阅读说明：本技术 一种临床级人脐带间充质干细胞资源库多级库的构建方法 (Construction method of clinical-grade human umbilical cord mesenchymal stem cell resource library multistage library ) 是由 刘中民 赵庆辉 汤红明 许啸 于 2021-10-21 设计创作，主要内容包括：本发明涉及一种临床级人脐带间充质干细胞资源库多级库的构建方法,属于细胞培养技术领域。本发明所述构建方法进行原料预处理、干细胞分离和扩增培养获得种子库,然后通过接种密度、培养时间等条件的设置实现主库、工作库和产品库的构建。本发明所述构建方法细胞活率高、质量可控,能够成功构建临床级四级干细胞资源库,即为种子库、主库、工作库和产品库。(The invention relates to a construction method of a clinical-grade human umbilical cord mesenchymal stem cell resource library multistage library, belonging to the technical field of cell culture. The construction method of the invention carries out raw material pretreatment, stem cell separation and amplification culture to obtain a seed bank, and then realizes the construction of a main bank, a working bank and a product bank by setting conditions such as inoculation density, culture time and the like. The construction method of the invention has high cell viability and controllable quality, and can successfully construct clinical grade four-stage stem cell resource libraries, namely a seed library, a main library, a working library and a product library.)

1. A construction method of a clinical-grade human umbilical cord mesenchymal stem cell resource library multistage library, wherein the multistage library comprises a seed library, a main library, a working library and a product library; the construction method comprises the following steps:

the umbilical cord is cut open, cleaned, blood vessels are removed, and the umbilical cord is sequentially soaked in the first flushing liquid and the second flushing liquid to obtain a pretreated umbilical cord; the first flushing liquid comprises a serum-free culture medium with the volume percentage of 80% and a streptomycin mixed liquid with the volume percentage of 20%; the second flushing liquid comprises a serum-free culture medium with the volume percentage of 90% and a streptomycin mixed liquid with the volume percentage of 10%;

cutting the pretreated umbilical cord into pieces with the thickness of 1-2 mm³Placing the tissue blocks in a 6-hole plate, carrying out adherent culture in a carbon dioxide incubator for 20min, adding a serum-free culture medium for culture, and digesting after the cell fusion rate reaches 80-90% to obtain primary cells;

the primary cells are processed according to the ratio of 0.8-1 ten thousand/cm²Inoculating the cells into a 6-hole plate, adding a serum-free culture medium, culturing for 3-4 days until the fusion degree reaches 90-95%, digesting, centrifuging, and removing the supernatant to obtain P0 generation cells;

the cell generation P0 is treated according to the ratio of 0.8-1 ten thousand/cm²Inoculating the strain to a culture bottle, adding a serum-free culture medium for culturing for 3-4 days until the fusion degree reaches 90-95%, digesting, centrifuging, and removing a supernatant to obtain P1 generation cells;

the cell generation P1 is treated according to the ratio of 0.8-1 ten thousand/cm²Inoculating the strain into a culture bottle, adding a serum-free culture medium for culturing for 2-3 days until the fusion degree reaches 90-95%, digesting, centrifuging, and removing a supernatant to obtain P2 generation cells; resuspending the cells of the P2 generation in a freezing medium, freezing and constructing a seed bank;

the cell generation P2 is treated according to the ratio of 0.5-0.8 ten thousand/cm²Inoculating the strain into a culture bottle, adding a serum-free culture medium for culturing for 3-4 days until the fusion degree reaches 90-95%, digesting, centrifuging, and removing a supernatant to obtain P3 generation cells; resuspending the cells of the P3 generation in a freezing medium, freezing and constructing to obtain a master library;

the cell generation P3 is treated according to the ratio of 0.5-0.8 ten thousand/cm²Inoculating the strain into a culture bottle, adding a serum-free culture medium for culturing for 3-4 days until the fusion degree reaches 90-95%, digesting, centrifuging, and removing a supernatant to obtain P4 generation cells; resuspending the P4 generation cells in a freezing medium, freezing and constructing to obtain a working library;

the cell generation P4 is treated according to the ratio of 0.5-0.8 ten thousand/cm²Inoculating the strain into a cell factory, adding a serum-free culture medium for culturing for 4-5 days until the fusion degree reaches 90-95%, digesting, centrifuging, and removing a supernatant to obtain P5 generation cells; and (4) resuspending the cells of the P5 generation in a freezing medium, freezing and constructing to obtain a product library.

2. The method of construction according to claim 1, wherein the umbilical cord is from a healthy donor; the healthy donor has no family infection history and no drug-taking history, and the items with negative detection comprise: antibodies to lymphocytic leukemia virus, antibodies to hepatitis c virus, antibodies to cytomegalovirus IgM, antibodies to hepatitis b virus surface, antibodies and antigens to human immunodeficiency virus, antibodies to treponema pallidum, nucleic acids from hepatitis b virus, nucleic acids from hepatitis c virus, nucleic acids from human immunodeficiency virus, and antibodies to EBV virus IgM.

3. The construction method according to claim 1, wherein the soaking time is 3-5 min.

4. The method of claim 1, wherein the tissue mass is placed in 20-25 per well of a 6-well plate.

5. The method of claim 1, wherein before obtaining the cells of the P0 generation and before obtaining the cells of the P1 generation, the method further comprises detecting endotoxin, mycoplasma, anaerobes and aerobes in the supernatant.

6. The construction method according to claim 1, wherein before the generation P2 cells, the generation P3 cells, the generation P4 cells and the generation P5 cells are obtained, endotoxin, mycoplasma, anaerobe and aerobe detection is carried out on the supernatant, and viability detection, flow detection and infectious disease nucleic acid detection are carried out on the cells; the flow assay comprises detecting CD73, CD90, CD105, CD34, CD45, HLA-DR, CD11b and CD 19; the indexes for detecting infectious disease nucleic acid include novel coronavirus nucleic acid, hepatitis B virus nucleic acid, hepatitis C virus nucleic acid, treponema pallidum antibody and human immunodeficiency virus nucleic acid.

