CN113832548B - Construction method of clinical-grade human umbilical cord mesenchymal stem cell resource library multilevel library - Google Patents
Construction method of clinical-grade human umbilical cord mesenchymal stem cell resource library multilevel library Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention relates to a method for constructing a clinical-grade human umbilical cord mesenchymal stem cell resource library multilevel library, belonging to the technical field of cell culture. The construction method of the invention comprises the steps of raw material pretreatment, stem cell separation, amplification culture to obtain a seed library, and then realizing the construction of a main library, a working library and a product library through the setting of conditions such as inoculation density, culture time and the like. The construction method has high cell activity and controllable quality, and can successfully construct a clinical-grade four-level stem cell resource library, namely a seed library, a main library, a working library and a product library.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to a method for constructing a clinical-grade human umbilical cord mesenchymal stem cell resource library multistage library.
Background
As the first large country of the global population, and as the developing country that is walking into the aging society, china is facing many challenges in terms of population health. These challenges are reflected in the ongoing rise in the incidence of tissue organ dysfunction due to the progressive shift from infectious to chronic and senile degenerative diseases, the increasingly pronounced trend of the younger onset of various tumors, and the various factors of genetic wound metabolism. For these diseases, traditional drug therapies and surgical therapies are largely ineffective, and cell, tissue or organ based replacement therapies are expected to be the best option for radical treatment of these diseases.
Human umbilical cord mesenchymal stem cells (Human Umbilical Cord MESENCHYMAL STEM CELLS, HUC-MSCs) are a type of pluripotent stem cells with higher differentiation potential that exist in neonatal umbilical cord tissue. Under certain conditions, it can differentiate into various functional tissues and organs. Due to the multi-directional differentiation potential, low immunogenicity and adhesion characteristics, and abundant and less ethical disputes of material sources, the cell has become one of ideal seed cells for regenerative medicine therapy and tissue engineering transplantation, and is increasingly favored by researchers. In view of this, it is particularly critical to build a clinical-grade hUC-MSCs (which can meet the relevant quality standards for clinic) resource library, which can not only serve the clinical research and application of stem cells, but also provide stem cell resources meeting the demands of scientific research for universities, scientific research institutions, enterprises and institutions, and the like.
At present, the preservation of the domestic stem cell bank on the umbilical cord stem cells is mainly scientific research, and the used preparation technology and detection technology cannot be unified and do not have a standardized method.
Disclosure of Invention
The invention aims to provide a method for constructing a clinical-grade human umbilical cord mesenchymal stem cell resource library multistage library. The construction method has high cell activity and controllable quality, can successfully construct a clinical-grade four-level stem cell resource library, namely a seed library, a main library, a working library and a product library, and can realize standardization of the preparation process.
The invention provides a method for constructing a multi-level library of a clinical-grade human umbilical mesenchymal stem cell resource library, wherein the multi-level library comprises a seed library, a main library, a working library and a product library; the construction method comprises the following steps:
Sectioning the umbilical cord, cleaning, removing blood vessels, and sequentially soaking in a first flushing fluid and a second flushing fluid to obtain a pretreated umbilical cord; the first flushing liquid comprises a serum-free culture medium with the volume percentage of 80% and a mixture of the green streptomycin with the volume percentage of 20%; the second flushing liquid comprises a serum-free culture medium with the volume percentage of 90% and a mixture of the green streptomycin with the volume percentage of 10%;
Cutting the pretreated umbilical cord into tissue blocks with the thickness of 1-2 mm 3, placing the tissue blocks in a 6-hole plate, performing wall-attached 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%, thus obtaining primary cells;
inoculating primary cells into a 6-hole plate according to the inoculation density of 0.8-1 ten thousand/cm 2, adding a serum-free culture medium, culturing for 3-4 d, digesting, centrifuging and removing the supernatant to obtain P0-generation cells when the fusion degree reaches 90-95%;
Inoculating the P0 generation cells into a culture flask according to the inoculation density of 0.8-1 ten thousand/cm 2, adding a serum-free culture medium for culturing for 3-4 d, and removing the supernatant after the fusion degree reaches 90-95%, and obtaining the P1 generation cells;
inoculating the P1 generation cells into a culture flask according to the inoculation density of 0.8-1 ten thousand/cm 2, adding a serum-free culture medium for culturing for 2-3 d, and removing the supernatant after the fusion degree reaches 90-95%, and obtaining the P2 generation cells; resuspending the P2 generation cells in a freezing solution, freezing and preserving to construct a seed library;
Inoculating the P2 generation cells into a culture flask according to the inoculation density of 0.5-0.8 ten thousand/cm 2, adding a serum-free culture medium for culturing for 3-4 d, and after the fusion degree reaches 90-95%, digesting, centrifuging and removing the supernatant to obtain the P3 generation cells; resuspending the P3 generation cells in a freezing solution, freezing and storing to construct a main library;
Inoculating the P3 generation cells into a culture flask according to the inoculation density of 0.5-0.8 ten thousand/cm 2, adding a serum-free culture medium for culturing for 3-4 d, and after the fusion degree reaches 90-95%, digesting, centrifuging and removing the supernatant to obtain the P4 generation cells; resuspending the P4 generation cells in a freezing solution, freezing and storing to construct a working library;
inoculating the P4 generation cells into a cell factory according to the inoculation density of 0.5-0.8 ten thousand/cm 2, adding a serum-free culture medium for culturing for 4-5 d, and after the fusion degree reaches 90-95%, digesting, centrifuging and removing the supernatant to obtain the P5 generation cells; resuspending the P5 generation cells in the frozen stock solution, freezing and storing to construct a product library.
