CN108251358B

CN108251358B - Multi-batch primary separation method of human mesenchymal stem cells from same donor source

Info

Publication number: CN108251358B
Application number: CN201711347861.7A
Authority: CN
Inventors: 袁茵; 鲁欣; 匡梅娜; 黄思瑞
Original assignee: Guangdong Pharmaceutical University
Current assignee: Guangdong Pharmaceutical University
Priority date: 2017-12-15
Filing date: 2017-12-15
Publication date: 2021-10-26
Anticipated expiration: 2037-12-15
Also published as: CN108251358A

Abstract

The invention discloses a multi-batch primary separation method of human mesenchymal stem cells from the same donor source, belonging to the field of cell culture. The method of the invention is to treat donor tissue to obtain 1-5mm³The donor tissue small blocks are soaked by fetal bovine serum or human AB serum, and then the conventional adherent culture of the tissue blocks is carried out. When the mesenchymal stem cells are harvested, the donor tissue block is subjected to plate-rotating continuous culture, and the culture supernatant of the tissue block is recovered and continuously incubated, so that high-quality and multi-batch stable separation of the human mesenchymal stem cells from the same donor is realized. The morphology, proliferation and differentiation capacity and immunophenotype of the cells obtained by the invention are kept unchanged among batches. Compared with the traditional tissue mass adherence method and the enzyme digestion method, the method disclosed by the invention avoids the waste of clinical specimen resources, saves manpower and material resources in clinical links, and also improves the separation efficiency and yield of the mesenchymal stem cells.

Description

Multi-batch primary separation method of human mesenchymal stem cells from same donor source

Technical Field

The invention belongs to the field of cell culture, and particularly relates to a multi-batch primary separation method of human mesenchymal stem cells from the same donor.

Background

Mesenchymal Stem Cells (MSCs) have high self-renewal and multi-directional differentiation ability, can be differentiated into mesenchymal cells such as fat, bone, cartilage, muscle and the like, have low immunogenicity and immunosuppressive effects, and are important seed cells for tissue engineering, cell replacement therapy, transplantation immunity, autoimmune disease and the like.

In the seventies of the last century, Friedenstein and colleagues first cultured mesenchymal stem cells from bone marrow. In recent years, researchers have subsequently isolated MSCs from other tissues, such as fat, deciduous teeth, and umbilical cord, placenta, amnion, and umbilical cord blood of newborns. Among them, umbilical cord tissue of newborn has the advantages of abundant MSCs, convenient material acquisition and no ethical dispute, and is considered as an ideal source for acquiring MSCs.

Human umbilical cord mesenchymal stem cells (hUC-MSCs) are fetal stem cells which are different from embryonic stem cells and adult stem cells, are novel multipotent mesenchymal stem cells, and have original tissue sources, stable biological properties, strong proliferation and differentiation capacity and low immunogenicity. The hUC-MSCs are mainly present in Wharton's jelly of umbilical cord and the endothelial lower layer of umbilical vein, and can be obtained by enzyme digestion and tissue mass adherence.

The following specifically describes the specific operation steps of the two methods of enzymatic digestion and tissue block adherence in the prior art.

(1) Enzyme digestion (Single enzyme method for example)

The cord was cut into pieces of about 2cm long, and the cord blood was removed by repeating washing 2 to 3 times with 1% double antibody-containing PBS buffer. The umbilical cord is cut off longitudinally, and three blood vessels are removed. Cutting the rest umbilical cord tissue into small pieces, placing into a small beaker, and cutting into pieces of 1-2mm³Size of tissue pieces. The tissue pieces were transferred to a 100ml sterile glass bottle containing a magnetic stirrer, collagenase II was added to a final concentration of 0.1%, and digestion was carried out with continuous magnetic gentle stirring in a water bath at 37 ℃ for 2 h. Adding PBS buffer solution to wash collagen, centrifuging at 2000rpm for 15min, discarding supernatant, and keeping precipitate. PBS was added to suspend the tissue, filtered through a nylon mesh, the cell filtrate was collected, centrifuged at 1000rpm for 5min, and the supernatant was discarded. The collected cells were resuspended in DMEM/F12 complete medium, blown evenly, counted, inoculated into a cell culture flask, and cultured in a cell culture box at 37 ℃ with 5% carbon dioxide and saturated humidity. After 24h, the solution was changed for the first time to remove the suspended cells. Changing the solution every 3 days later until the cell content reaches 80%After-90% fusion, conventional subculture was performed.

(2) Tissue block wall pasting method

The cord was cut into pieces of about 2cm long, and the cord blood was removed by repeating washing 2 to 3 times with 1% double antibody-containing PBS buffer. The umbilical cord is cut off longitudinally and the blood vessels are removed. The remaining umbilical cord tissue is cut into smaller tissue pieces (usually 1 mm)³) The culture medium is evenly spread in a culture vessel according to a certain distance, and the subsequent operation is divided into a turning drying method and a thin-layer culture method:

a method of 'tumble drying': the culture dish is turned over gently and placed in a carbon dioxide incubator at 37 ℃ for culture for 4-6 hours, when the tissue small blocks are dried up slightly, the culture dish is turned over slowly, a small amount of complete culture medium is added slowly to ensure that the culture solution can submerge the tissue small blocks, and the culture dish is placed back into the culture box again for standing culture;

the "thin layer culture" method: placing the culture dish with the tissue blocks right, standing for 30 minutes to make the tissue blocks adhere to the wall better, slowly adding a small amount of culture solution to cover the surface of the tissue blocks, and standing and culturing in a carbon dioxide incubator at 37 ℃.

