue digestive enzyme, and incubating and digesting at 37 deg.C; (c) filtering the tissue mass with a copper mesh, and grinding if necessary to facilitate filtration; (d) centrifuging the collected filtrate, separating mononuclear cells, suspending the obtained cells by using an MSC culture medium, and culturing in a 5% CO2 incubator at 37 ℃; (e) after the cells are scattered to form clones, selecting each clone cell, respectively culturing by using an MSC culture medium, and after the cells are fused, digesting and passaging by using pancreatin to obtain the placenta mesenchymal stem cells; and optionally one or more of the following steps: (f) detecting at least one of the following items for the placental mesenchymal stem cells obtained in step (e): cell viability, cell contamination, genetic disease, HLA-ABC/DR match; (g) freezing and storing the placenta mesenchymal stem cells obtained in the step (e) after passage in liquid nitrogen; (h) establishing a database of placental stem cells comprising the above information, and correlating the database with the cryopreserved cells of step (g). It is believed that high purity placental mesenchymal stem cells can be obtained by the method of the present invention. These methods are to be further improved in terms of process simplicity and/or purity and/or recovery of the extract. In addition, an invention publication CN107299082A (chinese patent application No. 201710653583.1, published 2018, 10 and 27) of the inventor team of the present application describes a method of obtaining placental mesenchymal stem cells, which has been shown to exhibit some excellent properties.
Although the prior art discloses some culture methods such as those described above in relation to placental mesenchymal stem cells, these methods mostly require the use of culture media containing relatively high concentrations of fetal bovine serum.
The art still expects to provide a new method for isolation culture and even subculture of the mesenchymal stem cells, and particularly to provide a new method without fetal calf serum for isolation culture and even subculture of the mesenchymal stem cells.
Disclosure of Invention
The invention aims to provide a novel method for preparing placenta mesenchymal stem cells, in particular to provide a method suitable for separating and obtaining adipose mesenchymal stem cells from placenta, and the method is expected to show the characteristics of high cell yield, high cell survival rate and the like and/or other excellent performances. The present inventors have surprisingly found that the technical effects of one or more aspects as described herein can be achieved using the process of the present invention. The present invention has been completed based on this finding.
To this end, the present invention provides in a first aspect a method for the isolated culture of primary placental mesenchymal stem cells, comprising the steps of:
(1) processing a placenta sample transported to a laboratory via a cold chain at 2-8 ℃ in a biosafety cabinet;
(2) cleaning with D-Hanks solution, cutting placenta chorion, and cutting to 0.25 + -0.05 cm 2 Cleaning D-Hanks once, filtering with 300 mesh filter screen to remove residual blood cells in tissue, adding 1% type II collagenase, and oscillating for digestion;
(3) after digestion, adding one-time volume of D-hanks liquid for dilution, centrifuging, and reserving the cell sediment at the bottom layer for the next operation;
(4) taking the cell sediment obtained in the step (3), adding primary complete culture medium for resuspension, sampling and counting, and inoculating according to the specified cell quantityPlanting into a culture bottle; placing CO 2 Culturing in an incubator;
(5) and (3) half-replacing the culture medium until the culture reaches the 4 th day, completely replacing the culture medium until the cell fusion degree reaches more than 80%, removing the old culture medium, cleaning the cells by using D-hanks liquid, adding recombinant pancreatin solution to digest the cells to make the cells fall off, adding D-hanks liquid to dilute, centrifuging, and re-suspending the cell precipitate by using the primary complete culture medium to obtain the primary placenta mesenchymal stem cells (namely the P0 generation).
The method according to the first aspect of the present invention, wherein in the step (2), 1% type II collagenase is added in an amount of 2 times the volume thereof for digestion with shaking for 30 min.
The method according to the first aspect of the present invention, wherein in the step (3), the centrifugation is performed under a condition of centrifugation at 100Xg for 5 min.
The method according to the first aspect of the present invention, wherein the inoculation into the culture flask in the step (4) according to the prescribed cell amount means that the cell amount is 1 to 5X 10 5 /cm 2 Inoculation into T225 flasks, for example, means 2X 10 cells 5 /cm 2 Inoculated into a T225 flask.
The process according to the first aspect of the present invention, wherein in the step (4), CO 2 The conditions for the culture in the incubator were: 5% CO 2 At 37 deg.C, saturated humidity.
The method according to the first aspect of the present invention, wherein in the step (5), 5ml of the recombinant pancreatin solution is added per vial for digesting the cells for 2 min.
The process according to the first aspect of the present invention, wherein in step (5), 25ml of D-hanks solution is added per bottle for dilution.
The method according to the first aspect of the present invention, wherein in the step (5), the centrifugation is carried out at 100Xg for 10 min.
The method according to the first aspect of the present invention wherein the formulation of the D-Hanks liquid is as follows: 8.0g NaCl, 0.4g KCl, 0.06g KH2PO4, 0.08g Na2HPO4.12H2O, 0.35g NaHCO3, water to 1000 ml. For example, the preparation method is as follows: dissolving the materials in 1000ml, and filtering with 0.22 μm microporous membrane for sterilization.
The method according to the first aspect of the invention, wherein said primary complete medium is prepared with DMEM-F12 medium as a matrix and comprises: 1% platelet lysate, 1% human serum albumin, 2. mu.g/ml recombinant insulin, 10ng/ml EGF, 20ng/ml bFGF.
The method according to the first aspect of the invention, wherein said primary complete medium is replaced by primary supplemented medium. The method according to the first aspect of the invention, wherein said primary supplementary medium is formulated in DMEM-F12 medium as a medium and comprises: 1% platelet lysate, 1% human serum albumin, 2. mu.g/ml recombinant insulin, 10ng/ml EGF, 20ng/ml bFGF, 0.12% thioglycerol, 1% fructose.