Preferably, the umbilical cord is from a healthy donor; the healthy donor has no family infection history and no drug absorption history, and the negative detection items comprise: lymphocytic leukemia virus antibodies, hepatitis C virus antibodies, cytomegalovirus IgM antibodies, hepatitis B virus surface antigens, human immunodeficiency virus antibodies and antigens, treponema pallidum antibodies, hepatitis B virus nucleic acids, hepatitis C virus nucleic acids, human immunodeficiency virus nucleic acids, and EBV virus IgM antibodies.
Preferably, the soaking time is 3-5 min respectively.
Preferably, the tissue pieces are placed in 20 to 25 wells per well of a 6-well plate.
Preferably, the detection of endotoxin, mycoplasma, anaerobic bacteria and aerobic bacteria is also included in the supernatant before the generation of P0 cells and the generation of P1 cells.
Preferably, the method further comprises the steps of detecting endotoxin, mycoplasma, anaerobic bacteria and aerobic bacteria in the supernatant, and detecting the viability, the flow type and the infectious disease nucleic acid of the cells before obtaining the cells of the generation P2, the cells of the generation P3, the cells of the generation P4 and the cells of the generation P5; the streaming assays include the assays for CD73, CD90, CD105, CD34, CD45, HLA-DR, CD11b and CD 19; the indexes of the infectious disease nucleic acid detection comprise 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 liquid;
the cryopreservation density of the P3 generation stem cells in the main library is 500 ten thousand stem cells per milliliter of cell cryopreservation liquid;
the cryopreservation density of the P4 generation stem cells in the working library is 1000 ten thousand stem cells per milliliter of cell cryopreservation liquid;
The cryopreservation density of the P5 generation stem cells in the product library is 1000 ten thousand stem cells per milliliter of cell cryopreservation liquid.
Preferably, the freezing conditions are as follows: keeping at 4deg.C for 15min, cooling to-7deg.C at 1deg.C/min, cooling to-60deg.C at 60deg.C/min, cooling to-30deg.C at 30deg.C/min, and cooling to-90deg.C at 1deg.C/min.
Preferably, the centrifugation conditions are: 2000-3000 rpm, 5min.
Preferably, before the freezing, the method further comprises: and labeling the frozen storage tube by attaching an antifreezing two-dimensional code. .
The invention provides a method for constructing a clinical-grade human umbilical cord mesenchymal stem cell resource library multilevel library. The construction method has high cell activity and controllable quality, and can successfully construct a clinical-grade four-level stem cell resource library, namely a seed library, a main library, a working library and a product library. Specifically, the invention realizes high cell activity rate through the screening mode of the donor, the separation mode of the primary cells, the control of the inoculation density and the setting of the freezing mode of the cells, and performs quality control and relevant detection in each link of library establishment by referring to the pre-clinical guidelines 'Chinese pharmacopoeia' and the like, thereby ensuring the quality control. The 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 stored, and a culture mode and a freezing mode are reasonably set according to the action of a reservoir area.
Drawings
FIG. 1 is a flow chart of the construction and detection method provided by the present invention.
Detailed Description
The invention provides a method for constructing a multi-level library of a clinical-grade human umbilical mesenchymal stem cell resource library, wherein the multi-level library comprises a seed library, a main library, a working library and a product library; the construction method comprises the following steps:
Sectioning the umbilical cord, cleaning, removing blood vessels, and sequentially soaking in a first flushing fluid and a second flushing fluid to obtain a pretreated umbilical cord; the first flushing liquid comprises a serum-free culture medium with the volume percentage of 80% and a mixture of the green streptomycin with the volume percentage of 20%; the second flushing liquid comprises a serum-free culture medium with the volume percentage of 90% and a mixture of the green streptomycin with the volume percentage of 10%;
Cutting the pretreated umbilical cord into tissue blocks with the thickness of 1-2 mm 3, placing the tissue blocks in a 6-hole plate, performing wall-attached 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%, thus obtaining primary cells;
inoculating primary cells into a 6-hole plate according to the inoculation density of 0.8-1 ten thousand/cm 2, adding a serum-free culture medium, culturing for 3-4 d, digesting, centrifuging and removing the supernatant to obtain P0-generation cells when the fusion degree reaches 90-95%;
Inoculating the P0 generation cells into a culture flask according to the inoculation density of 0.8-1 ten thousand/cm 2, adding a serum-free culture medium for culturing for 3-4 d, and removing the supernatant after the fusion degree reaches 90-95%, and obtaining the P1 generation cells;
inoculating the P1 generation cells into a culture flask according to the inoculation density of 0.8-1 ten thousand/cm 2, adding a serum-free culture medium for culturing for 2-3 d, and removing the supernatant after the fusion degree reaches 90-95%, and obtaining the P2 generation cells; resuspending the P2 generation cells in a freezing solution, freezing and preserving to construct a seed library;
Inoculating the P2 generation cells into a culture flask according to the inoculation density of 0.5-0.8 ten thousand/cm 2, adding a serum-free culture medium for culturing for 3-4 d, and after the fusion degree reaches 90-95%, digesting, centrifuging and removing the supernatant to obtain the P3 generation cells; resuspending the P3 generation cells in a freezing solution, freezing and storing to construct a main library;
Inoculating the P3 generation cells into a culture flask according to the inoculation density of 0.5-0.8 ten thousand/cm 2, adding a serum-free culture medium for culturing for 3-4 d, and after the fusion degree reaches 90-95%, digesting, centrifuging and removing the supernatant to obtain the P4 generation cells; resuspending the P4 generation cells in a freezing solution, freezing and storing to construct a working library;
inoculating the P4 generation cells into a cell factory according to the inoculation density of 0.5-0.8 ten thousand/cm 2, adding a serum-free culture medium for culturing for 4-5 d, and after the fusion degree reaches 90-95%, digesting, centrifuging and removing the supernatant to obtain the P5 generation cells; resuspending the P5 generation cells in the frozen stock solution, freezing and storing to construct a product library.