7. The construction method according to claim 1, wherein the cryopreservation density of the P2 generation stem cells in the seed bank is 300 ten thousand stem cells per milliliter of cell cryopreservation solution;

the cryopreservation density of the P3 generation stem cells in the master library is 500 ten thousand stem cells per milliliter of cell cryopreservation solution;

the cryopreservation density of the P4 generation stem cells in the working library is 1000 ten thousand stem cells per milliliter of cell cryopreservation solution;

the cryopreservation density of the P5 generation stem cells in the product library is 1000 ten thousand stem cells per milliliter of cell cryopreservation solution.

8. The construction method according to claim 1, wherein the conditions for cryopreservation are as follows: keeping the temperature at 4 ℃ for 15min, reducing the temperature at 1 ℃/min to-7 ℃, reducing the temperature at 60 ℃/min to-60 ℃, increasing the temperature at 30 ℃/min to-30 ℃, and reducing the temperature at 1 ℃/min to-90 ℃.

9. The method of construction according to claim 1, wherein the conditions of the centrifugation are: 2000-3000 r/min, 5 min.

10. The method of constructing as claimed in claim 1, further comprising, prior to freezing: and pasting an anti-freezing two-dimensional code on the freezing storage pipe for marking.

Technical Field

The invention relates to the technical field of cell culture, in particular to a construction method of a clinical-grade human umbilical cord mesenchymal stem cell resource library multistage library.

Background

As the first major world of the global population and as developing countries that are stepping into aging society, our country faces many challenges in terms of population health. These challenges are reflected in the increasing incidence of tissue and organ dysfunction caused by the transformation of infectious diseases into chronic diseases and senile degenerative diseases, the trend toward more and more obvious youthful onset of various tumors, genetic wound metabolism, and other factors. For these diseases, traditional drug therapy and surgical therapy are mostly ineffective, and cell, tissue or organ-based replacement therapy is expected to become the best choice for radically treating the diseases.

Human Umbilical Cord Mesenchymal Stem Cells (Human Umbilical Cord Mesenchymal Stem Cells, hUC-MSCs) are a type of pluripotent Stem Cells which exist in Umbilical Cord tissues of newborns and have high differentiation potential. Under certain conditions, can be differentiated into various functional tissues and organs. Due to the multidirectional differentiation potential, low immunogenicity and adhesion characteristics, as well as abundant material sources and few ethical disputes, the hybrid seed cell gradually becomes one of ideal seed cells for regenerative medical therapy and tissue engineering transplantation, and is more and more favored by researchers. In view of this, it is particularly critical to establish a clinical-grade hic-MSCs (which can reach the relevant quality standard for clinical use) resource library, which can not only serve clinical research and application of stem cells, but also provide stem cell resources meeting the requirements of scientific research for colleges, scientific research institutions, enterprises and public institutions, and the like.

At present, most of the preservation of umbilical cord stem cells in domestic stem cell banks is mainly carried out by scientific research, the preparation technology and the detection technology used by the domestic stem cell banks cannot be unified, and a standardized method is not provided.

Disclosure of Invention

The invention aims to provide a construction method of a clinical-grade human umbilical cord mesenchymal stem cell resource library multistage library. The construction method provided by the invention has the advantages of high cell viability and controllable quality, can successfully construct clinical grade four-level stem cell resource libraries, namely a seed library, a main library, a working library and a product library, and can realize standardization of a preparation process.

The invention provides a construction method of a clinical-grade human umbilical cord mesenchymal stem cell resource library multistage library, wherein the multistage library comprises a seed library, a main library, a working library and a product library; the construction method comprises the following steps:

the umbilical cord is cut open, cleaned, blood vessels are removed, and the umbilical cord is sequentially soaked in the first flushing liquid and the second flushing liquid to obtain a pretreated umbilical cord; the first flushing liquid comprises a serum-free culture medium with the volume percentage of 80% and a streptomycin mixed liquid with the volume percentage of 20%; the second flushing liquid comprises a serum-free culture medium with the volume percentage of 90% and a streptomycin mixed liquid with the volume percentage of 10%;

cutting the pretreated umbilical cord into pieces with the thickness of 1-2 mm³Placing the tissue blocks in a 6-hole plate, carrying out adherent culture in a carbon dioxide incubator for 20min, adding a serum-free culture medium for culture, and digesting after the cell fusion rate reaches 80-90% to obtain primary cells;

the primary cells are processed according to the ratio of 0.8-1 ten thousand/cm²Inoculating the cells into a 6-hole plate, adding a serum-free culture medium, culturing for 3-4 days until the fusion degree reaches 90-95%, digesting, centrifuging, and removing the supernatant to obtain P0 generation cells;

the cell generation P0 is treated according to the ratio of 0.8-1 ten thousand/cm²Inoculating the strain to a culture bottle, adding a serum-free culture medium for culturing for 3-4 days until the fusion degree reaches 90-95%, digesting, centrifuging, and removing a supernatant to obtain P1 generation cells;

the cell generation P1 is treated according to the ratio of 0.8-1 ten thousand/cm²Inoculating the strain into a culture bottle, adding a serum-free culture medium for culturing for 2-3 days until the fusion degree reaches 90-95%, digesting, centrifuging, and removing a supernatant to obtain P2 generation cells; resuspending the cells of the P2 generation in a freezing medium, freezing and constructing a seed bank;

the cell generation P2 is treated according to the ratio of 0.5-0.8 ten thousand/cm²Inoculating the strain into a culture bottle, adding a serum-free culture medium for culturing for 3-4 days until the fusion degree reaches 90-95%, digesting, centrifuging, and removing a supernatant to obtain P3 generation cells; resuspending the cells of the P3 generation in a freezing medium, freezing and constructing to obtain a master library;

the cell generation P3 is treated according to the ratio of 0.5-0.8 ten thousand/cm²Inoculating the strain into a culture bottle, adding a serum-free culture medium for culturing for 3-4 days until the fusion degree reaches 90-95%, digesting, centrifuging, and removing a supernatant to obtain P4 generation cells; resuspending P4 passage cells inFreezing the solution, and constructing to obtain a working library;

the cell generation P4 is treated according to the ratio of 0.5-0.8 ten thousand/cm²Inoculating the strain into a cell factory, adding a serum-free culture medium for culturing for 4-5 days until the fusion degree reaches 90-95%, digesting, centrifuging, and removing a supernatant to obtain P5 generation cells; and (4) resuspending the cells of the P5 generation in a freezing medium, freezing and constructing to obtain a product library.