The method according to the first aspect of the present invention, wherein said DMEM-F12 medium formula consists of: 116.6mg of anhydrous calcium chloride, 59.05mg of L-leucine, 0.042mg of linoleic acid, 0.0013mg of copper sulfate pentahydrate, 91.25mg of L-lysine hydrochloride, 0.105mg of lipoic acid, 0.05mg of ferric nitrate nonahydrate, 17.24mg of L-methionine, 8.1mg of phenol red, 0.417mg of ferrous sulfate heptahydrate, 35.48mg of L-phenylalanine, 0.081mg of 1, 4-butanediamine dihydrochloride, 311.8mg of potassium chloride, 26.25mg of L-serine, 55mg of sodium pyruvate, 28.64mg of magnesium chloride, 53.45mg of L-threonine, 0.0035mg of vitamin H, 48.84mg of anhydrous magnesium sulfate, 4.45mg of L-alanine, 2.24mg of D-calcium pantothenate, 7000mg of sodium chloride, 7.5mg of L-asparagine, 8.98mg of choline chloride, 54.35mg of anhydrous sodium dihydrogen phosphate, 6.65mg of L-aspartic acid, 2.65mg of L-cysteine, 56.52 mg of L-disodium hydrogen phosphate, 17.12 mg of L-17 mg of inositol phosphate, 6.52 mg of L-asparagine, Zinc sulfate heptahydrate 0.432mg, L-glutamic acid 7.35mg, nicotinamide 2.02mg, L-arginine hydrochloride 147.5mg, L-proline 17.25mg, pyridoxal hydrochloride 2mg, L-cystine hydrochloride 31.29mg, L-tryptophan 9.02mg, pyridoxine hydrochloride 0.031mg, L-glutamine 365mg, L-tyrosine 38.4mg, riboflavin 0.219mg, glycine 18.75mg, L-valine 52.85mg, thiamine hydrochloride 2.17mg, L-histidine hydrochloride 31.48mg, D-glucose 3151mg, thymidine 0.365mg, L-isoleucine 54.47mg, hypoxanthine 2mg, vitamin B12 mg, and a proper amount of water added to 1000 mL; preparation: dissolving the materials in 1000ml, and filtering with 0.22 μm microporous membrane for sterilization.
The method according to the first aspect of the present invention, further comprising detecting the primary placental mesenchymal stem cells obtained by the isolated culture. For example, detection of cell morphology and/or immunophenotypic identification. In one embodiment, the immunophenotyping is detection of CD73, CD90, CD105 and CD19, CD11b, CD31, CD45, HLADR, CD 34. The primary placental mesenchymal stem cells obtained by the invention are positive in CD73, CD90 and CD105 (all more than 98%), and negative in CD19, CD11b, CD31, CD45, HLADR and CD34 (all less than 2%).
In the present invention, "10 ^ 6" indicates the power of 6 of 10 when indicating the number of cells; in the present invention, "cm ^ 2" represents a square centimeter when representing a culture area; other cases involving "^" symbols, all have similar meanings; the meaning of this symbol is also well known in the art.
Of the various process steps described above, although specific steps are described in some detail or in language specific to the process steps described in the examples of the following detailed description, those skilled in the art will be able to fully appreciate the above-described process steps from the detailed disclosure of the invention as a whole.
Any embodiment of any aspect of the invention may be combined with other embodiments, as long as they do not contradict. Furthermore, in any embodiment of any aspect of the invention, any feature may be applicable to that feature in other embodiments, so long as they do not contradict. The invention is further described below.
All documents cited herein are incorporated herein by reference in their entirety and to the extent they do not conform to the teachings of the present invention, the statements made therein shall control. Further, the various terms and phrases used herein have the ordinary meaning as is known to those skilled in the art, and even though such terms and phrases are intended to be described or explained in greater detail herein, reference is made to the term and phrase as being inconsistent with the known meaning and meaning as is accorded to such meaning throughout this disclosure.
In the present invention, the term "placental mesenchymal stem cells" refers to mesenchymal stem cells derived from the placenta. Thus in the present invention, and in particular in the context relating to the present invention, the term "placental mesenchymal stem cells" may be used interchangeably with "placental stem cells", "mesenchymal stem cells", unless otherwise specifically indicated.
In the present invention, the term "PBS buffer" or "PBS" refers to a phosphate buffer. The general formulation and formulation of the PBS used in the context of the present invention, as well as their general properties such as pH or pH range, are well known to those skilled in the art, and these PBS buffers are typically commercially available preformulations (or preps), e.g. the PBS used in the context of the present invention is typically a commercial buffer at pH7.4(± 0.1), such as HyClone brand PBS buffer; in the present invention, the composition of PBS buffer solution in the classical application of the art includes 137mM sodium chloride, 2.7nM potassium chloride and 10mM phosphate, and PBS used in the present invention has the same composition as that in the present invention, unless otherwise specified.
The method of the invention separates and cultures the placenta mesenchymal stem cells, and the obtained placenta mesenchymal stem cells have very high survival rate and very high yield. The present methods exhibit superior technical effects of one or more aspects as described herein.
Drawings
FIG. 1: microscopic cell morphology of mesenchymal stem cells (100 ×).
FIG. 2: directional differentiation potential of cells, A) adipogenic control, B) adipogenic induction; C) osteogenic control, D) osteogenic induction; E) chondrogenic control, F) chondrogenic induction.
Detailed Description
The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention. The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible.
In the present invention, the DMEM-F12 medium used in the experiments had the following formulation, unless otherwise specified: 116.6mg of anhydrous calcium chloride, 59.05mg of L-leucine, 0.042mg of linoleic acid, 0.0013mg of copper sulfate pentahydrate, 91.25mg of L-lysine hydrochloride, 0.105mg of lipoic acid, 0.05mg of ferric nitrate nonahydrate, 17.24mg of L-methionine, 8.1mg of phenol red, 0.417mg of ferrous sulfate heptahydrate, 35.48mg of L-phenylalanine, 0.081mg of 1, 4-butanediamine dihydrochloride, 311.8mg of potassium chloride, 26.25mg of L-serine, 55mg of sodium pyruvate, 28.64mg of magnesium chloride, 53.45mg of L-threonine, 0.0035mg of vitamin H, 48.84mg of anhydrous magnesium sulfate, 4.45mg of L-alanine, 2.24mg of D-calcium pantothenate, 7000mg of sodium chloride, 7.5mg of L-asparagine, 8.98mg of choline chloride, 54.35mg of anhydrous sodium dihydrogen phosphate, 6.65mg of L-aspartic acid, 2.65mg of L-cysteine, 56.52 mg of L-disodium hydrogen phosphate, 17.12 mg of L-17 mg of inositol phosphate, 6.52 mg of L-asparagine, 0.432mg of zinc sulfate heptahydrate, 7.35mg of L-glutamic acid, 2.02mg of nicotinamide, 147.5mg of L-arginine hydrochloride, 17.25mg of L-proline, 2mg of pyridoxal hydrochloride, 31.29mg of L-cystine hydrochloride, 9.02mg of L-tryptophan, 0.031mg of pyridoxine hydrochloride, 365mg of L-glutamine, 38.4mg of L-tyrosine, 0.219mg of riboflavin, 18.75mg of glycine, 52.85mg of L-valine, 2.17mg of thiamine hydrochloride, 31.48mg of L-histidine hydrochloride, 3151mg of D-glucose, 0.365mg of thymidine, 54.47mg of L-isoleucine, 2mg of hypoxanthine, 0.68mg of vitamin B12 and adding a proper amount of water to 1000 mL; preparation: dissolving the materials in 1000ml, and filtering with 0.22 μm microporous membrane for sterilization.