And D, after the block paving is completed through the step A or the step B, absolutely standing for 1-7 days, then changing the liquid in a full amount every 3 days, observing the creeping-out condition of adherent cells at the periphery of the tissue block, and slightly taking and slightly placing during operation.

And 3-4 weeks, when the fusiform adherent cells climbing out from the periphery of the tissue block grow to 80% -90% of fusion under a microscope, adding pancreatin to digest the cells and the tissue block together. Collecting the mixed digestive juice, filtering by a 100-mesh filter screen, removing umbilical cord tissue blocks, and collecting filtered cells for subculture.

The disadvantages of the two methods are:

firstly, the method comprises the following steps: the number of cells obtained is limited. In the prior art, no matter the tissue block adherence method or the enzyme digestion method, after a batch of hUC-MSCs is obtained by using fresh umbilical cord tissue blocks, the umbilical cord tissue blocks are discarded as waste. In fact, due to the factors of culture area, enzyme digestion efficiency and the like inherent in the separation vessel, the mesenchymal stem cells rich in the umbilical cord tissue are difficult to be separated sufficiently and thoroughly after only one treatment and culture. Therefore, according to the prior technical scheme, the utilization efficiency of the clinically collected umbilical cord specimen and the separation yield of the hUC-MSCs are lower.

Secondly, the method comprises the following steps: the time required to isolate hUC-MSCs is long (current tissue mass adherence method). After the freshly collected umbilical cord is primarily processed into a tissue block, a slow adaptation process is provided for the isolated culture environment, and in addition, the tissue structure is firm and compact, and single cells are difficult to dissociate in a short time, so the time required from the primary plating culture of the umbilical cord tissue block to the first harvest of the hUC-MSCs is usually long. For example: li Xiao war^[1]When the traditional planting block method (namely a tissue block adherence method) is adopted to separate the human umbilical cord mesenchymal stem cells, cell climbing-out is observed in about 12 days, and the cells can be fused by 80% in about 16 days; also of the tissue mass adherence, the Lize bell^[2]Primary isolation of mesenchymal stem cells was performed using umbilical cords treated at different times after collection, and it was found that the average harvest time of primary cells was 17.33 days, 22.00 days, and 21.69 days for the treatment group within 24 hours, the treatment group within 24-48 hours, and the treatment group within 48-72 hours, respectively; he Shao Qing, etc^[3]The report also indicates that the time from the inoculation of the umbilical cord tissue mass to the first passage of cells is about 20 days. In conclusion, the time for obtaining the hUC-MSCs by the existing tissue mass adherence method is generally about 20 days, and sufficient mesenchymal stem cells are difficult to provide for relevant experimental research or clinical treatment in a short time.

Thirdly, the method comprises the following steps: the isolated cells have limited viability and high isolation costs (enzymatic digestion). Collagenase, pancreatin and other reagents are expensive, and the activity of the mesenchymal stem cells is easy to be reduced by enzymolysis treatment.

Reference to the literature

[1] Study of the lyze. human umbilical mesenchymal stem cell isolation culture method [ D ]. beijing: the institute of hematology of the Beijing institute of coordination and medicine, 2011: 20-21.

[2] Heshaoqing, luoshenyu, liuqiying, etc. separation and culture of human umbilical cord mesenchymal stem cells and differentiation into fat and osteoblasts [ J ]. chinese tissue engineering research and clinical rehabilitation, 2010, 14 (14): 2492-2496.

[3] Li war, maroon, schaikeni, et al improvement of primary culture method of human umbilical mesenchymal stem cells [ J ]. proceedings of Jiangsu university, 2014, 25 (5): 375-379.

Disclosure of Invention

The invention aims to provide a multi-batch primary separation method of human mesenchymal stem cells from the same donor source.

The technical scheme adopted by the invention is as follows:

a multi-batch primary isolation method of human mesenchymal stem cells of the same donor source comprises the following steps:

(1) aseptically collecting donor tissues and processing;

(2) cutting the treated donor tissue to obtain a tissue size of 1-5mm³A small mass of donor tissue;

(3) soaking the donor tissue small block with fetal calf serum or human AB serum for 5-20 min;

(4) inoculating the donor tissue small blocks into a cell culture plate, adding a proper amount of complete culture medium, and culturing;

(5) harvesting primary mesenchymal stem cells when the dissociated adherent cells grow to 80% -90% confluence;

(6) while harvesting the primary mesenchymal stem cells, recovering and retreating donor tissue small blocks and culture supernatant in a primary culture system; wherein the donor tissue small block is repeatedly soaked in the fetal calf serum or the human AB serum for 5-20min and subsequent treatment, and the mesenchymal stem cells are continuously harvested; transferring the primary culture supernatant to a new cell culture bottle for secondary incubation culture, and continuously harvesting the mesenchymal stem cells.

Preferably, the donor includes at least one of a neonate umbilical cord, a placenta, and an amniotic membrane.

In the embodiment of the method, the neonatal umbilical cord is used as a donor to carry out multi-batch primary separation on the mesenchymal stem cells. According to the common knowledge of technicians in the field, the method can also be applied to other compact tissues similar to the umbilical cord structure, such as the separation of mesenchymal stem cells from tissues such as placenta, amnion and the like. For different donors, such as placenta and amnion, the treatment method is different from that of the umbilical cord of the newborn, and the operation is performed according to the conventional method.

The technical scheme adopted by the invention is further explained by taking the donor as the umbilical cord of the newborn as an example:

a multi-batch primary isolation method of human umbilical cord mesenchymal stem cells of the same donor comprises the following steps:

(1) the umbilical cord of the newborn is aseptically collected and soaked in the buffer solution.