Preferably, the umbilical cord is from a healthy donor; the healthy donor has no family infection history and no drug-taking history, and the items with negative detection comprise: antibodies to lymphocytic leukemia virus, antibodies to hepatitis c virus, antibodies to cytomegalovirus IgM, antibodies to hepatitis b virus surface, antibodies and antigens to human immunodeficiency virus, antibodies to treponema pallidum, nucleic acids from hepatitis b virus, nucleic acids from hepatitis c virus, nucleic acids from human immunodeficiency virus, and antibodies to EBV virus IgM.

Preferably, the soaking time is 3-5 min respectively.

Preferably, 20-25 tissue blocks are placed in each hole of the 6-hole plate.

Preferably, before obtaining the cells of the P0 generation and obtaining the cells of the P1 generation, the method also comprises the detection of endotoxin, mycoplasma, anaerobe and aerobe on the supernatant.

Preferably, before obtaining the cells of the P2 generation, the P3 generation, the P4 generation and the P5 generation, the method also comprises the steps of detecting endotoxin, mycoplasma, anaerobe and aerobe of the supernatant, and detecting the survival rate, flow detection and infectious disease nucleic acid of the cells; the flow assay comprises detecting CD73, CD90, CD105, CD34, CD45, HLA-DR, CD11b and CD 19; the indexes for detecting infectious disease nucleic acid include novel coronavirus nucleic acid, hepatitis B virus nucleic acid, hepatitis C virus nucleic acid, treponema pallidum antibody and human immunodeficiency virus nucleic acid.

Preferably, the cryopreservation density of the P2 generation stem cells in the seed bank is 300 ten thousand stem cells per milliliter of cell cryopreservation solution;

the cryopreservation density of the P3 generation stem cells in the master library is 500 ten thousand stem cells per milliliter of cell cryopreservation solution;

the cryopreservation density of the P4 generation stem cells in the working library is 1000 ten thousand stem cells per milliliter of cell cryopreservation solution;

the cryopreservation density of the P5 generation stem cells in the product library is 1000 ten thousand stem cells per milliliter of cell cryopreservation solution.

Preferably, the freezing condition is as follows: keeping the temperature at 4 ℃ for 15min, reducing the temperature at 1 ℃/min to-7 ℃, reducing the temperature at 60 ℃/min to-60 ℃, increasing the temperature at 30 ℃/min to-30 ℃, and reducing the temperature at 1 ℃/min to-90 ℃.

Preferably, the centrifugation conditions are: 2000-3000 r/min, 5 min.

Preferably, before freezing, the method further comprises: and pasting an anti-freezing two-dimensional code on the freezing storage pipe for marking. .

The invention provides a construction method of a clinical-grade human umbilical cord mesenchymal stem cell resource library multistage library. The construction method of the invention has high cell viability and controllable quality, and can successfully construct clinical grade four-stage stem cell resource libraries, namely a seed library, a main library, a working library and a product library. Specifically, the method realizes high cell survival rate through the arrangement of a donor screening mode, a primary cell separation mode, inoculation density control and a cell cryopreservation mode, and performs quality control and related detection in each link of library construction according to the Chinese pharmacopoeia of the preclinical guidelines, and the like, thereby ensuring controllable quality. A clinical grade four-level stem cell resource library is established, namely a seed library, a main library, a working library and a product library, a plurality of generations of cells are preserved, and a culture mode and a freezing mode are reasonably set according to the function of a library area.

Drawings

FIG. 1 is a flow chart of a construction and detection method provided by the present invention.

Detailed Description

The invention provides a construction method of a clinical-grade human umbilical cord mesenchymal stem cell resource library multistage library, wherein the multistage library comprises a seed library, a main library, a working library and a product library; the construction method comprises the following steps:

the umbilical cord is cut open, cleaned, blood vessels are removed, and the umbilical cord is sequentially soaked in the first flushing liquid and the second flushing liquid to obtain a pretreated umbilical cord; the first flushing liquid comprises a serum-free culture medium with the volume percentage of 80% and a streptomycin mixed liquid with the volume percentage of 20%; the second flushing liquid comprises a serum-free culture medium with the volume percentage of 90% and a streptomycin mixed liquid with the volume percentage of 10%;

cutting the pretreated umbilical cord into pieces with the thickness of 1-2 mm³Placing the tissue blocks in a 6-hole plate, carrying out adherent culture in a carbon dioxide incubator for 20min, adding a serum-free culture medium for culture, and digesting after the cell fusion rate reaches 80-90% to obtain primary cells;

the primary cells are processed according to the ratio of 0.8-1 ten thousand/cm²Inoculating the cells into a 6-hole plate, adding a serum-free culture medium, culturing for 3-4 days until the fusion degree reaches 90-95%, digesting, centrifuging, and removing the supernatant to obtain P0 generation cells;

the cell generation P0 is treated according to the ratio of 0.8-1 ten thousand/cm²Inoculating the strain to a culture bottle, adding a serum-free culture medium for culturing for 3-4 days until the fusion degree reaches 90-95%, digesting, centrifuging, and removing a supernatant to obtain P1 generation cells;

the cell generation P1 is treated according to the ratio of 0.8-1 ten thousand/cm²Inoculating the strain into a culture bottle, adding a serum-free culture medium for culturing for 2-3 days until the fusion degree reaches 90-95%, digesting, centrifuging, and removing a supernatant to obtain P2 generation cells; resuspending the cells of the P2 generation in a freezing medium, freezing and constructing a seed bank;