The umbilical cord of the present invention is preferably from a healthy donor; the healthy donor needs no family history of infection and no history of drug absorption, and the negative detection items need to include: lymphocytic leukemia virus antibodies, hepatitis C virus antibodies, cytomegalovirus IgM antibodies, hepatitis B virus surface antigens, human immunodeficiency virus antibodies and antigens, treponema pallidum antibodies, hepatitis B virus nucleic acids, hepatitis C virus nucleic acids, human immunodeficiency virus nucleic acids, and EBV virus IgM antibodies. In the present invention, the age of the donor is preferably between 20 and 35. After screening to obtain the umbilical cord of the donor according to the above conditions, the present invention preferably shears 5-10 cm umbilical cord. After the umbilical cord is sheared, hemostatic clamps are preferably used for clamping two ends of the umbilical cord, and the umbilical cord is stored and transported at 4 ℃. Before the umbilical cord is pretreated, the hemostatic clamp is preferably opened, and the blood of the umbilical cord is washed clean by using physiological saline. After flushing, the umbilical cord is dissected, cleaned and the blood vessel is removed, and the umbilical cord is soaked in the first flushing fluid and the second flushing fluid in sequence to obtain a pretreated umbilical cord; the first flushing liquid comprises a serum-free culture medium with the volume percentage of 80% and a mixture of the green streptomycin with the volume percentage of 20%; the second flushing liquid comprises 80% of serum-free culture medium and 10% of a mixture of the green streptomycin by volume. The umbilical cord is preferably cut into 2cm strips prior to dissection. After the blood is dissected, the blood is preferably cleaned by using normal saline, and the cleaning times are preferably 2-3 times, so that the blood is ensured to be cleaned. The removal of blood vessels according to the invention is preferably the removal of 3 blood vessels, including 2 arteries and 1 vein. The raw materials for preparing the flushing fluid comprise serum-free culture medium and a mixture of the green streptomycin, the sources of the two raw materials are not particularly limited, and the flushing fluid is prepared by adopting conventional commercial products. In the present invention, the green streptomycin mixture is preferably a green streptomycin mixture (100×) dedicated for cell culture, available from Gibco under the model number 15140-122. In the present invention, the serum-free medium is preferably purchased from Gibco under the model number 8122159. According to the invention, the two components in the first flushing liquid and the second flushing liquid are arranged in different proportions for twice soaking, so that the blood can be washed off as much as possible, the climbing of primary cells from tissues is facilitated, and the adhesion of the tissue blocks is facilitated. In the present invention, the soaking time is preferably 3 to 5 minutes, respectively. The invention preferably uses the first flushing fluid to soak for 10 minutes and then uses the second flushing fluid to soak for 10 minutes.
After the pretreated umbilical cord is obtained, the pretreated umbilical cord is cut into tissue blocks with the diameter of 1-2 mm 3, the tissue blocks are placed in a 6-hole plate, the tissue blocks are subjected to wall-attached culture in a carbon dioxide incubator for 20min, a serum-free culture medium is added for formulation culture, and after the cell fusion rate reaches 80-90%, primary cells are obtained through digestion. In the present invention, the tissue pieces are placed in 6-well plates, preferably 20 to 25 per well. The present invention preferably places the tissue mass uniformly into each well of a 6-well plate. The invention is preferably carried out overnight after adding the serum-free medium, and the invention is preferably carried out after overnight culture, and the serum-free medium is preferably added again, so as to supplement the volatilized serum-free medium. In the present invention, 1mL of serum-free medium is preferably added to each well of the 6-well plate. After the serum-free culture medium is added, the invention continues to culture for 4-10 d, and digestion is carried out after the cell fusion rate reaches 80-90%. In the present invention, the time of the digestion is preferably 3 minutes. The digestion according to the present invention is preferably carried out at room temperature, preferably 20 to 25℃and the conditions for the digestion described hereinafter are the same as those described herein and will not be described in detail. The digests of the present invention are preferably purchased from GIBCO, model 25200-056. After primary cells are obtained, partial primary cells are preferably selected 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.
Inoculating primary cells into a 6-hole plate according to the inoculation density of 0.8-1 ten thousand/cm 2, adding a serum-free culture medium, culturing for 3-4 d, and removing supernatant after the fusion degree reaches 90-95%, digesting and centrifuging to obtain P0-generation cells. 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 the digestion solution is preferably added to each well. The sources of the serum-free medium and the digestive juice are preferably the same as above, and the sources of the serum-free medium and the digestive juice are also the same as above, and are not described in detail. In the present invention, the time of the digestion is preferably 3 minutes. In the present invention, the centrifugation conditions are: 2000-3000 rpm, 5min. In the present invention, detection of endotoxin, mycoplasma, anaerobic bacteria and aerobic bacteria is preferably further included in the supernatant before obtaining the P0-th cell.