(2) Under sterile conditions, the cord was cut into 1-5cm long pieces, washed thoroughly with buffer, and the residual cord blood was removed.

(3) The umbilical cord segments are longitudinally split, tiled and the blood vessels are removed.

(4) Cutting the cord tissue with blood vessel removed to obtain 1-5mm³The umbilical cord tissue pieces of (a).

Generally, the smaller the umbilical cord tissue mass is sheared, the more tissue sections (created by the artificial shear) are likely to climb out of the cells. Therefore, about 1mm is generally adopted in the prior art³The tissue pieces are plated for culture. However, in the invention, the umbilical cord tissue small blocks are soaked for 5-20min by using fetal bovine serum or human AB serum, and the soaking treatment can obviously improve the adherent viability of the tissue blocks and the separation yield and speed of mesenchymal stem cells. Therefore, the invention expands the volume range of the umbilical cord tissue block to 1-5mm³. The tissue block with the specification of the invention is not easy to float in the subsequent culture, has high adherence rate or cell separation success rate, and is more suitable for tissue block method separation of mesenchymal stem cells.

(5) Soaking the small pieces of umbilical cord tissue with fetal calf serum or human AB serum for 5-20 min.

The operation of the link can obviously improve the nutrition condition around the tissue block in the subsequent culture microenvironment, reduce the floating of the tissue block, improve the adherent viability of the tissue block and contribute to improving the separation yield and speed of the mesenchymal stem cells, which is shown in table 1.

In addition, the invention proves that similar separation effect can be achieved by replacing fetal calf serum with human AB serum, and a mesenchymal stem cell product without heterologous animal protein can be obtained, so that the possibility of applying the technology of the invention to clinic is provided.

(6) The umbilical cord tissue pieces are inoculated into a cell culture plate, and an appropriate amount of complete medium is added for culture.

The culture of the invention can adopt a 'turnover drying' method and a 'thin layer culture' method. And can be selected as desired.

(7) Primary mesenchymal stem cells were harvested when the dissociated adherent cells grew to 80% -90% confluence.

(8) Recovering and retreating umbilical cord tissue small blocks and culture solution in a primary culture system while harvesting the mesenchymal stem cells; wherein the umbilical cord tissue small pieces are repeatedly soaked in the fetal calf serum or the human AB serum for 5-20min and subsequent treatment, and the mesenchymal stem cells are continuously harvested; transferring the original culture supernatant to a new cell culture bottle for incubation culture again, and continuously harvesting the mesenchymal stem cells.

The process realizes the repeated recycling of the umbilical cord tissue blocks and the culture supernatant thereof, greatly improves the utilization efficiency of the umbilical cord specimen and the yield of the hUC-MSCs from the same donor source, and realizes the multi-batch primary separation of the human umbilical cord mesenchymal stem cells from the same donor. In the invention, the recovered and reused umbilical cord tissue small blocks are soaked by fetal calf serum or human AB serum before being repeatedly paved, so that the cells climbed out by the repeatedly cultured umbilical cord tissue small blocks are faster and more than those in the primary culture.

The invention not only recycles the umbilical cord tissue block, but also recycles the culture supernatant of the original strain, and primary cells with the same amount as or even more than that of the tissue block in single culture can be obtained by re-incubating the original culture supernatant, which is probably because part of the cells are directly released into the culture solution from the tissue block after the previous round of culture, and the cells scattered in the cells are not attached to the wall yet and exist in the culture supernatant in a suspension manner; in addition, a large number of adherent cells which have already formed clones secrete certain growth factors and the like into the culture supernatant, and promote the adherence and proliferation of the above-mentioned free cells.

The operation of the method is carried out in a sterile environment, a proper amount of antibiotic needs to be added into the used buffer solution to prevent pollution, and the culture environment of the human umbilical cord mesenchymal stem cells is a cell culture box with the temperature of 37 ℃, the carbon dioxide content of 5 percent and the saturated humidity. These are all common general knowledge of those skilled in the art.

Preferably, after the umbilical cord of the newborn is aseptically collected in the step (1), the umbilical cord is immediately soaked in a buffer solution containing antibiotics; such as HBSS buffer with 1% cyan/streptomycin or PBS buffer with 1% cyan/streptomycin.

Preferably, the buffer in step (2) is HBSS buffer or PBS buffer containing appropriate amount of antibiotic (such as 1% penicillin/streptomycin).

Preferably, the umbilical cord segments are longitudinally split and tiled in step (3), blood vessels (2 arteries and 1 vein) are removed, and umbilical cord tissue except the blood vessels is reserved.

Preferably, the cord tissue from which blood vessels have been removed in step (4) is cut into pieces of 2-5mm³The umbilical cord tissue pieces of (a). More preferably 3-4mm³The umbilical cord tissue pieces of (a).

Generally, the smaller the tissue mass is sheared, the more tissue sections (created by the artificial shear) may climb out of the cells. But the applicant researches and discovers that: 1mm³The small pieces of umbilical cord tissue are too small to float easily during culture. Preferably 2-5mm³More preferably 3-4mm, of the umbilical cord tissue³The umbilical cord tissue small blocks of the specification are superior to 1mm in adherence effect³And the separation quantity and quality are considered, and the separation quantity and the separation quality are not too large.

Preferably, the volume percent concentration of the fetal calf serum in the step (5) is 10% -100%.

More preferably, the volume percent concentration of fetal bovine serum in step (5) is 100%.