the cell generation P2 is treated according to the ratio of 0.5-0.8 ten thousand/cm²Inoculating the strain into a culture bottle, adding a serum-free culture medium for culturing for 3-4 days until the fusion degree reaches 90-95%, digesting, centrifuging, and removing a supernatant to obtain P3 generation cells; resuspending the cells of the P3 generation in a freezing medium, freezing and constructing to obtain a master library;

the cell generation P3 is treated according to the ratio of 0.5-0.8 ten thousand/cm²Inoculating the strain into a culture bottle, adding a serum-free culture medium for culturing for 3-4 days until the fusion degree reaches 90-95%, digesting, centrifuging, and removing a supernatant to obtain P4 generation cells; resuspending the P4 generation cells in a freezing medium, freezing and constructing to obtain a working library;

the number of P4 generation cells is 0.5-0.8 ten thousand/cm²Inoculating the strain into a cell factory, adding a serum-free culture medium for culturing for 4-5 days until the fusion degree reaches 90-95%, digesting, centrifuging, and removing a supernatant to obtain P5 generation cells; and (4) resuspending the cells of the P5 generation in a freezing medium, freezing and constructing to obtain a product library.

The umbilical cord of the invention is preferably from a healthy donor; the healthy donor needs no family infectious disease history and no drug taking history, and the items which are detected to be negative need to include: antibodies to lymphocytic leukemia virus, antibodies to hepatitis c virus, antibodies to cytomegalovirus IgM, antibodies to hepatitis b virus surface, antibodies and antigens to human immunodeficiency virus, antibodies to treponema pallidum, nucleic acids from hepatitis b virus, nucleic acids from hepatitis c virus, nucleic acids from human immunodeficiency virus, and antibodies to EBV virus IgM. In the present invention, the age of the donor is preferably 20 to 35. After the umbilical cord of the donor is obtained by screening according to the conditions, the umbilical cord of 5-10 cm is preferably cut. After the umbilical cord is cut, the two ends of the umbilical cord are preferably clamped by hemostatic clamps and stored and transported at 4 ℃. Before the umbilical cord is pretreated, the invention preferably opens the hemostatic clamp and washes the blood of the umbilical cord clean by using physiological saline. After washing, the umbilical cord is cut open, washed, blood vessels are removed, and the umbilical cord is sequentially soaked in a first washing liquid and a second washing liquid to obtain a pretreated umbilical cord; the first flushing liquid comprises a serum-free culture medium with the volume percentage of 80% and a streptomycin mixed liquid with the volume percentage of 20%; the second flushing liquid comprises a serum-free culture medium with the volume percentage of 80% and a streptomycin mixed liquid with the volume percentage of 10%. In the invention, before the dissection, the umbilical cord is preferably cut into strips of 2 cm. After the dissection, the blood collection device is preferably cleaned by using normal saline, the cleaning frequency is preferably 2-3 times, and the blood is ensured to be cleaned. The removal of blood vessels according to the present invention is preferably a removal of 3 blood vessels, including 2 arteries and 1 vein. The raw materials for preparing the flushing fluid comprise a serum-free culture medium and a streptomycin mixing solution, and the sources of the serum-free culture medium and the streptomycin mixing solution are not particularly limited and can be prepared by adopting conventional commercial products. In the present invention, the penicillin streptomycin mixed solution is preferably penicillin streptomycin mixed solution (100 ×) dedicated for cell culture, purchased from Gibco and having model number 15140-122. In the present invention, the serum-free medium is preferably purchased from Gibco, model 8122159. The first washing liquid and the second washing liquid are arranged in different proportions and soaked for two times, so that blood can be washed away as much as possible, primary cells can climb out of tissues, and the tissue blocks can adhere to the walls. In the invention, the soaking time is preferably 3-5 min respectively. The invention preferably uses the first washing liquid to soak for 10min and then uses the second washing liquid to soak for 10 min.

After obtaining the pretreated umbilical cord, the invention cuts the pretreated umbilical cord into pieces with the diameter of 1-2 mm³And placing the tissue blocks in a 6-hole plate, carrying out adherent culture in a carbon dioxide incubator for 20min, adding a serum-free culture medium formulation for culture, and digesting after the cell fusion rate reaches 80-90% to obtain primary cells. In the invention, 20-25 tissue blocks are placed in each hole of a 6-hole plate. The present invention preferably places the tissue mass evenly into each well of a 6-well plate. After the serum-free culture medium is added, the overnight culture is preferably carried out, and after the overnight culture, the serum-free culture medium is preferably added to supplement the volatile serum-free culture medium. In the present invention, 1mL of serum-free medium is preferably added to each well of the 6-well plate. And (3) adding a serum-free culture medium, continuing to culture for 4-10 days, and digesting after the cell fusion rate reaches 80-90%. In the present invention, the time for the digestion is preferably 3 min. The digestion is preferably carried out at room temperature, the room temperature is preferably 20-25 ℃, and the digestion conditions are the same as those in the following description, and are not described in detail later. The digestive juice of the present invention is preferably purchased from GIBCO, model 25200-. After the primary cells are obtained, part of the primary cells are preferably taken for flow detection. In the present invention, the flow assay preferably detects CD73, CD90, CD105, CD34, CD45 and HLA-DR to determine whether the purity of the stem cells meets the characteristics of the stem cells.

The primary cells are processed according to the ratio of 0.8-1 ten thousand/cm²Inoculating the strain to a 6-well plate, adding a serum-free culture medium, culturing for 3-4 days until the fusion degree reaches 90-95%, digesting, centrifuging, and removing supernatant to obtain the strainBy P0 passages. In the present invention, 1.5mL of serum-free medium is preferably added to each well of the 6-well plate, and 0.3mL of digestion solution is preferably added to each well. The sources of the serum-free culture medium and the digestive juice are preferably the same as those described above, and the sources of the serum-free culture medium and the digestive juice described below are also the same as those described above, and are not described in detail below. In the present invention, the time for the digestion is preferably 3 min. In the present invention, the conditions of the centrifugation are: 2000-3000 r/min, 5 min. In the present invention, before the generation of P0 cells is obtained, it is preferable to further include the detection of endotoxin, mycoplasma, anaerobic bacteria and aerobic bacteria in the supernatant.