Inoculating the P0 generation cells into a culture flask according to the inoculation density of 0.8-1 ten thousand/cm 2, adding a serum-free culture medium for culturing for 3-4 d, and after the fusion degree reaches 90-95%, digesting, centrifuging and removing the supernatant to obtain the 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 addition amount of the digestive juice is preferably 1mL, and the time for digestion is preferably 3min. In the present invention, the centrifugation conditions are: 2000-3000 rpm, 5min. In the present invention, detection of endotoxin, mycoplasma, anaerobic bacteria and aerobic bacteria is preferably further included in the supernatant before obtaining the P1-generation cells.
Inoculating the P1 generation cells into a culture flask according to the inoculation density of 0.8-1 ten thousand/cm 2, adding a serum-free culture medium for culturing for 2-3 d, and removing the supernatant after the fusion degree reaches 90-95%, and obtaining the P2 generation cells; resuspension the P2 generation cells in the frozen stock solution, freezing and storing to construct a seed library. In the present invention, the culture flask is preferably a T75 culture flask. Each flask is preferably charged with 15mL of serum-free medium. In the present invention, the volume of the digestion solution used for the digestion is preferably 2mL per flask of T75 flask. The time of digestion according to the invention is preferably 3 minutes. In the present invention, the centrifugation conditions are: 2000-3000 rpm, 5min. In the present invention, before obtaining the P2-generation cells, the detection of endotoxin, mycoplasma, anaerobic bacteria and aerobic bacteria, and the detection of the cell viability, flow type detection and infectious disease nucleic acid detection are preferably further included; the flow assay includes the detection of CD73, CD90, CD105, CD34, CD45 and HLA-DR (the present invention increases the number of surface markers, more accurate results can be obtained); the indexes of the infectious disease nucleic acid detection comprise 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 detection, flow detection and infectious disease nucleic acid detection. In the present invention, the activity rate detection preferably maintains the activity rate at 90% or more, and the flow detection requires: CD73 is positive, CD90 is positive, CD105 is positive, CD34 is negative, CD45 is negative, HLA-DR is negative. The detection of infectious disease nucleic acid requires that the above indicators are negative. In the invention, the cryopreservation density of the P2 generation stem cells in the seed pool is preferably 300 ten thousand stem cells per milliliter of cell cryopreservation liquid. In the present invention, the conditions for the cryopreservation are preferably: keeping at 4deg.C for 15min, cooling to-7deg.C at 1deg.C/min, cooling to-60deg.C at 60deg.C/min, cooling to-30deg.C at 30deg.C/min, and cooling to-90deg.C at 1deg.C/min. In the present invention, before the freezing, the method preferably further comprises: and labeling the frozen storage tube by attaching an antifreezing two-dimensional code.
Inoculating the P2 generation cells into a culture flask according to the inoculation density of 0.5-0.8 ten thousand/cm 2, adding a serum-free culture medium for culturing for 3-4 d, and after the fusion degree reaches 90-95%, digesting, centrifuging and removing the supernatant to obtain the P3 generation cells; and re-suspending the P3 generation cells in the frozen stock solution, freezing and storing to construct a main library. In the present invention, the culture flask is preferably a T175 culture flask. Each flask is preferably charged with 25mL of serum-free medium. When T175 flasks are used, the addition of digestive juice per flask is preferably 4mL. In the present invention, the time of the digestion is preferably 3 minutes. In the present invention, the conditions of the centrifugation are preferably: 2000-3000 rpm, 5min. In the invention, before the P3 generation cells are obtained, the detection of endotoxin, mycoplasma, anaerobic bacteria and aerobic bacteria, the detection of the activity rate, the flow detection and the detection of infectious disease nucleic acid are also carried out on the supernatant; the flow assay includes the detection of CD73, CD90, CD105, CD34, CD45 and HLA-DR (the present invention increases the number of surface markers, more accurate results can be obtained); the indexes of the infectious disease nucleic acid detection comprise 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 detection, flow detection and infectious disease nucleic acid detection. In the present invention, the activity rate detection preferably maintains the activity rate at 90% or more, and the flow detection requires: CD73 positive, CD90 positive, CD105 positive, CD34 negative, CD45 negative and HLA-DR negative. The detection of infectious disease nucleic acid requires that the above indicators are negative. In the invention, the cryopreservation density of the P3 generation stem cells in the main library is preferably 1000 ten thousand stem cells per milliliter of cell cryopreservation liquid. In the present invention, the conditions for the cryopreservation are preferably: keeping at 4deg.C for 15min, cooling to-7deg.C at 1deg.C/min, cooling to-60deg.C at 60deg.C/min, cooling to-30deg.C at 30deg.C/min, and cooling to-90deg.C at 1deg.C/min. In the present invention, before the freezing, the method preferably further comprises: and labeling the frozen storage tube by attaching an antifreezing two-dimensional code.