In the present invention, Platelet lysates used in the experiments can be readily purchased from the market, and as not specifically indicated herein, PLTGold Human Platelet Lysate from Sigma-Aldrich, having the product number SCM151, was used in the experiments.
In the present invention, collagenase type II used in the experiments can be easily obtained from the market, and as not specifically mentioned, used in the experiments herein is from Gibco.
In the present invention, bFGF (basic fibroblast growth factor) used in the test is readily available from the market, and as not specifically mentioned, it is used in the test herein from Sigma-Aldrich under the product number GF 003.
In the present invention, EGF (epidermal growth factor) used in the test is readily available from the market, and as not specifically mentioned herein, it is used in the test from Gibco under the reference PHG 0311L.
In the present invention, recombinant insulin used in the test is readily available from the market, and as not specifically mentioned, it is used in the test herein from Solarbio, having a product number of I8830.
In the present invention, the recombinant pancreatin solution used in the test can be easily obtained from the market, and as not specifically mentioned, the recombinant pancreatin solution of 2000u/ml concentration available from Rambox corporation, cat # RT2S01, is used in the test herein.
In the present invention, the D-Hanks solution used in the test, unless otherwise specified, was formulated and prepared as follows: 8.0g NaCl, 0.4g KCl, 0.06g KH2PO4, 0.08g Na2HPO4.12H2O, 0.35g NaHCO3 and water to 1000 ml; preparation: dissolving the materials in 1000ml, and filtering and sterilizing with 0.22 μm microporous membrane.
In the present invention, the primary complete medium used in the experiments was prepared with DMEM-F12 medium as a medium and contained, unless otherwise specified: 1% platelet lysate, 1% human serum albumin, 2. mu.g/ml recombinant insulin, 10ng/ml EGF, 20ng/ml bFGF.
In the present invention, the primary supplement medium used in the experiment was prepared with DMEM-F12 medium as a medium and contained: 1% platelet lysate, 1% human serum albumin, 2. mu.g/ml recombinant insulin, 10ng/ml EGF, 20ng/ml bFGF, 0.12% thioglycerol, 1% fructose.
In a specific experiment of the present invention, the prepared stem cells of a certain generation were sampled, nucleated cells, i.e., MSC cells were counted using a sysmex hemocytometer, cell viability was detected by trypan blue staining, and the samples were taken for microbial detection.
Example 1: isolation culture of primary placenta mesenchymal stem cells
(1) Placenta donated by volunteers transported to the laboratory via a cold chain AT 2-8 ℃ (sample AT) is processed in a biosafety cabinet;
(2) cleaning with D-Hanks solution, cutting placenta chorion, and cutting to 0.25 + -0.05 cm 2 Cleaning D-Hanks once, filtering with 300 mesh filter screen to remove residual blood cells in tissue, adding 2 times volume of 1% type II collagenase, and performing oscillatory digestion for 30 min;
(3) after digestion, adding one-time volume of D-hanks liquid for dilution, centrifuging at 100Xg for 5min, and leaving the cell sediment at the bottom layer for the next operation;
(4) taking the cell sediment obtained in the step (3), adding primary complete culture medium for heavy suspension, sampling and counting, and performing 2X 10 5 /cm 2 Inoculating into a T225 bottle; placing CO 2 Incubator (5% CO) 2 At 37 ℃, saturated humidity);
(5) and (3) culturing until the culture reaches the 4 th day, half replacing the culture solution until the culture reaches the 7 th day, completely replacing the culture solution until the cell fusion degree reaches more than 80 percent, removing the old culture medium, cleaning the cells by using D-hanks solution, adding 5ml of recombinant pancreatin solution into each bottle for digesting the cells for 2min to make the cells fall off, adding 25ml of D-hanks solution into each bottle for diluting, centrifuging at 100Xg for 10min, and re-suspending the cell precipitate by using a primary complete culture medium to obtain the primary placental mesenchymal stem cells (namely P0 generation).
The above steps (1) to (5) are performed at 2X 10 in step (4) 5 /cm 2 After inoculation into T225 flasks, a nucleated cell number (mean of 5 replicates) of 1.47 × 10^6(n ═ 5) was obtained per flask (this data can be referred to herein as cell harvest), and a cell viability of 94.6% (n ═ 5).
Example 1 a: isolation culture of primary placenta mesenchymal stem cells
The operations and materials from step (1) to step (3) are continued in example 1;
(4) the cell pellet obtained in step (3) of example 1 was taken, added to primary supplement medium for resuspension, sampled and counted in 2X 10 5 /cm 2 Inoculating into a T225 bottle; placing CO 2 Incubator (5% CO) 2 At 37 ℃, saturated humidity);
(5) and (3) performing half-liquid replacement until the culture reaches the 4 th day, performing full-liquid replacement until the cell fusion degree reaches more than 80% when the culture reaches the 7 th day, removing the old culture medium, cleaning the cells by using D-hanks liquid, adding 5ml of recombinant pancreatin solution into each bottle for digesting the cells for 2min to make the cells fall off, adding 25ml of D-hanks liquid into each bottle for dilution, centrifuging for 10min at 100Xg, and re-suspending the cell precipitate by using a primary supplement culture medium to obtain the primary placenta mesenchymal stem cells (namely P0 generation).
Example 1a Steps (1) to (5) described above were carried out at 2X 10 in step (4) 5 /cm 2 After inoculation into T225 flasks, a nucleated cell number (mean of 5 replicates) of 7.38 × 10^6(n ═ 5) was obtained per flask (this data can be referred to herein as cell harvest), and a cell viability of 92.6% (n ═ 5).