The cell generation P0 is treated according to the ratio of 0.8-1 ten thousand/cm²Inoculating the cells into a culture bottle, adding a serum-free culture medium for culturing for 3-4 days until the fusion degree reaches 90-95%, digesting, centrifuging, and removing the supernatant to obtain P1 generation cells. In the present invention, the culture flask is preferably a T25 culture flask. Preferably, 5mL of serum-free medium is added to each flask. In the present invention, when a T25 flask is used, the amount of the digestion solution to be added is preferably 1mL, and the digestion time is preferably 3 min. In the present invention, the conditions of the centrifugation are: 2000-3000 r/min, 5 min. In the present invention, before the generation of P1 cells is obtained, it is preferable to further include the detection of endotoxin, mycoplasma, anaerobic bacteria and aerobic bacteria in the supernatant.

The cell generation P1 is treated according to the ratio of 0.8-1 ten thousand/cm²Inoculating the strain into a culture bottle, adding a serum-free culture medium for culturing for 2-3 days until the fusion degree reaches 90-95%, digesting, centrifuging, and removing a supernatant to obtain P2 generation cells; and (4) resuspending the cells of the P2 generation in a freezing medium, freezing and constructing a seed bank. In the present invention, the culture flask is preferably a T75 culture flask. Each flask is preferably filled with 15mL of serum-free medium. In the present invention, the volume of the digestion solution used for the digestion is preferably 2mL per T75 flask. The digestion time in the present invention is preferably 3 min. In the present invention, the conditions of the centrifugation are: 2000-3000 r/min, 5 min. In the present invention, before obtaining the P2 generation cells, it is preferable to further perform endotoxin, mycoplasma, anaerobe and aerobe detection on the supernatant, and perform viability detection, flow detection and infectious disease detection on the cellsDetecting nucleic acid; the flow detection comprises the detection of CD73, CD90, CD105, CD34, CD45 and HLA-DR (the invention increases the number of surface markers, and can obtain more accurate results); the indexes for detecting infectious disease nucleic acid include novel coronavirus nucleic acid, hepatitis B virus nucleic acid, hepatitis C virus nucleic acid, treponema pallidum antibody and human immunodeficiency virus nucleic acid. The invention preferably selects 20 ten thousand cells for activity rate detection, flow detection and infectious disease nucleic acid detection. In the invention, the activity rate detection preferably keeps the activity rate above 90%, and the flow detection requires: CD73 was positive, CD90 was positive, CD105 was positive, CD34 was negative, CD45 was negative, HLA-DR was negative. The infectious disease nucleic acid detection requires that all the indexes are negative. In the present invention, the cryopreservation density of the P2 generation stem cells in the seed bank is preferably 300 ten thousand stem cells per milliliter of cell cryopreservation solution. In the present invention, the conditions for the cryopreservation are preferably: keeping the temperature at 4 ℃ for 15min, reducing the temperature at 1 ℃/min to-7 ℃, reducing the temperature at 60 ℃/min to-60 ℃, increasing the temperature at 30 ℃/min to-30 ℃, and reducing the temperature at 1 ℃/min to-90 ℃. In the present invention, before the cryopreservation, it is preferable to further include: and pasting an anti-freezing two-dimensional code on the freezing storage pipe for marking.

The cell generation P2 is treated according to the ratio of 0.5-0.8 ten thousand/cm²Inoculating the strain into a culture bottle, adding a serum-free culture medium for culturing for 3-4 days until the fusion degree reaches 90-95%, digesting, centrifuging, and removing a supernatant to obtain P3 generation cells; and (4) resuspending the cells of the P3 generation in a freezing medium, freezing and constructing to obtain a master library. In the present invention, the culture flask is preferably a T175 flask. Each flask is preferably filled with 25mL of serum-free medium. When T175 flasks are used, the preferred amount of digestive juice added to each flask is 4 mL. In the present invention, the time for the digestion is preferably 3 min. In the present invention, the conditions of the centrifugation are preferably: 2000-3000 r/min, 5 min. In the invention, before obtaining the P3 generation cells, the method also comprises the steps of detecting endotoxin, mycoplasma, anaerobe and aerobe of the supernatant, and detecting the survival rate, flow detection and infectious disease nucleic acid of the cells; the flow assay includes detection of CD73, CD90, CD105, CD34, CD45 and HLA-DR (the present invention increases the number of surface markers and can achieve more accurate detectionThe exact result); the indexes for detecting infectious disease nucleic acid include novel coronavirus nucleic acid, hepatitis B virus nucleic acid, hepatitis C virus nucleic acid, treponema pallidum antibody and human immunodeficiency virus nucleic acid. The invention preferably selects 20 ten thousand cells for activity rate detection, flow detection and infectious disease nucleic acid detection. In the invention, the activity rate detection preferably keeps the activity rate above 90%, and the flow detection requires: CD73 was positive, CD90 was positive, CD105 was positive, CD34 was negative, CD45 was negative and HLA-DR was negative. The infectious disease nucleic acid detection requires that all the indexes are negative. In the present invention, the cryopreservation density of the P3 generation stem cells in the master library is preferably 1000 ten thousand stem cells per ml of cell cryopreservation solution. In the present invention, the conditions for the cryopreservation are preferably: keeping the temperature at 4 ℃ for 15min, reducing the temperature at 1 ℃/min to-7 ℃, reducing the temperature at 60 ℃/min to-60 ℃, increasing the temperature at 30 ℃/min to-30 ℃, and reducing the temperature at 1 ℃/min to-90 ℃. In the present invention, before the cryopreservation, it is preferable to further include: and pasting an anti-freezing two-dimensional code on the freezing storage pipe for marking.