Inoculating the P3 generation cells into a culture flask according to the inoculation density of 0.5-0.8 ten thousand/cm 2, adding a serum-free culture medium for culturing for 3-4 d, and after the fusion degree reaches 90-95%, digesting, centrifuging and removing the supernatant to obtain the P4 generation cells; and re-suspending the P4 generation cells in the frozen stock solution, freezing and storing to construct a working library. In the present invention, the culture flask is preferably a T175 culture flask. Each flask is preferably charged with 25mL of serum-free medium. When T175 flasks are used, the addition of digestive juice per flask is preferably 4mL. In the present invention, the time of the digestion is preferably 3 minutes. In the present invention, the centrifugation conditions are: 2000-3000 rpm, 5min. In the invention, before obtaining the P4 generation cells, the method also comprises the steps of detecting endotoxin, mycoplasma, anaerobic bacteria and aerobic bacteria of supernatant, and detecting the activity rate, the flow type detection and infectious disease nucleic acid of the cells; the flow assay includes the detection of CD73, CD90, CD105, CD34, CD45, HLA-DR, CD11b and CD19 (the invention increases the number of surface markers, more accurate results can be obtained); the indexes of the infectious disease nucleic acid detection comprise 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 detection, flow detection and infectious disease nucleic acid detection. In the present invention, the activity rate detection preferably maintains the activity rate at 90% or more, and the flow detection requires: CD73 is positive, CD90 is positive, CD105 is positive, CD34 is negative, CD45 is negative, HLA-DR is negative, CD11b is negative and CD19 is negative. The detection of infectious disease nucleic acid requires that the above indicators are negative. In the invention, the cryopreservation density of the P4 generation stem cells in the working library is preferably 1000 ten thousand stem cells per milliliter of cell cryopreservation liquid. In the invention, the freezing conditions are as follows: keeping at 4deg.C for 15min, cooling to-7deg.C at 1deg.C/min, cooling to-60deg.C at 60deg.C/min, cooling to-30deg.C at 30deg.C/min, and cooling to-90deg.C at 1deg.C/min. In the invention, before the freezing, the method further comprises the following steps: and labeling the frozen storage tube by attaching an antifreezing two-dimensional code.
Inoculating the P4 generation cells into a cell factory according to the inoculation density of 0.5-0.8 ten thousand/cm 2, adding a serum-free culture medium for culturing for 4-5 d, and after the fusion degree reaches 90-95%, digesting, centrifuging and removing the supernatant to obtain the P5 generation cells; resuspending the P5 generation cells in the frozen stock solution, freezing and storing to construct 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 digestion time is preferably 3min. What is the amount of medium added? In the present invention, before obtaining the P5-generation cells, the detection of endotoxin, mycoplasma, anaerobic bacteria and aerobic bacteria, and the detection of the cell viability, flow type detection and infectious disease nucleic acid detection are preferably further included; the flow assay includes the detection of CD73, CD90, CD105, CD34, CD45, HLA-DR, CD11b and CD19 (the invention increases the number of surface markers, more accurate results can be obtained); the indexes of the infectious disease nucleic acid detection comprise 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 detection, flow detection and infectious disease nucleic acid detection. In the present invention, the activity rate detection preferably maintains the activity rate at 90% or more, and the flow detection requires: CD73 is positive, CD90 is positive, CD105 is positive, CD34 is negative, CD45 is negative, HLA-DR is negative, CD11b is negative and CD19 is negative. The detection of infectious disease nucleic acid requires that the above indicators are negative. In the invention, the cryopreservation density of the P5 generation stem cells in the product library is preferably 1000 ten thousand stem cells per milliliter of cell cryopreservation liquid. In the invention, 1000 ten thousand P5 generation cells are preferably selected, and 10 cells are frozen per 100 ten thousand cells to be used as quality control products. In the invention, the freezing conditions are as follows: keeping at 4deg.C for 15min, cooling to-7deg.C at 1deg.C/min, cooling to-60deg.C at 60deg.C/min, cooling to-30deg.C at 30deg.C/min, and cooling to-90deg.C at 1deg.C/min. In the present invention, the centrifugation conditions are: 2000-3000 rpm, 5min. In the invention, before the freezing, the method further comprises the following steps: and labeling the frozen storage tube by attaching an antifreezing two-dimensional code.
The construction method provided by the invention consists of collection, separation, culture and freezing storage, and the clinical-grade human umbilical cord mesenchymal stem cell library consists of a seed library, a main library, a working library and a product library.
The specific composition is preferably as follows:
seed library: p2 generation stem cells, 2ml cryopreservation tube, 300 ten thousand stem cells/tube, 1ml cell cryopreservation solution;
and (3) a main library: p3 generation stem cells, 2ml cryopreservation tube, 500 ten thousand stem cells/tube, 1ml cell cryopreservation solution;
The working library: p4 generation stem cells, 2ml cryopreservation tube, 1000 ten thousand stem cells/tube, 1ml cell cryopreservation solution;
product library: p5 generation stem cells, 5ml cryopreservation tube, 5000 ten thousand stem cells/tube, 5ml cell cryopreservation solution;
the structure is as follows: level 4 library structure, seed library: and P2 generation cells are used as main library raw materials.
And (3) a main library: and P3 generation cells serving as a raw material of a working library.
The working library: p4 generation cell used as raw material of product warehouse or directly used for clinic
Product library: p5 generation cells are used directly in clinic.
The invention provides a construction method of a clinical-grade human umbilical mesenchymal stem cell resource library, which is characterized in that the cell inoculation density of each stage is set, and through multiple verification tests, cells can reach a specified fusion degree in a specified time, and the generation number of cell expansion can be controlled more accurately in a traditional proportional expansion mode, so that the uniformity of cell quality is achieved. The primary cell separation method can lead the primary cells to be separated within a specified time, and improves the cell activity rate in a cell bank (at present, the experiment is carried out on about 20 umbilical cords in one month in a hospital, and more than 90% of umbilical cords can separate stem cells). By detecting each index, namely, detecting the cell safety index: the detection of mycoplasma, endotoxin, anaerobe, aerobe and infectious disease index is required to be negative. Cell dryness index: the activity rate is above 90%, and the flow detection is 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. The detection link ensures that the warehouse-in cells can meet the detection requirement 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, detected key points, a large-scale product library concept and a two-dimensional code management concept of each step of the construction method, and lays a foundation for future stem cell clinical application.