Preferably, the concentration of human AB serum in step (5) is between 10% and 100% by volume.

More preferably, the concentration of human AB serum in step (5) is 100% by volume.

The use of xenogeneic fetal bovine serum for the culture of human MSCs inevitably introduces heterologous animal proteins (immunogens or pathogens) into cell preparations used for human therapy, affecting the quality and safety of clinical applications of MSCs. The invention establishes a substitution scheme of soaking human umbilical cord tissue blocks by using human AB serum and separately culturing hUC-MSCs, can avoid potential risks and disadvantages of culturing human MSCs by using fetal calf serum, and lays a foundation for the application of the invention in clinical treatment.

The fetal calf serum and the human AB serum are both commercially available products.

Preferably, in step (5), the umbilical cord tissue pieces are soaked with fetal bovine serum or human AB serum for 5-20min, more preferably 10-20 min.

Furthermore, this procedure does not result in premature cell differentiation, since the fetal calf serum or human AB serum is soaked with freshly processed pieces of umbilical cord tissue, not "cells" (from which no mesenchymal stem cells have been freed), plus the short treatment time (5-20 minutes, the time to induce cell differentiation is usually "days"). In fact, the applicant's studies have identified the proliferative capacity, immunophenotype, adipogenic and osteogenic differentiation capacity of each batch of cells, and the results have shown no effect.

Preferably, in step (6), the umbilical cord tissue pieces are seeded into the cell culture plate, and the cell culture plate with the tissue pieces laid thereon is placed in CO₂After the culture is carried out for 4-6h in the incubator by reversing, the cell culture plate is turned over; an appropriate amount of complete medium was added to the cell culture plate and the culture was continued.

In the prior art, the tissue block method is used for primary cell isolation, and the tissue block method comprises a turnover drying method and a thin layer culture method. The "tumble dry" method is preferred in the present invention. The research of the invention finds that the soaked umbilical cord tissue small pieces can be tightly attached to the culture plate and can not fall off during the back-off treatment. The reason is that: firstly, most of umbilical cord tissues remained after blood vessels are removed are Wharton's jelly structures, are very viscous substances and are easily attached to the surface of a plastic vessel; secondly, the fetal calf serum contains various nutritional ingredients such as protein and lipid, and the pure fetal calf serum has certain viscosity and is used for soaking umbilical cord small pieces taking Wharton's jelly as a main ingredient, so that the adhesion force of the umbilical cord small pieces can be further increased.

After the culture treatment is carried out through the back-off, the tissue small pieces can be better adhered to the culture plate, and then the tissue small pieces are not easy to float when a complete culture medium is added, so that the yield of the hUC-MSCs is improved.

Preferably, in step (6), the umbilical cord tissue small blocks are inoculated into the cell culture plate, air-dried (for example, for 20-35min) under the aseptic condition until the edges of the tissue small blocks are slightly dried, and then the cell culture plate paved with the tissue small blocks is subjected to inverted culture for 4-6 h.

In order to enhance the plate attaching effect of the tissue blocks and prevent the tissue blocks from falling down in the next step of reversing, the small tissue blocks are inoculated into the cell culture plate and need to be properly air-dried. In addition, the effect of air-drying still lies in, avoids when next step back-off, and the serum of parcel in tissue piece surface flows to the lid of culture plate when the last step soaks and handles, causes the pollution.

This is a detail of the operation and is not mentioned in many publications, but the applicant believes that this step is added to the actual operation to facilitate the subsequent operation.

Preferably, in step (6), the umbilical cord tissue pieces are inoculated into a cell culture plate, allowed to stand for 20-40 minutes, and then an appropriate amount of complete medium is added to continue the culture. That is, the present invention may be used for cell culture by the "thin layer culture" method.

Preferably, the complete medium is DMEM/F12 medium, and 10% fetal bovine serum or 10% human AB serum, 1% double antibody (double antibody is cyan/streptomycin).

Preferably, the cell culture is CO at 37 ℃₂And (4) absolutely standing for 7 days in an incubator, changing the liquid every 3 days after 7 days, observing the creeping-out condition of adherent cells at the periphery of the tissue block, and slightly taking and slightly placing during operation.

Preferably, the umbilical cord tissue small pieces and culture supernatant are recovered and reprocessed while the primary mesenchymal stem cells are harvested; wherein the umbilical cord tissue small pieces are repeatedly soaked in the fetal calf serum or the human AB serum for 5-20min and subsequent treatment, and the mesenchymal stem cells are continuously harvested. The invention carries out repeated culture on the umbilical cord tissue small blocks for two times (in addition to the first culture of the tissue small blocks, the total number of the umbilical cord of the same donor is 3 batches of primary isolated culture), the number of primary cells obtained by the repeated culture of each tissue block is about 1.6 times of that of the first culture of the tissue block on average, and the earliest emergence time and the first passage time of the primary cells are respectively shortened to 2.4 days on average and 6.9 days on average (see tables 2 and 3).

Preferably, when the mesenchymal stem cells are harvested, the culture solution in the original culture system is collected, the culture solution is filtered by a 100-mesh filter screen to remove micro tissue debris which can remain in the culture solution, the filtered original culture supernatant is transferred to a new cell culture bottle and is placed in a carbon dioxide incubator at 37 ℃ for culture, and the mesenchymal stem cells are continuously harvested. The invention carries out repeated culture of two batches of culture supernatants, the number of primary cells obtained by culturing the culture supernatants in each time is about 1.5 times of that of the tissue blocks in the first culture, and the earliest appearance time and the first passage time of the primary cells are respectively shortened to 2.5 days in average and 7.3 days in average (see tables 2 and 3).