The cell generation P3 is treated according to the ratio of 0.5-0.8 ten thousand/cm²Inoculating the strain into a culture bottle, adding a serum-free culture medium for culturing for 3-4 days until the fusion degree reaches 90-95%, digesting, centrifuging, and removing a supernatant to obtain P4 generation cells; and (4) resuspending the P4 generation cells in a freezing medium, freezing and constructing to obtain a working library. In the present invention, the culture flask is preferably a T175 flask. Each flask is preferably filled with 25mL of serum-free medium. When T175 flasks are used, the preferred amount of digestive juice added to each flask is 4 mL. In the present invention, the time for the digestion is preferably 3 min. In the present invention, the conditions of the centrifugation are: 2000-3000 r/min, 5 min. In the invention, before obtaining the P4 generation cells, the method also comprises the steps of detecting endotoxin, mycoplasma, anaerobe and aerobe of the supernatant, and detecting the survival rate, flow detection and infectious disease nucleic acid of the cells; the flow detection comprises the detection of CD73, CD90, CD105, CD34, CD45, HLA-DR, CD11b and CD19 (the invention increases the number of surface markers, and can obtain more accurate results); the index for detecting nucleic acid of infectious disease comprises novel coronavirus nucleic acid, hepatitis B virus nucleic acid, hepatitis C virus nucleic acid, and treponema pallidumAntibodies and human immunodeficiency virus nucleic acids. The invention preferably selects 20 ten thousand cells for activity rate detection, flow detection and infectious disease nucleic acid detection. In the invention, the activity rate detection preferably keeps the activity rate above 90%, and the flow detection requires: CD73 was positive, CD90 was positive, CD105 was positive, CD34 was negative, CD45 was negative, HLA-DR was negative, CD11b was negative and CD19 was negative. The infectious disease nucleic acid detection requires that all the indexes are negative. In the present invention, the cryopreservation density of the P4 generation stem cells in the working library is preferably 1000 ten thousand stem cells per ml of cell cryopreservation solution. In the invention, the cryopreservation conditions are as follows: keeping the temperature at 4 ℃ for 15min, reducing the temperature at 1 ℃/min to-7 ℃, reducing the temperature at 60 ℃/min to-60 ℃, increasing the temperature at 30 ℃/min to-30 ℃, and reducing the temperature at 1 ℃/min to-90 ℃. In the invention, before freezing, the method further comprises the following steps: and pasting an anti-freezing two-dimensional code on the freezing storage pipe for marking.

The cell generation P4 is treated according to the ratio of 0.5-0.8 ten thousand/cm²Inoculating the strain into a cell factory, adding a serum-free culture medium for culturing for 4-5 days until the fusion degree reaches 90-95%, digesting, centrifuging, and removing a supernatant to obtain P5 generation cells; and (4) resuspending the cells of the P5 generation in a freezing medium, freezing and constructing to obtain a product library. The cell factory according to the invention is preferably a 10-layer cell factory. In the present invention, 100mL of the digestion solution is preferably added to each cell factory, and the time for digestion is preferably 3 min. What is the amount of medium added? In the invention, before obtaining the P5 generation cells, the method preferably further comprises the steps of detecting endotoxin, mycoplasma, anaerobe and aerobe of the supernatant, and detecting the survival rate, flow detection and infectious disease nucleic acid of the cells; the flow detection comprises the detection of CD73, CD90, CD105, CD34, CD45, HLA-DR, CD11b and CD19 (the invention increases the number of surface markers, and can obtain more accurate results); the indexes for detecting infectious disease nucleic acid include novel coronavirus nucleic acid, hepatitis B virus nucleic acid, hepatitis C virus nucleic acid, treponema pallidum antibody and human immunodeficiency virus nucleic acid. The invention preferably selects 20 ten thousand cells for activity rate detection, flow detection and infectious disease nucleic acid detection. In the invention, the activity rate detection preferably keeps the activity rate above 90%, and the flow detection requires: CD73 positive, CD90Positive, CD105 positive, CD34 negative, CD45 negative, HLA-DR negative, CD11b negative and CD19 negative. The infectious disease nucleic acid detection requires that all the indexes are negative. In the present invention, the cryopreservation density of the P5 generation stem cells in the product library is preferably 1000 ten thousand stem cells per ml of cell cryopreservation solution. In the present invention, it is preferable to collect 1000 ten thousand P5 generation cells and freeze 10 cells per 100 ten thousand cells in each tube as a quality control material. In the invention, the cryopreservation conditions are as follows: keeping the temperature at 4 ℃ for 15min, reducing the temperature at 1 ℃/min to-7 ℃, reducing the temperature at 60 ℃/min to-60 ℃, increasing the temperature at 30 ℃/min to-30 ℃, and reducing the temperature at 1 ℃/min to-90 ℃. In the present invention, the conditions of the centrifugation are: 2000-3000 r/min, 5 min. In the invention, before freezing, the method further comprises the following steps: and pasting an anti-freezing two-dimensional code on the freezing storage pipe for marking.

The construction method provided by the invention comprises the steps of collection, separation, culture and cryopreservation, and the clinical-grade human umbilical cord mesenchymal stem cell bank comprises a seed bank, a master bank, a working bank and a product bank.

The specific composition is preferably as follows:

seed bank: p2 generation stem cells, 2ml of cryopreservation tubes, 300 ten thousand stem cells/tube, and 1ml of cell cryopreservation solution;

a master library: p3 generation stem cells, 2ml of cryopreservation tubes, 500 ten thousand stem cells/tube, and 1ml of cell cryopreservation solution;

a working library: p4 generation stem cells, 2ml of cryopreservation tubes, 1000 ten thousand of stem cells/tube, and 1ml of cell cryopreservation solution;

product library: p5 generation stem cells, 5ml of cryopreservation tubes, 5000 ten thousand stem cells/tube, and 5ml of cell cryopreservation liquid;

the structure is as follows: level 4 library architecture, seed library: p2 passage cells as stock.

A master library: p3 passage cells as the stock of the working bank.

A working library: p4 generation cell used as raw material of product library or directly used in clinic

Product library: the P5 generation cells were used directly in clinic.