Example 1 b: primary placental mesenchymal stem cells were obtained as in example 1a, except that the primary supplemented medium was supplemented with thioglycerol and isolated for culture. The primary placental mesenchymal stem cells of example 1b had a cell harvest of 1.41 × 10^6(n ═ 5) and a cell viability of 91.1% (n ═ 5).
Example 1 c: primary placental mesenchymal stem cells were obtained as in example 1a, except that no fructose was added to the primary supplemented medium and isolated and cultured. The primary placental mesenchymal stem cells of example 1b had a cell harvest of 1.21 x 10^6(n ═ 5) and a cell viability of 93.7% (n ═ 5).
In this document, the embodiment 1a, the embodiment 1b and the embodiment 1c can also be referred to as an auxiliary example of the embodiment 1, while the embodiment 1 can be referred to as a main example, and they can be referred to as an example family.
Compared with the example 1, the cell viability rates of the example 1a, the example 1b and the example 1c are basically the same and are all in the range of 91-95%; however, the cell harvest was about 5.02 times that of example 1a, about 0.96 times that of example 1b, and about 0.82 times that of example 1c for example 1 a; these unexpected findings indicate that by adding a small amount of cheap thioglycerol and fructose to the primary complete culture medium, not only can primary stem cells with substantially equivalent cell viability be obtained, but also the harvesting yield of the cells is promoted to be increased by about 5 times, and the harvesting yield of the cells is increased substantially without changing the production cost. However, when only thioglycerol or only fructose was added, although the cell viability rate did not change, the cell harvest did not increase, and even when only thioglycerol was additionally added, the cell harvest was significantly reduced.
In terms of cell viability, the main examples 2-8 and their respective subsidiary a, b, c examples also show substantially the same results as those of the above-mentioned example 1 and its subsidiary examples (example 1a, example 1b, example 1c), and the cell viability is in the range of 90-95%, for example, the cell viability of the primary mesenchymal stem cells obtained in example 2 and the fourth of example 2a, example 2b, example 2c is 91.6%, 94.2%, 92.6%, 93.1%.
In terms of cell harvest, the main examples 2-8 and their respective auxiliary examples a, b, c also show substantially the same trend and even result as the above-mentioned example 1 and its auxiliary examples (example 1a, example 1b, example 1c), the cell harvest of the main examples 2-8 is in the range of (1.31-1.64) × 10^6, the cell harvest of the group a auxiliary examples is 4.83-5.61 times of their respective main examples, the cell harvest of the group b auxiliary examples is 0.93-1.16 times of their respective main examples, and the cell harvest of the group c auxiliary examples is 0.73-0.87 times of their respective main examples; for example, the cell harvest yields (n ═ 5) of examples 2, 2a, 2b and 2c were 1.56X 10^6, 8.38X 10^6(5.37 times), 1.74X 10^6(1.12 times) and 1.32X 10^6(0.85 times), respectively.
The primary placental mesenchymal stem cells obtained from examples 1-8 and their respective appendixes a, b and c were tested, and the morphology was normal, and immunophenotyping showed that each primary placental mesenchymal stem cell was positive for CD73, CD90 and CD105 (both greater than 98%, for example, CD73 obtained from example 1 was greater than 99.2%), negative for CD19, CD11b, CD31, CD45, HLADR and CD34 (both less than 2%, for example, CD19 obtained from example 1 was less than 0.86%).
Example 2: isolation culture of primary placenta mesenchymal stem cells
(1) Placenta from volunteer donations (sample BT) transported to the laboratory via the cold chain at 2-8 ℃ was processed in a biosafety cabinet;
(2) cleaning with D-Hanks solution, cutting placenta chorion, and cutting to 0.25 + -0.05 cm 2 Cleaning D-Hanks once, filtering with 300 mesh filter screen to remove residual blood cells in tissue, adding 2 times volume of 1% type II collagenase, and performing oscillatory digestion for 30 min;
(3) after digestion, adding one-time volume of D-hanks liquid for dilution, centrifuging for 5min at 100Xg, and reserving the cell sediment at the bottom layer for next operation;
(4) taking the cell sediment obtained in the step (3), adding primary complete culture medium for heavy suspension, sampling and counting, and performing 2X 10 5 /cm 2 Inoculating into a T225 bottle; placing CO 2 Incubator (5% CO) 2 37 ℃, saturated humidity);
(5) and (3) half-changing the culture solution when the culture is carried out till the 4 th day, completely changing the culture solution when the culture is carried out till the 7 th day, removing the old culture medium after the cell fusion degree reaches more than 80% (10 th-11 th day), cleaning the cells by using D-hanks solution, adding 5ml of recombinant pancreatin solution into each bottle to digest the cells for 2min so as to make the cells fall off, adding 25ml of D-hanks solution into each bottle for dilution, centrifuging at 100Xg for 10min, and re-suspending the cell precipitate by using a primary complete culture medium to obtain the placenta primary mesenchymal stem cells (namely, the generation P0).
Example 2 a: isolation culture of primary placenta mesenchymal stem cells
The operations and materials from step (1) to step (3) were continued in example 2;
(4) taking the cell pellet obtained in step (3) of example 2, adding primary supplement medium to resuspend, sampling and counting according to 2X 10 5 /cm 2 Inoculating into a T225 bottle; placing CO 2 Incubator (5% CO) 2 At 37 ℃, saturated humidity);
(5) and (3) performing half-liquid replacement until the culture reaches the 4 th day, performing full-liquid replacement until the cell fusion degree reaches more than 80% when the culture reaches the 7 th day, removing the old culture medium, cleaning the cells by using D-hanks liquid, adding 5ml of recombinant pancreatin solution into each bottle for digesting the cells for 2min to make the cells fall off, adding 25ml of D-hanks liquid into each bottle for dilution, centrifuging for 10min at 100Xg, and re-suspending the cell precipitate by using a primary supplement culture medium to obtain the primary placenta mesenchymal stem cells (namely P0 generation).
Example 2 b: primary placental mesenchymal stem cells were obtained as in example 2a, except that the primary supplemented medium was supplemented with thioglycerol and isolated for culture.
Example 2 c: primary placental mesenchymal stem cells were obtained as in example 2a, except that no fructose was added to the primary supplemented medium and isolated and cultured.