The invention has the beneficial effects that:

the invention improves the prior art of separating hUC-MSCs by using a tissue block adherence method, solves the problems of low tissue block separation efficiency and the like by links such as serum coating, umbilical cord tissue block 'transfer plate' continuous culture, tissue block culture supernatant recovery incubation and the like, and realizes high-quality and multi-batch stable separation of hUC-MSCs from the same donor.

The number of the hUC-MSCs obtained by the method is far higher than that of the hUC-MSCs obtained by the traditional tissue mass adherence method. Specifically, the single-time separation yield of primary cells of the umbilical cord tissue block can be increased by 3-4 times by adopting pure FBS or human AB serum soaking treatment (see table 1); compared with the single culture yield of the tissue block treated by serum soaking, the serum soaking combined with multi-batch culture (the tissue block and the culture supernatant) can improve the total yield of primary cells by 7-8 times (see tables 2 and 3); serum soaking in combination with multiple batches of culture (tissue blocks and culture supernatants) increased the overall yield of primary cells by 24-34 fold compared to the single culture yield of tissue blocks not soaked with serum (see tables 1-3).

The invention significantly shortens the time of primary isolation, and when the umbilical cord tissue mass or the original culture supernatant is used for repeated culture, the cells can climb out within 48-72 h.

The shape, proliferation and differentiation ability and immunophenotype of the cells obtained by the method are kept unchanged among batches.

In addition, the invention proves that similar separation effect can be achieved by replacing fetal calf serum with human AB serum, which provides possibility for applying the invention to clinic.

In conclusion, compared with the traditional tissue block adherence method and the enzyme digestion method, the method disclosed by the invention avoids the waste of clinical umbilical cord specimen resources, not only saves the manpower and material resources in the clinical link, but also improves the separation efficiency and yield of the hUC-MSCs, and can provide sufficient high-quality mesenchymal stem cells with the same donor source for related research in a shorter time.

Drawings

FIG. 1 shows three batches of separation of the same donor hUC-MSCs observed with a light microscope;

FIG. 2 is a graph showing growth curves of three batches of the same donor hUC-MSCs (all cells of P4 generation);

FIG. 3 shows the cell cycle of three lots of the same donor hUC-MSCs;

FIG. 4 is a flow assay of three batches of the same donor hUC-MSCs immunophenotype;

FIG. 5 shows the adipogenic differentiation capacity identification (X100) of hUC-MSCs in each batch;

FIG. 6 shows the capability of differentiating osteogenic cells of hUC-MSCs in batches (. times.100).

Detailed Description

The fetal bovine serum used in the present invention was purchased from the Biotechnology corporation of Hangzhou in Zhejiang (cat. No. 13011-.

The technical scheme adopted by the invention is as follows:

(1) the umbilical cord of the newborn was aseptically collected and immediately soaked in HBSS buffer containing 1% double antibody (double antibody is penicillin/streptomycin).

(2) Starting a super clean bench, soaking the umbilical cord in HBSS buffer solution containing 1% double antibody (the double antibody is cyan/streptomycin), fixing one end of the umbilical cord by a hemostatic forceps, shearing the umbilical cord into a plurality of small sections with the length of 1-5cm by a surgical scissors, fully washing in HBSS, and removing residual umbilical cord blood.

(3) Replacing HBSS buffer solution, and soaking the umbilical cord segment in the previous step; taking each umbilical cord segment, longitudinally splitting the umbilical cord segment, tiling and stripping 3 blood vessels inside the umbilical cord segment, and reserving umbilical cord tissues except the blood vessels.

(4) Collecting the removed umbilical cord tissue into a 10ml small beaker, and cutting the umbilical cord tissue into pieces of 3-4mm with an ophthalmic scissors³Tissue patch of (2).

(5) Soaking the umbilical cord tissue small pieces in the previous step with Fetal Bovine Serum (FBS) or human AB serum of different concentrations for 5-20 min.

(6) And (3) uniformly inoculating the small umbilical cord tissue blocks into 6-hole cell culture plates, placing 5 umbilical cord tissue blocks in each hole, and performing sterile air drying in a super clean bench for 20-35min until the edges of the tissue blocks are slightly dry.

(7) The tissue block-spread plates were inverted and incubated in a 37 ℃ carbon dioxide incubator (5% CO)₂Saturated humidity) for 4-6h, and then turning the culture plate to be upright.

(8) To each well of the 6-well plate was slowly added 2ml of DMEM/F12 complete medium (containing 10% fetal bovine serum or 10% human AB serum, 1% double antibody) and the incubation was continued in a carbon dioxide incubator (5% CO) at 37 deg.C₂Saturated humidity), and allowed to stand absolutely for 7 days.

(9) And after 7 days, changing the liquid in a full amount every 3 days, observing the creeping-out condition of adherent cells at the periphery of the tissue block, and slightly taking and slightly placing during operation.

(10) When adherent cells dissociated from the tissue blocks are 80-90% fused under the microscope, recovering and reprocessing the umbilical cord tissue small blocks and the culture solution in the original culture system; wherein the umbilical cord tissue small pieces are repeatedly soaked in the fetal calf serum or the human AB serum for 5-20min and subsequent treatment, and the mesenchymal stem cells are continuously harvested; collecting original culture solution, filtering with 100 mesh filter screen, transferring culture supernatant into new cell culture flask, and placing at 37 deg.CCarbon-changing incubator (5% CO)₂Saturated humidity), and continuously harvesting the mesenchymal stem cells.