The invention provides a construction method of a clinical-grade human umbilical cord mesenchymal stem cell resource library multistage library, which can lead cells to reach the specified fusion degree in the specified time through setting the cell inoculation density of each stage and multiple verification tests, and can more accurately control the generation of cell amplification compared with the traditional proportional amplification mode so as to achieve the unification of cell quality. The primary cell separation method can separate primary cells within a specified time, and improves the cell viability in a later cell bank (at present, the hospital performs tests according to 20 cases of umbilical cords in one month, and more than 90 percent of umbilical cords can separate stem cells). The cell safety indexes are detected by detecting each index, namely: the detection of mycoplasma, endotoxin, anaerobe, aerobe and infectious disease indexes are all required to be negative. Cell dryness index: the survival rate is more than 90%, and the flow detection shows that the CD73 is positive, the CD90 is positive, the CD105 is positive, the CD34 is negative, the CD45 is negative, the HLA-DR is negative, the CD11b is negative, and the CD19 is negative. The detection link ensures that the warehoused cells can meet the detection requirements of Chinese pharmacopoeia on biological agents. The setting of the freezing condition of the cells ensures the survival rate of the cells after freezing. The invention provides key parameters, detection key points, a large-scale product library concept and a two-dimension code management concept of each step of the construction method, and lays a foundation for clinical application of stem cells in the future.

The construction method of the clinical-grade human umbilical cord mesenchymal stem cell resource library multistage library is further described in detail with reference to specific examples, and the technical scheme of the invention includes but is not limited to the following examples.

Example 1

A seed bank establishment scheme:

donor screening: donor test items: the kit is used for detecting the human immunodeficiency virus (EBV) IgM antibody, the lymphoblastic leukemia virus (HCLV) antibody, the Hepatitis C Virus (HCV) antibody, the cytomegalovirus (HCMV) IgM antibody, the hepatitis B virus surface antigen, the human immunodeficiency virus antibody and the antigen, the treponema pallidum antibody, the hepatitis B virus nucleic acid, the hepatitis C virus nucleic acid, the human immunodeficiency virus nucleic acid and the EBV IgM antibody, and is a healthy female with no family infection history, no virus suction history and the age of 20-35.

Collecting raw materials: cutting 5-10 cm of umbilical cord, clamping two ends of the umbilical cord by using disposable hemostatic clamps, respectively, filling the umbilical cord into a medical self-sealing bag, sticking a donor bar code, filling into a 4 ℃ transfer box, filling an infectious disease detection report, an informed consent and acquisition record list into a material bag, and transferring to a preparation room.

Pretreatment of raw materials: opening the hemostatic clamp, washing blood with normal saline, cutting the umbilical cord into strips with the length of 2cm by using a medical scalpel, splitting the umbilical cord by using a scissors, washing the umbilical cord for 2-3 times by using the normal saline, and washing the blood. Using forceps, 3 vessels (2 arteries, 1 vein) in the umbilical cord were removed, and 2X flush was prepared: 30ml of 80% serum-free medium + 20% double antibody (for penicillin streptomycin mixed solution (100 ×) cell culture), 1 × rinsing solution was prepared: 30ml of 90% serum-free medium and 10% double antibody in total, and soaking for 10min by using 2X flushing fluid and then soaking for 10min by using 1X flushing fluid.

Stem cell isolation: cutting the umbilical cord into 1-2 mm pieces by using a scalpel³And (3) uniformly placing 20-25 tissue blocks in each hole of a 6-hole plate, placing the tissue blocks in a carbon dioxide incubator for 20min to adhere to the wall, adding 1ml of serum-free culture medium at 37 ℃ into each hole, placing the tissue blocks in the carbon dioxide incubator overnight, and adding 1ml of the culture medium the next day.

Amplification culture: after the fusion rate of the primary cells reaches 80-90%, digesting the primary cells for 3min at room temperature by using a digestive juice, taking 5-10 ten thousand cells for flow detection, and performing flow detection according to the inoculation density of 0.8-1 ten thousand/cm²Transferring into 6-well plate, adding 1.5ml of serum-free culture medium into each well, culturing in carbon dioxide incubator for 3-4 days until the fusion degree reaches 90-95%, digesting with 0.3ml of digestive fluid at room temperature for 3min, centrifuging at 2000-3000 rpm for 5min, removing supernatant, collecting supernatant, detecting endotoxin, mycoplasma, anaerobic bacteria and aerobic bacteria, and inoculating cells (P0 generation) at an inoculation density of 0.8-1 ten thousand/cm²Inoculating the cells into a T25 culture bottle, adding 5ml of serum-free culture medium into each bottle, culturing for 3-4 days until the fusion degree reaches 90-95%, digesting for 3min at room temperature by using 1ml of digestive juice, centrifuging for 5min at 2000-3000 r/min of heavy suspension, removing supernatant, taking supernatant, performing the same detection as primary culture, and detecting the cells (P1 generation) according to the inoculation density of 0.8-1 ten thousand/cm²Inoculating into T75 culture flask, adding culture medium 15ml into each flask until the fusion degree reaches 90-95%, and using digestive juiceDigesting 2ml of the mixture at room temperature for 3min, centrifuging the heavy suspension for 5min at 2000-3000 rpm, removing the supernatant, taking the supernatant to perform anaerobic bacteria, aerobic bacteria, mycoplasma and endotoxin detection, taking 20 ten thousand cells to perform survival rate, counting, flow detection, novel coronavirus nucleic acid detection, hepatitis B virus nucleic acid detection, hepatitis C virus nucleic acid detection, treponema pallidum antibody detection and human immunodeficiency virus nucleic acid detection. Taking 1000 ten thousand cells (P2 generation), and inoculating at a density of 0.5-0.8 ten thousand/cm²Inoculated into 10T 175 flasks, and the remainder resuspended at 300 million cells per ml of the cryopreservative, using the cryopreservation procedure: keeping the temperature at 4 ℃ for 15min, reducing the temperature at 1 ℃/min to-7 ℃, reducing the temperature at 60 ℃/min to-60 ℃, increasing the temperature at 30 ℃/min to-30 ℃, and reducing the temperature at 1 ℃/min to-90 ℃. 5-10 pipes are frozen, and each pipe is pasted with an anti-freezing two-dimensional code.