The method for constructing the clinical-grade human umbilical cord mesenchymal stem cell resource library multistage library is described in further detail below with reference to specific embodiments, and the technical scheme of the invention includes but is not limited to the following embodiments.
Example 1
The seed bank establishment scheme comprises the following steps:
Donor screening: donor detection project: lymphocytic leukemia virus antibody detection, hepatitis C virus antibody, cytomegalovirus IgM antibody, hepatitis B virus surface antigen, human immunodeficiency virus antibody and antigen, treponema pallidum antibody, hepatitis B virus nucleic acid, hepatitis C virus nucleic acid, human immunodeficiency virus nucleic acid, EBV virus IgM antibody detection is negative, no family infection history, no drug absorption history, healthy females between 20 and 35 ages.
Collecting raw materials: cutting the umbilical cord by 5-10 cm, respectively clamping two ends of the umbilical cord by using a disposable hemostatic clamp, placing the umbilical cord into a medical self-sealing bag, pasting a donor bar code, placing into a 4 ℃ transfer box, placing into a material bag for transferring to a preparation room, and carrying out infectious disease detection report, informed consent and acquisition record.
Pretreatment of raw materials: the hemostatic clamp is opened, normal saline is used for flushing blood, a medical surgical knife is used for cutting the umbilical cord into strips with the length of 2cm, scissors are used for cutting the umbilical cord, the normal saline is used for flushing for 2-3 times, and the blood is cleaned. 3 vessels (2 arteries, 1 vein) in the umbilical cord were removed using forceps, and a 2X rinse solution was prepared: 30ml of 80% serum-free medium+20% double antibody (for use in cell culture of Streptomyces lividans (100X)) was prepared, followed by 1X washing: the total of 90% serum-free culture medium and 10% double antibody is 30ml, and the total is soaked for 10min by using 2X flushing fluid and then soaked for 10min by using 1X flushing fluid.
Stem cell isolation: cutting the umbilical cord into tissue blocks with the thickness of 1-2 mm 3 by using a surgical knife, uniformly placing 20-25 tissue blocks into each hole of a 6-hole plate, placing into a carbon dioxide incubator for 20min for adherence, adding 1ml of serum-free culture medium with the temperature of 37 ℃ into each hole, placing into the carbon dioxide incubator overnight, and adding 1ml of culture medium the next day.
Amplification culture: after the primary cell fusion rate reaches 80% -90%, digesting for 3min at room temperature by using a digestive juice, taking 5-10 ten thousand cells for flow detection, transferring the cells into a 6-hole plate according to the inoculation density of 0.8-1 ten thousand/cm 2, adding 1.5ml of serum-free culture medium into each hole, placing the cells into a carbon dioxide incubator for culturing for 3-4 days until the fusion degree reaches 90-95%, digesting for 3min at room temperature by using 0.3ml of digestive juice, centrifuging for 5min at 2000-3000 rpm, removing supernatant, taking supernatant as detection of endotoxin, mycoplasma, anaerobic bacteria and aerobic bacteria, inoculating cells (P0 generation) into T25 culture bottles according to the inoculation density of 0.8-1 ten thousand/cm 2, adding 5ml of serum-free culture medium into each bottle for 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 rpm, removing supernatant, taking supernatant for detection the same as primary detection, inoculating cells (P1 generation) into T75 culture bottles according to the inoculation density of 0.8-1 ten thousand/cm 2, adding 15ml of culture medium into each bottle, digesting for 3min at room temperature by using 2ml of digestive juice until the fusion degree reaches 90-95%, centrifuging for 5min at 2000-3000 rpm, removing supernatant, taking supernatant for anaerobic bacteria, aerobic bacteria, mycoplasma and endotoxin detection, taking 20 ten thousand cells for activity rate, counting, detecting in a flow type, and detecting novel coronavirus nucleic acid, hepatitis B virus nucleic acid, hepatitis C virus nucleic acid, treponema pallidum antibody and human immunodeficiency virus nucleic acid. Taking 1000 ten thousand cells (P2 generation), inoculating the 1000 ten thousand cells into 10T 175 culture flasks according to the inoculation density of 0.5-0.8 ten thousand/cm 2, adding the rest 300 ten thousand cells into the frozen solution for re-suspension according to the per milliliter frozen solution, and using a frozen program: keeping at 4deg.C for 15min, cooling to-7deg.C at 1deg.C/min, cooling to-60deg.C at 60deg.C/min, cooling to-30deg.C at 30deg.C/min, and cooling to-90deg.C at 1deg.C/min. Freezing 5-10 tubes, and attaching an antifreezing two-dimensional code to each tube.