The specific method comprises the following steps: opening the 6-hole plate under sterile environment, taking the elbow ophthalmic forceps to carefully clamp the umbilical cord tissue blocks in the plate holes one by one from the bottom of the culture plate, and repeatedly soaking the fetal calf serum or human AB serum for 5-20min and subsequent treatment (step 5-10). Then, the old culture solution in the original culture system is collected and filtered by a 100-mesh filter screen to remove the residual umbilical cord tissue debris, and the filtered original culture supernatant is directly transferred to a new cell culture bottle and placed in a carbon dioxide incubator (5% CO) at 37 DEG C₂Saturated humidity) (without adding fresh complete medium, filtering and then directly culturing). After the recovery of the umbilical cord tissue blocks and the original culture solution is finished, the primary cells which are kept in an adherent state at the bottom of the original culture plate are digested by 0.25 percent pancreatin according to a conventional method, and the harvest of the mesenchymal stem cells is finished.

(11) And identifying the cell morphology, immunophenotype, proliferation and differentiation capacity of each batch of the hUC-MSCs. The specific content comprises the following steps: observing the obtained cell morphology under an inverted microscope, measuring a cell growth curve by a cell counting method, detecting a cell cycle and a cell immunophenotype by flow cytometry, and evaluating the adipogenic and osteogenic differentiation capacity of each batch of cells.

The present invention will be further described with reference to the following examples, but is not limited thereto. In the following examples of the present invention, mesenchymal stem cells obtained by culturing or incubating tissue pieces and original culture supernatant without passaging are collectively referred to as primary cells.

Example 1 Multi-batch Primary isolation method of human mesenchymal Stem cells from the same Donor

The method is as described above, wherein, in the step (5), the umbilical cord tissue small blocks in the previous step are soaked for 15min by adopting Fetal Bovine Serum (FBS) or human AB serum with different concentrations, and primary cells are obtained after plating culture.

Primary cells (primary culture cells) refer to the earliest passage of cells directly originating from ex vivo cultured tissues and the like. In the invention, the mesenchymal stem cells which are obtained after the tissue small blocks and the original culture supernatant are cultured or incubated and are not subjected to passage are collectively called primary cells.

Statistical analysis was performed on the earliest appearance time of primary cells, the 1 st passage time, the tissue block culture success rate (number of tissue blocks from which cells were successfully dissociated/total number of inoculated tissue blocks × 100%), and the number of primary cells (number of cells isolated per cm of umbilical cord) of each group, and the results are shown in table 1.

TABLE 1 time and yield for first adherent isolation of Primary cells from differently pretreated umbilical cord tissue blocks

P <0.05, P <0.01, P <0.001, compared to the serum-free soaked group; wherein, in the 100% FBS soaking group vs serum-free soaking group, the average number of primary cells is about 3.6 times of that in the serum-free soaking group; the 100% human AB serum soaked vs serum-free soaked group had an average of about 4.1 times the number of primary cells as compared to the serum-free soaked group.

As can be seen from Table 1, in the case of a single culture, the immersion treatment of the umbilical cord tissue mass with pure FBS or human AB serum can significantly shorten the initial appearance time and harvest time of the primary cells, improve the culture success rate of the tissue mass (cells are migrated out around the tissue mass), and increase the yield of the primary cells by 3-4 times. The reasons for this may be: the concentration of nutrients such as adhesion protein, growth factors and the like in pure fetal calf serum or human AB serum is obviously higher than that of a complete culture medium only containing 10-50% of fetal calf serum, so that the operation of soaking and treating a tissue block by using the pure serum is more meaningful.

Example 2 Multi-batch Primary isolation method of human mesenchymal Stem cells from the same Donor

The method is as described above, and in step (5), the umbilical cord tissue small pieces in the previous step are soaked for 15min by using 100% fetal calf serum. And (4) comparing the separation time and the yield of each batch of the primary hUC-MSCs. Wherein, the group A is an umbilical cord tissue block primary culture group; the B-t group is mesenchymal stem cells (primary cells) obtained by culturing the umbilical cord tissue blocks recovered after the first culture for the 2 nd time; the B-s group is mesenchymal stem cells (primary cells) obtained after the supernatant fluid of the first culture of the tissue block is singly incubated; c-t group is mesenchymal stem cells (primary cells) obtained by 3 rd culture of umbilical cord tissue pieces (recovered from B-t group); group C-s are mesenchymal stem cells (primary cells) obtained after incubation of the supernatant of the 2 nd culture of the tissue mass (recovered from group B-t) alone. The earliest appearance time, 1 st passage time and the number of primary cells of each group were counted. The results are shown in Table 2.

TABLE 2 isolation time and yield of different batches of hUC-MSCs from the same donor source^△

^△Before the adherent culture of the tissue block, 100 percent fetal calf serum is soaked in advance. P compared to the first culture group<0.05，**P<0.01。

As can be seen in Table 2, a larger number of primary cells can be obtained in a shorter time than in the case of the umbilical cord tissue pieces cultured for the first time, regardless of whether they are repeatedly cultured or the culture supernatant. Specifically, the average total cell yield of 3 cultures before and after the tissue block together with 2 cultures of culture supernatant was about 7.5 times the average yield of a single culture of the tissue block (soaked with FBS) and 27.2 times the average yield of a single culture of the tissue block without serum soaking (table 1, "serum-free soaking group").

In the present invention, 3 batches of cells were repeatedly cultured. Theoretically, 4 batches and 5 batches are also possible. The 3 times of culture are mainly considered that in the practical application process, the probability of microbial contamination is increased along with the increase of the repeated culture times and the prolongation of the in vitro maintenance time.