Example 3: isolation culture of primary placenta mesenchymal stem cells
(1) Placenta from volunteer donations transported to the laboratory via the cold chain at 2-8 ℃ (sample CT) was processed in a biosafety cabinet;
(2) washing with D-Hanks solution, cutting placenta chorion, and cutting to 0.25 + -0.05 cm 2 Cleaning D-Hanks once, filtering with 300 mesh filter screen to remove residual blood cells in tissue, adding 2 times volume of 1% type II collagenase, and performing oscillatory digestion for 30 min;
(3) after digestion, adding one-time volume of D-hanks liquid for dilution, centrifuging for 5min at 100Xg, and reserving the cell sediment at the bottom layer for next operation;
(4) taking the cell sediment obtained in the step (3), adding primary complete culture medium for heavy suspension, sampling and counting, and performing 2X 10 5 /cm 2 Inoculating into a T225 bottle; placing CO 2 Incubator (5% CO) 2 At 37 ℃, saturated humidity);
(5) and (3) half-changing the culture solution when the culture is carried out till the 4 th day, completely changing the culture solution when the culture is carried out till the 7 th day, removing the old culture medium after the cell fusion degree reaches more than 80% (10 th-11 th day), cleaning the cells by using D-hanks solution, adding 5ml of recombinant pancreatin solution into each bottle to digest the cells for 2min so as to make the cells fall off, adding 25ml of D-hanks solution into each bottle for dilution, centrifuging at 100Xg for 10min, and re-suspending the cell precipitate by using a primary complete culture medium to obtain the placenta primary mesenchymal stem cells (namely, the generation P0).
Example 3 a: isolation culture of primary placenta mesenchymal stem cells
The operations and materials from step (1) to step (3) are continued in example 3;
(4) the fine powder obtained in step (3) of example 3 was takenPrecipitating cells, adding primary supplement medium for resuspension, sampling and counting according to 2 × 10 5 /cm 2 Inoculating into a T225 bottle; placing CO 2 Incubator (5% CO) 2 At 37 ℃, saturated humidity);
(5) and (3) performing half-liquid replacement until the culture reaches the 4 th day, performing full-liquid replacement until the cell fusion degree reaches more than 80% when the culture reaches the 7 th day, removing the old culture medium, cleaning the cells by using D-hanks liquid, adding 5ml of recombinant pancreatin solution into each bottle for digesting the cells for 2min to make the cells fall off, adding 25ml of D-hanks liquid into each bottle for dilution, centrifuging for 10min at 100Xg, and re-suspending the cell precipitate by using a primary supplement culture medium to obtain the primary placenta mesenchymal stem cells (namely P0 generation).
Example 3 b: primary placental mesenchymal stem cells were obtained as in example 3a, except that the primary supplemented medium was supplemented with thioglycerol and isolated for culture.
Example 3 c: the procedure of example 3a was followed except that fructose was not added to the primary supplemental medium, and isolated to obtain primary placental mesenchymal stem cells.
Example 4: isolation culture of primary placenta mesenchymal stem cells
(1) Placenta donated by volunteers (sample DT) cold-chain transported to the laboratory at 2-8 ℃ was processed in a biosafety cabinet;
(2) cleaning with D-Hanks solution, cutting placenta chorion, and cutting to 0.25 + -0.05 cm 2 Cleaning D-Hanks once, filtering with 300 mesh filter screen to remove residual blood cells in tissue, adding 2 times volume of 1% type II collagenase, and performing oscillatory digestion for 30 min;
(3) after digestion, adding one-time volume of D-hanks liquid for dilution, centrifuging at 100Xg for 5min, and leaving the cell sediment at the bottom layer for the next operation;
(4) taking the cell sediment obtained in the step (3), adding primary complete culture medium for heavy suspension, sampling and counting, and performing 2X 10 5 /cm 2 Inoculating into a T225 bottle; placing CO 2 Incubator (5% CO) 2 At 37 ℃, saturated humidity);
(5) and (3) culturing until the culture reaches the 4 th day, half replacing the culture solution until the culture reaches the 7 th day, completely replacing the culture solution until the cell fusion degree reaches more than 80 percent, removing the old culture medium, cleaning the cells by using D-hanks solution, adding 5ml of recombinant pancreatin solution into each bottle for digesting the cells for 2min to make the cells fall off, adding 25ml of D-hanks solution into each bottle for diluting, centrifuging at 100Xg for 10min, and re-suspending the cell precipitate by using a primary complete culture medium to obtain the primary placental mesenchymal stem cells (namely P0 generation).
Example 4 a: isolation culture of primary placenta mesenchymal stem cells
The operations and materials from step (1) to step (3) were continued in example 4;
(4) taking the cell pellet obtained in step (3) of example 4, adding primary supplement medium to resuspend, sampling and counting according to 2X 10 5 /cm 2 Inoculating into a T225 bottle; placing CO 2 Incubator (5% CO) 2 At 37 ℃, saturated humidity);
(5) and (3) half-replacing the culture medium when the culture is carried out till the 4 th day, completely replacing the culture medium when the culture is carried out till the 7 th day, removing the old culture medium after the cell fusion degree reaches more than 80% (10 th-11 th day), cleaning the cells by using D-hanks liquid, adding 5ml of recombinant pancreatin solution into each bottle to digest the cells for 2min so as to make the cells fall off, adding 25ml of D-hanks liquid into each bottle for dilution, centrifuging at 100Xg for 10min, and re-suspending the cell sediment by using a primary supplement culture medium to obtain the placenta primary mesenchymal stem cells (namely, the generation P0).
Example 4 b: primary placental mesenchymal stem cells were obtained as in example 4a, except that the primary supplemented medium was supplemented with thioglycerol and isolated for culture.
Example 4 c: the procedure of example 4a was followed except that fructose was not added to the primary supplemental medium, and isolated to obtain primary placental mesenchymal stem cells.