Establishing a main library scheme:

10T 175 culture bottles filled with P2 cells are obtained in the seed bank establishing process, the culture is carried out for 3-4 days until the fusion degree reaches 90-95%, 4ml of digestive juice is added, room temperature digestion is carried out for 3min, the heavy suspension is centrifuged for 5min at 2000-3000 r/min, the supernatant is removed, the supernatant is taken as a detection item of the same seed bank, 20 ten thousand cells are taken as a detection item of the same seed bank, the survival rate, the counting and the flow type are carried out, 2000 ten thousand cells are taken, and the inoculation density is 0.5-0.8 ten thousand/cm²The cells were inoculated into 20T 175 flasks and the remainder were resuspended in 500 million cells per ml of the culture using the same seed bank cryopreservation procedure. And (5) freezing 10-15 pipes, and sticking an anti-freezing two-dimensional code to each pipe.

The working library establishment scheme comprises the following steps:

and (3) obtaining 20T 175 culture bottles filled with P3 cells in the process of establishing the main library, culturing for 3-4 days until the fusion degree reaches 90-95%, adding 4ml of digestive juice into each bottle, digesting for 3min at room temperature, centrifuging for 5min at 2000-3000 r/min of heavy suspension, removing supernatant, taking supernatant, detecting anaerobic bacteria, aerobic bacteria, mycoplasma and endotoxin, and taking 20 ten thousand cells for viability, counting and flowing. Taking 2000 ten thousand cells, and inoculating according to the density of 0.5-0.8 ten thousand/cm²The cells were seeded into 2 10-layered cell factories, and the rest were resuspended in 1000 million cells per ml of the cryopreservative by adding the cryopreservative, using the same seed bank cryopreservation procedure. And (5) freezing 10-15 pipes, and sticking an anti-freezing two-dimensional code to each pipe.

A product library establishment scheme:

2 10 layers of cell factories containing P4 cells are obtained in the process of establishing a working library, the factories are cultured for 4-5 days, when the fusion degree reaches 90-95%, 100ml of digestive juice is added into each factory for digesting for 3min at room temperature, 2000-3000 r/min of heavy suspension is centrifuged for 5min, the supernatant is removed, the supernatant is taken for detecting anaerobic bacteria, aerobic bacteria, mycoplasma and endotoxin, and 20 ten thousand cells are taken for survival rate, counting and flow. And (3) taking 1000 ten thousand cells, freezing and storing 10 cells per tube of 100 ten thousand cells to serve as a quality control product, adding the freezing medium into the rest 1000 ten thousand cells per milliliter of the freezing medium for resuspension, and using the freezing and storing program of the same seed bank. And (5) freezing 10-15 pipes, and sticking an anti-freezing two-dimensional code to each pipe.

1% of the total donor number was extracted for detection

The detection method comprises the following steps:

endotoxin: gel method

Mycoplasma: PCR method

Anaerobic bacteria and aerobic bacteria: culture method

The survival rate: MTT method

Flow type: flow cytometry

Infectious disease nucleic acid detection: PCR

The above detection methods are subject to the reagent and equipment operation instructions.

The recovery method comprises the following steps:

and (3) thawing the cells by using a cell resuscitator, centrifuging for 5min at 3000 r/min, removing supernatant of frozen stock solution, collecting the supernatant for detection, adding 2ml of serum-free basal medium into the cells, and carrying out cell detection after carrying out basal rotation.

The flow chart of the construction and detection of the present invention is shown in FIG. 1.

And (3) detection results:

TABLE 1 Activity Rate test results

Donor	Fruit before frozen storage	Fruit after frozen storage
			Donor 1 (seed bank)	99％	90％
Donor 1 (Master library)	98％	92％
			Donor 1 (working library)	99％	93％
Donor 1 (product store)	99％	90％
			Donor 2 (seed bank)	98％	95％
Donor 2 (Master library)	97％	95％
			Donor 2 (working library)	98％	93％
Donor 2 (product store)	96％	90％
			Donor 3 (seed bank)	99％	93％
Donor 3 (Master library)	99％	95％
			Donor 3 (working library)	98％	92％
Donor 3 (product store)	99％	88％

As shown in Table 1, the cell viability reaches 96-99% before cryopreservation, and the cell viability is high. After freezing, the cell survival rate can be reduced by 2-11% compared with the cell before freezing in the processes of temperature reduction, rewarming and centrifugation of cell fruiting programs.

The higher survival rate before freezing indicates that the method for constructing the seed bank is effective, can extract stem cells from umbilical cord tissues and culture cells with better activity, and the activity of most of the cells can reach more than 90 percent after freezing.

Compared with the prior art, the cell viability of the whole cell bank is improved.

TABLE 2 flow assay results

The flow detection items of the seed bank and the main bank comprise 6 surface markers, namely CD73, CD90, CD105, CD34, CD45 and HLA-DR, and all detection results before and after the frozen storage are positive for CD73, positive for CD90, positive for CD105, negative for CD34, negative for CD45 and negative for HLA-DR. The flow-through test items of the working library and the product library comprise 8 surface markers, namely CD73, CD90, CD105, CD34, CD45, HLA-DR, CD11b and CD19, and all test results before and after the frozen storage are positive for CD73, positive for CD90, positive for CD105, negative for CD34, negative for CD45, negative for HLA-DR, negative for CD11b and negative for CD 19.

As shown in Table 2, the cells according to the flow results are consistent with the characteristics of the stem cells, which indicates that the cell bank construction method of the invention can separate and culture the stem cells, and the characteristics of the stem cells are not changed during the storage process.

TABLE 3 endotoxin, Mycoplasma, anaerobe, and aerobe test results

As shown in Table 3, the results of detection of endotoxin, mycoplasma, anaerobe and aerobe before and after cryopreservation were negative. The cell bank construction method of the present invention is demonstrated to ensure the safety of cells and to prevent the cells from being contaminated during the preservation process. Compared with the prior art, the method adopts the partition management (namely setting the seed bank, the main bank, the working bank and the product bank) and the gas-phase liquid nitrogen tank for storage, thereby avoiding the cross contamination among cells and ensuring the safety of the cells.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.