Three batches of the same donor primary hUC-MSCs of example 2 were subjected to correlation analysis. The results are as follows.

1. Morphological detection of three batches of same donor hUC-MSCs

The results are shown in FIG. 1. In FIG. 1, group A (tissue block first culture) is umbilical cord tissue blocks cultured by first adherence to day 7 and spindle-shaped primary cells dissociated from the umbilical cord tissue blocks; b-t group (tissue block 2 nd culture), umbilical cord tissue block and a large number of primary cells around the umbilical cord tissue block at the time of 2 nd adherent culture for 7 days; b-s group (group A culture supernatant is re-incubated), and culture supernatant obtained when the 1 st primary cell is first passaged is recovered and singly incubated for 7 days to obtain a large amount of adherent cells; c-t group (3 rd culture of tissue block), umbilical cord tissue block and a large number of primary cells around the umbilical cord tissue block at 7 days of 3 rd adherent culture; and C-s group (B-t group culture supernatant is re-incubated), and culture supernatant obtained when the 2 nd culture group (B-t group) of the tissue block is subjected to first passage is recovered and is singly incubated for 7 days to obtain a large amount of adherent cells. The brown opaque portion is a mass of umbilical cord tissue.

In the initial adherent culture, a small number of fusiform, short rod-shaped adherent cells were observed to climb out from the edge of the brownish yellow umbilical cord tissue patch under an inverted microscope at 1 week of inoculation culture (fig. 1. a). In the case of repeated cultures of

batches

2 and 3, adherent cells grown to 80% confluence were obtained from the tissue block and supernatant groups up to 1 week of culture (FIG. 1.B-t, B-s, C-t, C-s). These umbilical cord-derived cells exhibited typical morphological characteristics of MSCs, both in primary culture systems and after passage: single cells are in a fibroid shape, have strong refractivity and abundant cytoplasm, and have polarity distribution; the cells grow in a vortex-like manner in parallel arrangement when they are combined.

2. Growth curves of three batches of the same donors hUC-MSCs

Cell growth curves were plotted using cytometry and the results are shown in figure 2.

As can be seen in FIG. 2, the growth curves for the batches of hUC-MSCs were similar in morphology: the 1-2d cells are in a latent stage and do not obviously proliferate; 3-5d cells enter a logarithmic growth phase, and cell proliferation is accelerated; after 6d, a plateau phase was entered and cell growth arrested.

3. Cell cycle analysis of three batches of the same donors hUC-MSCs

The cell cycle of each batch of hUC-MSCs was determined by flow cytometry, and the experiment was repeated 4 times, with the results shown in FIG. 3.

The top panel of FIG. 3 is a representative cell cycle map (all P4 generation cells); the lower part is a statistical analysis chart. As shown in FIG. 3, there was no significant difference in the proportion of cells in the G0/G1 phase or (S + G2/M) phase among the groups of cells. Indicating that the cells obtained by repeated culturing of multiple batches had normal proliferation capacity.

4. Immunophenotype of three batches of the same donors hUC-MSCs

The cell surface markers were detected by flow-based assay on batches of P4 cells. As shown in fig. 4, each batch of human umbilical cord-derived cells uniformly expressed the surface markers CD73, CD90, CD105 related to stromal cells, and did not express the surface marker CD34 of hematopoietic stem cells and the human leukocyte common antigen CD45, which are consistent with the phenotypic characteristics of mesenchymal stem cells.

5. Adipogenic and osteogenic differentiation capacity of three batches of same donor hUC-MSCs

5.1 measurement of adipogenic differentiation Capacity

Taking each batch of human umbilical cord cells after the generation of P4 for adipogenic induction differentiation, adding an adipogenic differentiation induction liquid to almost stop the cell growth, gradually increasing and mutually fusing small lipid droplets in cytoplasm after 72 hours, and changing the cell shape from fusiform to round or oval, wherein part of cell nucleus is deviated. After 2-3 weeks of continuous induction, oil red O staining revealed massive lipid precipitation in the cells, and the results are shown in FIG. 5.

In FIG. 5, O is undyed; a is the 1 st batch hUC-MSCs oil red O staining; b-t is hUC-MSCs oil red O staining from the tissue mass of the 2 nd batch; b-s is oil red O staining of hUC-MSCs (human umbilical cord mesenchymal stem cells) from the supernatant of the 1 st batch; c-t is hUC-MSCs oil red O staining from the 3 rd tissue block source; c-s is the oil red O staining of hUC-MSCs from the supernatant of batch 2. FIG. 5 results demonstrate that multiple batch isolation culture did not affect the in vitro adipogenic differentiation capacity of the obtained hUC-MSCs.

5.2 osteogenic differentiation Capacity test

And taking each batch of human umbilical cord cells after the generation of P4 for osteogenic induced differentiation. During the process of osteogenic directional induction differentiation, the volume of cells is gradually increased, the cells are transformed from fibrous shape to short spindle shape, scaly shape, polygonal shape and the like, the particulate matter in cytoplasm is gradually increased, the cells form short processes which are connected with each other to grow in a colony shape, and the cells have a typical osteoblast shape. In the anaphase of osteogenesis induction, the cells in the center of cell nodules gradually fuse and lose cell structures, calcium deposition can be seen among the cells, and the cells are red after alizarin red staining, and the result is shown in FIG. 6.