Example 5: isolation culture of primary placenta mesenchymal stem cells
(1) Placenta from volunteer donations (sample ET) transported to the laboratory via the cold chain at 2-8 ℃ was processed in a biosafety cabinet;
(2) cleaning with D-Hanks solution, cutting placenta chorion, and cutting to 0.25 + -0.05 cm 2 Size, D-Hanks Wash once and filter with 300 mesh filter to remove residual blood fines from the tissueAdding 1% type II collagenase with 2 times volume for 30 min;
(3) after digestion, adding one-time volume of D-hanks liquid for dilution, centrifuging at 100Xg for 5min, and leaving the cell sediment at the bottom layer for the next operation;
(4) taking the cell sediment obtained in the step (3), adding primary complete culture medium for heavy suspension, sampling and counting, and performing 2X 10 5 /cm 2 Inoculating into a T225 bottle; placing CO 2 Incubator (5% CO) 2 37 ℃, saturated humidity);
(5) and (3) half-changing the culture solution when the culture is carried out till the 4 th day, completely changing the culture solution when the culture is carried out till the 7 th day, removing the old culture medium after the cell fusion degree reaches more than 80% (10 th-11 th day), cleaning the cells by using D-hanks solution, adding 5ml of recombinant pancreatin solution into each bottle to digest the cells for 2min so as to make the cells fall off, adding 25ml of D-hanks solution into each bottle for dilution, centrifuging at 100Xg for 10min, and re-suspending the cell precipitate by using a primary complete culture medium to obtain the placenta primary mesenchymal stem cells (namely, the generation P0).
Example 5 a: isolation culture of primary placenta mesenchymal stem cells
The operations and materials of the steps (1) to (3) are continued in the example 5;
(4) taking the cell pellet obtained in step (3) of example 5, adding primary supplement medium to resuspend, sampling and counting according to 2X 10 5 /cm 2 Inoculating into a T225 bottle; placing CO 2 Incubator (5% CO) 2 At 37 ℃, saturated humidity);
(5) and (3) half-replacing the culture medium when the culture is carried out till the 4 th day, completely replacing the culture medium when the culture is carried out till the 7 th day, removing the old culture medium after the cell fusion degree reaches more than 80% (10 th-11 th day), cleaning the cells by using D-hanks liquid, adding 5ml of recombinant pancreatin solution into each bottle to digest the cells for 2min so as to make the cells fall off, adding 25ml of D-hanks liquid into each bottle for dilution, centrifuging at 100Xg for 10min, and re-suspending the cell sediment by using a primary supplement culture medium to obtain the placenta primary mesenchymal stem cells (namely, the generation P0).
Example 5 b: primary placental mesenchymal stem cells were obtained as in example 5a, except that the primary supplemented medium was supplemented with thioglycerol and isolated for culture.
Example 5 c: primary placental mesenchymal stem cells were obtained as in example 5a, except that no fructose was added to the primary supplemented medium and isolated and cultured.
Example 6: isolation culture of primary placenta mesenchymal stem cells
(1) Placenta from volunteer donors (sample FT) transported to the laboratory via the cold chain at 2-8 ℃ was processed in a biosafety cabinet;
(2) cleaning with D-Hanks solution, cutting placenta chorion, and cutting to 0.25 + -0.05 cm 2 Cleaning D-Hanks once, filtering with 300 mesh filter screen to remove residual blood cells in tissue, adding 2 times volume of 1% type II collagenase, and performing oscillatory digestion for 30 min;
(3) after digestion, adding one-time volume of D-hanks liquid for dilution, centrifuging for 5min at 100Xg, and reserving the cell sediment at the bottom layer for next operation;
(4) taking the cell sediment obtained in the step (3), adding primary complete culture medium for heavy suspension, sampling and counting, and performing 2X 10 5 /cm 2 Inoculating into a T225 bottle; placing CO 2 Incubator (5% CO) 2 37 ℃, saturated humidity);
(5) and (3) half-changing the culture solution when the culture is carried out till the 4 th day, completely changing the culture solution when the culture is carried out till the 7 th day, removing the old culture medium after the cell fusion degree reaches more than 80% (10 th-11 th day), cleaning the cells by using D-hanks solution, adding 5ml of recombinant pancreatin solution into each bottle to digest the cells for 2min so as to make the cells fall off, adding 25ml of D-hanks solution into each bottle for dilution, centrifuging at 100Xg for 10min, and re-suspending the cell precipitate by using a primary complete culture medium to obtain the placenta primary mesenchymal stem cells (namely, the generation P0).
Example 6 a: isolation culture of primary placenta mesenchymal stem cells
The operations and materials of the steps (1) to (3) are continued in the example 6;
(4) the cell pellet obtained in step (3) of example 6 was taken, added to primary supplement medium for resuspension, sampled and counted in 2X 10 5 /cm 2 Inoculating into a T225 bottle; placing CO 2 Incubator (5% CO) 2 At 37 ℃, saturated humidity);
(5) and (3) half-replacing the culture medium when the culture is carried out till the 4 th day, completely replacing the culture medium when the culture is carried out till the 7 th day, removing the old culture medium after the cell fusion degree reaches more than 80% (10 th-11 th day), cleaning the cells by using D-hanks liquid, adding 5ml of recombinant pancreatin solution into each bottle to digest the cells for 2min so as to make the cells fall off, adding 25ml of D-hanks liquid into each bottle for dilution, centrifuging at 100Xg for 10min, and re-suspending the cell sediment by using a primary supplement culture medium to obtain the placenta primary mesenchymal stem cells (namely, the generation P0).
Example 6 b: primary placental mesenchymal stem cells were obtained as in example 6a, except that the primary supplemented medium was supplemented with thioglycerol and isolated for culture.
Example 6 c: primary placental mesenchymal stem cells were obtained as in example 6a, except that no fructose was added to the primary supplemented medium and isolated and cultured.
Example 7: isolation culture of primary placenta mesenchymal stem cells
(1) Placenta from volunteer donations (sample GT) transported to the laboratory via the cold chain at 2-8 ℃ was processed in a biosafety cabinet;
(2) cleaning with D-Hanks solution, cutting placenta chorion, and cutting to 0.25 + -0.05 cm 2 Cleaning D-Hanks once, filtering with 300 mesh filter screen to remove residual blood cells in tissue, adding 2 times volume of 1% type II collagenase, and performing shake digestion for 30 min;
(3) after digestion, adding one-time volume of D-hanks liquid for dilution, centrifuging for 5min at 100Xg, and reserving the cell sediment at the bottom layer for next operation;
(4) taking the cell sediment obtained in the step (3), adding primary complete culture medium for heavy suspension, sampling and counting, and performing 2X 10 5 /cm 2 Inoculating into a T225 bottle; placing CO 2 Incubator (5% CO) 2 At 37 ℃, saturated humidity);
(5) and (3) culturing until the culture reaches the 4 th day, half replacing the culture solution until the culture reaches the 7 th day, completely replacing the culture solution until the cell fusion degree reaches more than 80 percent, removing the old culture medium, cleaning the cells by using D-hanks solution, adding 5ml of recombinant pancreatin solution into each bottle for digesting the cells for 2min to make the cells fall off, adding 25ml of D-hanks solution into each bottle for diluting, centrifuging at 100Xg for 10min, and re-suspending the cell precipitate by using a primary complete culture medium to obtain the primary placental mesenchymal stem cells (namely P0 generation).