The main library establishment scheme comprises the following steps:
10T 175 culture flasks containing P2 cells are obtained in the seed bank establishment process, culturing is carried out for 3-4 days, when the fusion degree reaches 90-95%, 4ml of digestive juice is added for digestion for 3min at room temperature, 2000-3000 rpm of suspension is added for centrifugation for 5min, supernatant is removed, the supernatant is taken as a detection item of the same seed bank, 20 ten thousand cells are taken as a living rate, counting and flow type, 2000 ten thousand cells are taken, the cells are inoculated into 20T 175 culture flasks according to the inoculation density of 0.5-0.8 ten thousand/cm 2, the rest of the cells are resuspended according to every milliliter of frozen solution, 500 ten thousand cells are added into the frozen solution for resuspension, and the frozen procedure of the same seed bank is used. Freezing 10-15 tubes, and attaching an antifreezing two-dimensional code to each tube.
The working base establishment scheme comprises the following steps:
Obtaining 20T 175 culture bottles filled with P3 cells in the process of establishing a 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 rpm, removing supernatant, taking supernatant to detect anaerobes, aerobe, mycoplasma and endotoxin, taking 20 ten thousand cells as the activation rate, counting and flowing. 2000 ten thousand cells are taken and inoculated into 2 10-layer cell factories according to the inoculation density of 0.5-0.8 ten thousand/cm 2, and the rest 1000 ten thousand cells are resuspended according to every milliliter of frozen stock solution and added with the frozen stock solution for resuspension, and the frozen stock program of the same seed bank is used. Freezing 10-15 tubes, and attaching an antifreezing two-dimensional code to each tube.
The product library establishment scheme comprises the following steps:
2 cell factories containing P4 cells are obtained in the working library establishment process, the culture is carried out for 4-5 days, the fusion degree reaches 90-95%, 100ml of digestive juice is added into each factory, the digestion is carried out for 3min at room temperature, 2000-3000 rpm of heavy suspension is centrifuged for 5min, the supernatant is removed, the supernatant is taken for detecting anaerobe, aerobe, mycoplasma and endotoxin, and 20 ten thousand cells are taken as the activity rate, the count and the flow. Taking 1000 ten thousand cells, freezing and preserving 10 per 100 ten thousand cells per tube, taking the rest of 1000 ten thousand cells per milliliter of frozen solution as quality control product, adding the frozen solution for re-suspension, and using the frozen storage program of the same seed library. Freezing 10-15 tubes, and attaching an antifreezing two-dimensional code to each tube.
1% Of the total donor number was withdrawn for detection
The detection method comprises the following steps:
Endotoxin: gel method
Mycoplasma: PCR method
Anaerobic bacteria, aerobic bacteria: culture method
The activity rate is as follows: MTT method
Flow type: flow cytometry
Detection of infectious disease nucleic acid: PCR
The above detection method is based on the reagent and the operation instructions of the device.
The resuscitation method comprises the following steps:
Thawing the cells by using a cell resuscitator, centrifuging at 3000 rpm for 5min, removing supernatant of the frozen stock, collecting supernatant for detection, adding 2ml of serum-free basic culture medium into the cells, and performing cell detection after re-spinning.
A flow chart of the construction and detection of the present invention is shown in fig. 1.
Detection result:
TABLE 1 Activity detection results
| Donor(s) | Results before freezing | Freezing and preserving the result |
| Donor 1 (seed pool) | 99％ | 90％ |
| Donor 1 (Main reservoir) | 98％ | 92％ |
| Donor 1 (working warehouse) | 99％ | 93％ |
| Donor 1 (product warehouse) | 99％ | 90％ |
| Donor 2 (seed pool) | 98％ | 95％ |
| Donor 2 (Main reservoir) | 97％ | 95％ |
| Donor 2% working warehouse | 98％ | 93％ |
| Donor 2 (product warehouse) | 96％ | 90％ |
| Donor 3 (seed pool) | 99％ | 93％ |
| Donor 3 (Main reservoir) | 99％ | 95％ |
| Donor 3 (working warehouse) | 98％ | 92％ |
| Donor 3 (product warehouse) | 99％ | 88％ |
As shown in Table 1, the cell viability was 96 to 99% before freezing, and the viability was high. After freezing, the cell results are subjected to procedures of cooling, re-heating and centrifugation, so that the cell activity rate is reduced by 2-11% compared with that before freezing.
The higher activity rate before freezing indicates that the method for constructing the seed bank is effective, stem cells can be extracted from umbilical cord tissues, cells with better activity can be cultured, and the activity of the cells can reach more than 90% after freezing.
Compared with the prior art, the cell viability of the whole cell bank is improved.
TABLE 2 stream detection results
The seed library and the main library have the flow detection items comprising 6 surface markers, namely CD73, CD90, CD105, CD34, CD45 and HLA-DR, wherein all detection results before freezing and after freezing are positive for CD73, positive for CD90, positive for CD105, negative for CD34, negative for CD45 and negative for HLA-DR. The flow detection items of the working library and the product library comprise 8 surface markers, namely CD73, CD90, CD105, CD34, CD45, HLA-DR, CD11b and CD19, wherein all detection results before freezing and after freezing 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 flow results show that the cells conform to the characteristics of stem cells, which means that the cell bank construction method of the present invention can isolate and culture stem cells, and the characteristics of stem cells do not change during storage.