In fig. 6: o is undyed; a is alizarin red staining of batch 1 hUC-MSCs; b-t is the alizarin red staining of hUC-MSCs from the tissue mass of the 2 nd batch; b-s is alizarin red staining of hUC-MSCs from supernatant of batch 1; c-t is alizarin red staining of hUC-MSCs from the 3 rd tissue block; c-s is alizarin red staining of hUC-MSCs from supernatant of batch 2. The results in FIG. 6 demonstrate that the in vitro osteogenic differentiation capacity of the hUC-MSCs is not affected by multi-batch isolated culture.

Example 3 Multi-batch Primary isolation method of human mesenchymal Stem cells from the same Donor

The method is as described above, and in step (5), 100% human AB serum is used to soak the umbilical cord tissue pieces in the previous step for 15 min. And (4) comparing the separation time and the yield of each batch of the primary hUC-MSCs. Wherein, the group A is an umbilical cord tissue block primary culture group; the B-t group is mesenchymal stem cells (primary cells) obtained by culturing the umbilical cord tissue blocks recovered after the first culture for the 2 nd time; the B-s group is mesenchymal stem cells (primary cells) obtained after the supernatant fluid of the first culture of the tissue block is singly incubated; c-t group is mesenchymal stem cells (primary cells) obtained by 3 rd culture of umbilical cord tissue pieces (recovered from B-t group); group C-s are mesenchymal stem cells (primary cells) obtained after incubation of the supernatant of the 2 nd culture of the tissue mass (recovered from group B-t) alone. The earliest appearance time, 1 st passage time and the number of primary cells of each group were counted. The results are shown in Table 3.

TABLE 3 isolation time and yield of different batches of hUC-MSCs from the same donor^△

	Earliest appearance time of Primary cell (d)	Passage time 1 (d)	Number of primary cells (× 10)⁴One/cm)
				Group A (tissue block first culture)	7.75±1.71	11.8±1.71	6.76±0.29
Group B-t (tissue block 2 nd time culture)	2.25±0.50^*	6.50±0.58^*	10.6±0.77^*
				Group B-s (group A culture supernatant re-incubation)	1.75±0.50^*	6.75±0.96^*	10.2±0.81^*
C-t group (tissue block 3 rd time culture)	2.50±0.58^*	7.00±0.82^*	10.8±0.82^**
				C-s group (B-t group culture supernatant re-incubation)	2.25±0.96^*	6.50±0.58^*	9.86±0.83^*

^△Before the adherent culture of the tissue block, the tissue block is soaked in 100 percent human AB serum in advance. P compared to the first culture group<0.05，**P<0.01。

As can be seen from Table 3, similar to the results for the tissue mass soaked with fetal bovine serum (Table 2), the umbilical cord tissue mass soaked with human AB serum and its culture supernatant can be used for at least two batches of culture reuse, with the earliest emergence time of primary cells, 1 st passage time, and the number of primary cell harvests being also superior to those of the single culture group. Specifically, the average total cell yield of 3 cultures before and after the tissue block together with 2 supernatant cultures was about 7.1 times the average yield of a single culture of the tissue block (soaked with human AB serum) and 29 times the average yield of a single culture of the tissue block without serum soaking (table 1, "serum-free soaking group").

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1.A multi-batch primary isolation method of human mesenchymal stem cells from the same donor is characterized by comprising the following steps:

aseptically collecting donor tissues and processing;

cutting the treated donor tissue to obtain 2-5mm³Donor tissue of (2)Small blocks;

soaking donor tissue small pieces with 100% human AB serum for 5-20 min;

inoculating the donor tissue small blocks into a cell culture plate, adding a proper amount of complete culture medium, and culturing;

harvesting primary mesenchymal stem cells when the dissociated adherent cells grow to 80% -90% confluence;

while harvesting the primary mesenchymal stem cells, recovering and retreating donor tissue small blocks and culture supernatant in a primary culture system; wherein the small pieces of donor tissue are repeatedly soaked in the 100% human AB serum for 5-20min and then are subjected to subsequent treatment, and mesenchymal stem cells are continuously harvested; transferring the primary culture supernatant to a new cell culture bottle for secondary incubation culture, and continuously harvesting the mesenchymal stem cells.

2. The multi-batch primary isolation method of claim 1, characterized in that: the donor comprises at least one of umbilical cord, placenta and amnion of newborn.

3. The multi-batch primary isolation method of claim 2, characterized in that: when the donor is a neonate umbilical cord, the treatment method is as follows: aseptically collecting umbilical cord of newborn, soaking in buffer solution, cutting umbilical cord into small segments with length of 1-5cm under aseptic environment, washing with buffer solution, and removing residual umbilical cord blood; the umbilical cord segment is longitudinally split and the blood vessel is removed.

4. The multi-batch primary isolation method of claim 3, characterized in that: the buffer solution is HBSS buffer solution or PBS buffer solution containing appropriate amount of antibiotic.

5. The multi-batch primary isolation method of claim 1, characterized in that: inoculating the donor tissue small blocks into a cell culture plate, reversely culturing the cell culture plate paved with the tissue small blocks for 4-6h, and turning the cell culture plate to be upright; an appropriate amount of complete medium was added to the cell culture plate and the culture was continued.

6. The multi-batch primary isolation method of claim 1, characterized in that: inoculating the donor tissue small blocks into a cell culture plate, air-drying under an aseptic condition until the edges of the tissue small blocks are slightly dry, and then reversely culturing the cell culture plate paved with the tissue small blocks for 4-6 h.

7. The multi-batch primary isolation method of claim 1, characterized in that: inoculating the donor tissue small block into a cell culture plate, standing for 20-40 minutes, adding a proper amount of complete culture medium, and culturing.