Example 7 a: isolation culture of primary placenta mesenchymal stem cells
The operations and materials from step (1) to step (3) were continued in example 7;
(4) taking the cell pellet obtained in step (3) of example 7, adding primary supplement medium to resuspend, sampling and counting according to 2X 10 5 /cm 2 Inoculating into a T225 bottle; placing CO 2 Incubator (5% CO) 2 At 37 ℃, saturated humidity);
(5) and (3) performing half-liquid replacement until the culture reaches the 4 th day, performing full-liquid replacement until the cell fusion degree reaches more than 80% when the culture reaches the 7 th day, removing the old culture medium, cleaning the cells by using D-hanks liquid, adding 5ml of recombinant pancreatin solution into each bottle for digesting the cells for 2min to make the cells fall off, adding 25ml of D-hanks liquid into each bottle for dilution, centrifuging for 10min at 100Xg, and re-suspending the cell precipitate by using a primary supplement culture medium to obtain the primary placenta mesenchymal stem cells (namely P0 generation).
Example 7 b: primary placental mesenchymal stem cells were obtained as in example 7a, except that the primary supplemented medium was supplemented with thioglycerol and isolated for culture.
Example 7 c: the procedure of example 7a was followed except that fructose was not added to the primary supplemental medium, and isolated to obtain primary placental mesenchymal stem cells.
Example 8: isolation culture of primary placenta mesenchymal stem cells
(1) Placenta donated by volunteers transported to the laboratory via the cold chain at 2-8 ℃ (sample HT) is processed in a biosafety cabinet;
(2) washing with D-Hanks solution, cutting placenta chorion, and cutting to 0.25 + -0.05 cm 2 Cleaning D-Hanks once, filtering with 300 mesh filter screen to remove residual blood cells in tissue, adding 2 times volume of 1% type II collagenase, and performing oscillatory digestion for 30 min;
(3) after digestion, adding one-time volume of D-hanks liquid for dilution, centrifuging at 100Xg for 5min, and leaving the cell sediment at the bottom layer for the next operation;
(4) Taking the cell sediment obtained in the step (3), adding primary complete culture medium for heavy suspension, sampling and counting, and performing 2X 10 5 /cm 2 Inoculating into a T225 bottle; placing CO 2 Incubator (5% CO) 2 At 37 ℃, saturated humidity);
(5) and (3) culturing until the culture reaches the 4 th day, half replacing the culture solution until the culture reaches the 7 th day, completely replacing the culture solution until the cell fusion degree reaches more than 80 percent, removing the old culture medium, cleaning the cells by using D-hanks solution, adding 5ml of recombinant pancreatin solution into each bottle for digesting the cells for 2min to make the cells fall off, adding 25ml of D-hanks solution into each bottle for diluting, centrifuging at 100Xg for 10min, and re-suspending the cell precipitate by using a primary complete culture medium to obtain the primary placental mesenchymal stem cells (namely P0 generation).
Example 8 a: isolation culture of primary placenta mesenchymal stem cells
The operations and materials from the step (1) to the step (3) are continued in the example 8;
(4) the cell pellet obtained in step (3) of example 8 was taken, added to primary supplement medium for resuspension, sampled and counted in 2X 10 5 /cm 2 Inoculating into a T225 bottle; placing CO 2 Incubator (5% CO) 2 37 ℃, saturated humidity);
(5) and (3) performing half-liquid replacement until the culture reaches the 4 th day, performing full-liquid replacement until the cell fusion degree reaches more than 80% when the culture reaches the 7 th day, removing the old culture medium, cleaning the cells by using D-hanks liquid, adding 5ml of recombinant pancreatin solution into each bottle for digesting the cells for 2min to make the cells fall off, adding 25ml of D-hanks liquid into each bottle for dilution, centrifuging for 10min at 100Xg, and re-suspending the cell precipitate by using a primary supplement culture medium to obtain the primary placenta mesenchymal stem cells (namely P0 generation).
Example 8 b: the procedure of example 8a was followed except that thioglycerol was not added to the primary supplement medium, and isolated and cultured to obtain primary placental mesenchymal stem cells.
Example 8 c: the procedure of example 8a was followed except that fructose was not added to the primary supplemental medium, and isolated to obtain primary placental mesenchymal stem cells.
The subculture of the mesenchymal stem cells of generation P0 prepared in examples 1 to 8 and their respective attached a, b and c shows that all the cells can be continuously passaged to more than P10 generation, the cells can still maintain stable continuous proliferation capacity, and the cell viability of each generation is stable to more than 89% during continuous passage, for example, the cell viability of a batch of mesenchymal stem cells of generation P0 obtained in example 1a is stable to 94.1% when the batch of mesenchymal stem cells of generation P10 is continuously passaged.
Test example 1: detection of mesenchymal stem cells
Typical characteristics of mesenchymal stem cells include: microscopic fusiform and adherent growth, flow cytometry identification shows that CD73, CD90 and CD105 are all positive and CD19, CD11b, CD31, CD45, HLADR and CD34 are all negative, and a directional differentiation potential test shows that the cells present the differentiation potential of osteogenesis, chondrogenesis and adipogenesis.
The mesenchymal stem cells prepared in the embodiments 1 to 8 and the auxiliary a, b and c examples thereof are detected by using a method known in the field, and the results are as follows: all mesenchymal stem cells exhibited spindle and adherent growth (e.g., microscopic cell morphology of the mesenchymal stem cells of generation P0 obtained in example 1a is shown in fig. 1), CD73, CD90 and CD105 of all mesenchymal stem cells were greater than 98% (e.g., CD73 ═ 99.6%, CD90 ═ 99.4%, CD105 ═ 99.9% of a batch of the mesenchymal stem cells of generation P0 obtained in example 1 a), CD19, CD11b, CD31, CD45 of all mesenchymal stem cells, HLADR and CD34 are less than 2% (for example, the batch of the P0-generation mesenchymal stem cells obtained in example 1a has CD19 ═ 0.32%, CD11b ═ 0.14%, CD31 ═ 0.24%, CD45 ═ 0.17%, HLADR ═ 0.03%, and CD34 ═ 0.26%), and the directed differentiation potential test shows that all the mesenchymal stem cells have osteogenic, chondrogenic and adipogenic differentiation potential (for example, the batch of the P0-generation mesenchymal stem cells obtained in example 1a has osteogenic, chondrogenic and adipogenic differentiation potential results shown in fig. 2).