TABLE 3 endotoxin, mycoplasma, anaerobic and aerobic detection results
As shown in Table 3, endotoxin, mycoplasma, anaerobic bacteria and aerobic bacteria were detected as negative before and after freezing. The cell bank construction method disclosed by the invention ensures the safety of cells, and does not pollute the cells in the preservation process of the cells. Compared with the prior art, the partition management (namely, setting a seed warehouse, a main warehouse, a working warehouse and a product warehouse) and the gas-phase liquid nitrogen tank are adopted for storage, so that cross contamination among cells is avoided, and the safety of the cells is ensured.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (7)
1. A method for constructing a multi-level library of clinical human umbilical cord mesenchymal stem cell resource library, wherein the multi-level library comprises a seed library, a main library, a working library and a product library; the construction method comprises the following steps:
Sectioning the umbilical cord, cleaning, removing blood vessels, and sequentially soaking in a first flushing fluid and a second flushing fluid to obtain a pretreated umbilical cord; the first flushing liquid comprises a serum-free culture medium with the volume percentage of 80% and a mixture of the green streptomycin with the volume percentage of 20%; the second flushing liquid comprises a serum-free culture medium with the volume percentage of 90% and a mixture of the green streptomycin with the volume percentage of 10%;
Cutting the pretreated umbilical cord into tissue blocks with the thickness of 1-2 mm 3, placing the tissue blocks in a 6-hole plate, performing wall-attached 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%, thus obtaining primary cells;
inoculating primary cells into a 6-hole plate according to the inoculation density of 0.8-1 ten thousand/cm 2, adding a serum-free culture medium, culturing for 3-4 d, digesting, centrifuging and removing the supernatant to obtain P0-generation cells when the fusion degree reaches 90-95%;
Before obtaining the P0 generation cells and the P1 generation cells, detecting endotoxin, mycoplasma, anaerobe and aerobe in the supernatant;
Inoculating the P0 generation cells into a culture flask according to the inoculation density of 0.8-1 ten thousand/cm 2, adding a serum-free culture medium for culturing for 3-4 d, and removing the supernatant after the fusion degree reaches 90-95%, and obtaining the P1 generation cells;
inoculating the P1 generation cells into a culture flask according to the inoculation density of 0.8-1 ten thousand/cm 2, adding a serum-free culture medium for culturing for 2-3 d, and removing the supernatant after the fusion degree reaches 90-95%, and obtaining the P2 generation cells; resuspending the P2 generation cells in a freezing solution, freezing and preserving to construct a seed library;
Inoculating the P2 generation cells into a culture flask according to the inoculation density of 0.5-0.8 ten thousand/cm 2, adding a serum-free culture medium for culturing for 3-4 d, and after the fusion degree reaches 90-95%, digesting, centrifuging and removing the supernatant to obtain the P3 generation cells; resuspending the P3 generation cells in a freezing solution, freezing and storing to construct a main library;
Inoculating the P3 generation cells into a culture flask according to the inoculation density of 0.5-0.8 ten thousand/cm 2, adding a serum-free culture medium for culturing for 3-4 d, and after the fusion degree reaches 90-95%, digesting, centrifuging and removing the supernatant to obtain the P4 generation cells; resuspending the P4 generation cells in a freezing solution, freezing and storing to construct a working library;
Inoculating the P4 generation cells into a cell factory according to the inoculation density of 0.5-0.8 ten thousand/cm 2, adding a serum-free culture medium for culturing for 4-5 d, and after the fusion degree reaches 90-95%, digesting, centrifuging and removing the supernatant to obtain the P5 generation cells; resuspending the P5 generation cells in a frozen stock solution, freezing and storing to construct a product library;
Before obtaining the P2 generation cells, the P3 generation cells, the P4 generation cells and the P5 generation cells, detecting endotoxin, mycoplasma, anaerobic bacteria and aerobic bacteria of the supernatant, and detecting the survival rate, the flow detection and the infectious disease nucleic acid of the cells; the streaming assays include the assays for CD73, CD90, CD105, CD34, CD45, HLA-DR, CD11b and CD 19; the indexes of the infectious disease nucleic acid detection comprise novel coronavirus nucleic acid, hepatitis B virus nucleic acid, hepatitis C virus nucleic acid, treponema pallidum antibody and human immunodeficiency virus nucleic acid;
the freezing conditions are as follows: keeping at 4deg.C for 15min, cooling to-7deg.C at 1deg.C/min, cooling to-60deg.C at 60deg.C/min, cooling to-30deg.C at 30deg.C/min, and cooling to-90deg.C at 1deg.C/min.
2. The method of claim 1, wherein the umbilical cord is from a healthy donor; the healthy donor has no family infection history and no drug absorption history, and the negative detection items comprise: lymphocytic leukemia virus antibodies, hepatitis C virus antibodies, cytomegalovirus IgM antibodies, hepatitis B virus surface antigens, human immunodeficiency virus antibodies and antigens, treponema pallidum antibodies, hepatitis B virus nucleic acids, hepatitis C virus nucleic acids, human immunodeficiency virus nucleic acids, and EBV virus IgM antibodies.
3. The method according to claim 1, wherein the soaking time is 3 to 5 minutes, respectively.
4. The method of claim 1, wherein the tissue pieces are placed in 20-25 wells per well of a 6-well plate.
5. The method of claim 1, wherein the cryopreservation density of 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 main library is 500 ten thousand stem cells per milliliter of cell cryopreservation liquid;
the cryopreservation density of the P4 generation stem cells in the working library is 1000 ten thousand stem cells per milliliter of cell cryopreservation liquid;
The cryopreservation density of the P5 generation stem cells in the product library is 1000 ten thousand stem cells per milliliter of cell cryopreservation liquid.
6. The method of claim 1, wherein the centrifugation conditions are: 2000-3000 rpm, 5min.
7. The method of claim 1, wherein prior to freezing, further comprising: and labeling the frozen storage tube by attaching an antifreezing two-dimensional code.
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