The above-described embodiments are merely preferred embodiments for fully illustrating the present application, and the scope of the present application is not limited thereto. The equivalent substitution or change made by the person skilled in the art on the basis of the present application is within the protection scope of the present application. The protection scope of this application is subject to the claims.

Claims (14)

1. The method for separating and culturing the primary placenta mesenchymal stem cells comprises the following steps:
(1) processing a placenta sample transported to a laboratory via a cold chain at 2-8 ℃ in a biosafety cabinet;
(2) cleaning with D-Hanks solution, cutting placenta chorion, cutting into pieces, cleaning with D-Hanks solution, filtering with 300 mesh filter screen to remove residual blood cells, adding 1% collagenase II, and performing oscillatory digestion;
(3) after digestion, adding one-time volume of D-hanks liquid for dilution, centrifuging, and reserving the cell sediment at the bottom layer for the next operation;
(4) taking the cell sediment obtained in the step (3), adding a primary supplement culture medium for resuspension, sampling and counting, and inoculating the cell sediment into a culture bottle according to the specified cell quantity; placing CO 2 Culturing in an incubator;
(5) half-replacing the culture medium until the culture reaches the 4 th day, completely replacing the culture medium until the cell fusion degree reaches more than 80%, removing the old culture medium, cleaning the cells by using D-hanks liquid, adding recombinant pancreatin solution to digest the cells to make the cells fall off, adding the D-hanks liquid to dilute, centrifuging, and resuspending the cell sediment by using a primary supplementary culture medium to obtain primary placenta mesenchymal stem cells;
wherein the primary supplementary culture medium is prepared by taking DMEM-F12 as a matrix and comprises: 1% platelet lysate, 1% human serum albumin, 2. mu.g/ml recombinant insulin, 10ng/ml EGF, 20ng/ml bFGF, 0.12% thioglycerol, 1% fructose.
2. The method according to claim 1, wherein in the step (2), 1% collagenase type II is added in an amount of 2 times the volume of the mixture for digestion with shaking for 30 min.
3. The method according to claim 1, wherein in step (2), the placenta sample is minced to 0.25 ± 0.05cm 2 Size.
4. The method according to claim 1, wherein in step (3), the centrifugation is performed at 100Xg for 5 min.
5. The method according to claim 1, wherein the step (4) of inoculating the culture flask with the predetermined amount of cells is performed in an amount of 1 to 5X 10 cells 5 /cm 2 Inoculated into a T225 flask.
6. The method according to claim 1, wherein in the step (4), the inoculation into the culture flask in accordance with the specified cell amount means that the inoculation is carried out in accordance with the cell amount of 2X 10 5 /cm 2 Inoculated into a T225 flask.
7. The method according to claim 1, wherein in the step (4), CO 2 The conditions for the culture in the incubator were: 5% CO 2 At 37 deg.C, saturated humidity.
8. The method according to claim 1, wherein in the step (5), 5ml of the recombinant pancreatin solution is added per vial for digesting the cells for 2 min.
9. The process of claim 1, wherein in step (5), 25ml of D-hanks solution is added per bottle for dilution.
10. The method according to claim 1, wherein in step (5), centrifugation is performed at 100Xg for 10 min.
11. The method of claim 1 wherein the D-Hanks liquid is formulated as follows: 8.0g NaCl, 0.4g KCl, 0.06g KH2PO4, 0.08g Na2HPO4.12H2O, 0.35g NaHCO3, water to 1000 ml.
12. The method of claim 11, wherein the D-Hanks solution is formulated as follows: dissolving each material by 1000ml, and filtering and sterilizing by a 0.22 mu m microporous filter membrane to obtain the product.
13. The method according to claim 1, wherein said DMEM-F12 medium formulation consists of: 116.6mg of anhydrous calcium chloride, 59.05mg of L-leucine, 0.042mg of linoleic acid, 0.0013mg of copper sulfate pentahydrate, 91.25mg of L-lysine hydrochloride, 0.105mg of lipoic acid, 0.05mg of ferric nitrate nonahydrate, 17.24mg of L-methionine, 8.1mg of phenol red, 0.417mg of ferrous sulfate heptahydrate, 35.48mg of L-phenylalanine, 0.081mg of 1, 4-butanediamine dihydrochloride, 311.8mg of potassium chloride, 26.25mg of L-serine, 55mg of sodium pyruvate, 28.64mg of magnesium chloride, 53.45mg of L-threonine, 0.0035mg of vitamin H, 48.84mg of anhydrous magnesium sulfate, 4.45mg of L-alanine, 2.24mg of D-calcium pantothenate, 7000mg of sodium chloride, 7.5mg of L-asparagine, 8.98mg of choline chloride, 54.35mg of anhydrous sodium dihydrogen phosphate, 6.65mg of L-aspartic acid, 2.65mg of L-cysteine, 56.52 mg of L-disodium hydrogen phosphate, 17.12 mg of L-17 mg of inositol phosphate, 6.52 mg of L-asparagine, 0.432mg of zinc sulfate heptahydrate, 7.35mg of L-glutamic acid, 2.02mg of nicotinamide, 147.5mg of L-arginine hydrochloride, 17.25mg of L-proline, 2mg of pyridoxal hydrochloride, 31.29mg of L-cystine hydrochloride, 9.02mg of L-tryptophan, 0.031mg of pyridoxine hydrochloride, 365mg of L-glutamine, 38.4mg of L-tyrosine, 0.219mg of riboflavin, 18.75mg of glycine, 52.85mg of L-valine, 2.17mg of thiamine hydrochloride, 31.48mg of L-histidine hydrochloride, 3151mg of D-glucose, 0.365mg of thymidine, 54.47mg of L-isoleucine, 2mg of hypoxanthine, 0.68mg of vitamin B12 and a proper amount of water added to 1000 mL.
14. The method according to claim 1, further comprising performing cell morphology detection and/or immunophenotyping on the isolated and cultured primary placental mesenchymal stem cells; said immunophenotypic identification refers to the detection of CD73, CD90, CD105 and CD19, CD11b, CD31, CD45, HLADR, CD 34.
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