CN112048470A - Method for preparing clinical-grade mesenchymal stem cell preparation by utilizing human induced pluripotent stem cells - Google Patents

Abstract

The invention provides a method for preparing a clinical mesenchymal stem cell preparation by utilizing human induced pluripotent stem cells, belonging to the technical field of cell culture. The method for preparing the clinical-grade mesenchymal stem cell preparation by utilizing the human induced pluripotent stem cells comprises the following steps: inducing and differentiating iPSC to iPSC-MSC, amplifying the iPSC-MSC and preparing an iPSC-MSC preparation, wherein the identification of the characteristics of the iPSC-MSC and the quality control of the iPSC-MSC preparation are also included. The clinical-grade mesenchymal stem cell preparation prepared by the invention has the advantages of providing a mesenchymal stem cell source with unlimited source, stable quality, high homogeneity, relatively controllable property and the like for individual requirements of various patients, realizes large-scale industrialization, and has the characteristic of wide clinical application market prospect.

Description

Method for preparing clinical-grade mesenchymal stem cell preparation by utilizing human induced pluripotent stem cells

Technical Field

The invention belongs to the technical field of cell culture, and particularly relates to a method for preparing a clinical-grade mesenchymal stem cell preparation by utilizing human induced pluripotent stem cells.

Background

Mesenchymal Stem Cells (MSCs) cultured in organ tissues and in vitro have natural high heterogeneity, are not only the basis for exerting multi-functional activity, but also limiting factors influencing the experimental repeatability, MSC quality and clinical efficacy of MSCs, and cause more variables and difficulties for standardized and standardized assessment of safety and effectiveness of MSC treatment. Human Induced Pluripotent Stem Cells (iPSCs) based on a cell reprogramming technology and related research remarkably improve understanding of stem cell maintenance pluripotency, desiccation and molecular regulation mechanisms thereof, and simultaneously provide an unlimited source of patient-specific individualized stem cells. With the improvement and optimization of non-integrated iPSC induction technology, ipscs free of viral vectors and tumorigenic genes c-myc are currently available. The iPSC-MSC still has the advantages of iPSC, can be induced from individual skin fibroblasts or various tissue cells, and avoids the related problems of rejection, disease transmission, ethics and the like related to allograft; the iPSC can be passed infinitely in principle, so the source of the MSC can be endless; theoretically, MSCs derived from a single iPSC cell clone are more homogeneous. Based on the characteristics, the iPSC can provide a stable and reliable infinite MSC source suitable for individual requirements of various patients, can be used for large-scale preparation and production of in vitro infinite amplification, has stable and relatively controllable quality of final products, and can achieve optimal treatment effect aiming at specific clinical diseases and individual requirements.

However, the defects and clinical needs of the prior art at present still have the problems of limited source, unstable quality and low homogeneity of the mesenchymal stem cells in the process of cell transplantation treatment and tissue engineering for articular cartilage damage and degenerative diseases.

Disclosure of Invention

In view of the above, the present invention aims to provide a method for preparing clinical-grade mesenchymal stem cell preparation by using human induced pluripotent stem cells, which satisfies the market demand for mesenchymal stem cell sources with stable and reliable quality and suitable for individual demands of various patients, and establishes large-scale preparation and production technology for in vitro infinite amplification.

The invention provides a method for preparing a clinical-grade mesenchymal stem cell preparation by utilizing human induced pluripotent stem cells, which comprises the following steps:

1) when the human iPSC grows to 80-90% in the 6-well plate, replacing the iPSC-MSC induction culture medium for culturing for 8-12 days, and replacing the culture medium every 2-3 days to obtain adherent cells, and digesting to obtain iPSC-MSC single cells;

2) the iPSC-MSC single cells are arranged according to 8 multiplied by 10⁴cell/cm²Inoculating to 25cm²In a cell culture bottle, defined as iPSC-MSC-P0, replacing the iPSC-MSC growth culture medium for culture for 4-7 days, replacing the culture medium once every 2-3 days, digesting, and replacing by 4 multiplied by 10⁴cell/cm²Inoculating to 25cm²In a cell culture bottle, defined as iPSC-MSC-P1, continuously culturing for 4-7 d in iPSC-MSC growth culture medium, changing liquid every 2-3 d, digesting, and performing 2 × 10⁴cell/cm²Inoculating to 75cm²In the cell culture bottle, when the cells grow to 80-90% every 4-7 days later, the number is 1 multiplied by 10⁴cell/cm²Inoculating to 175cm²Carrying out passage culture in a cell culture bottle at a ratio of 1: 3-1: 4 to obtain a large amount of iPSC-MSC-P3 cells;

the iPSC-MSC induction culture medium is a DMEM culture medium containing 2-5% of human platelet lysate in volume concentration, 1% of 100 multiplied by trace nonessential amino acid solution in volume concentration, 1% of 100 multiplied by glutamine additive in volume concentration and 1.0 g/LD-glucose;

the iPSC-MSC growth medium is a DMEM medium containing 2-5% of human platelet lysate in volume concentration, 1% of 100 multiplied by trace nonessential amino acid solution in volume concentration, 1% of 100 multiplied by glutamine additive in volume concentration and 4.5 g/LD-glucose;

3) clinical grade serum-free Recovery will be used^TMThe frozen stock solution of Cell Culture Freezing Medium is 1 × 10⁶～1×10¹⁰The iPSC-MSC-P3 cells are subpackaged at the concentration of each mL, and the prepared mesenchymal stem cell preparation is stored in a cell bank.

Preferably, the enzyme for digestion in step 1) or step 2) is TrypLE^TMSelecting; the digestion time is 5-8 min.

Preferably, the digestion is followed by centrifugation; the rotating speed of the centrifugation is 1000-1500 rpm; the centrifugation time is 3-7 min.

Preferably, the temperature of the culture in the step 2) is 36-38 ℃.

Preferably, as in step 1)The culture method of the human iPSC comprises the steps of inoculating a human iPSC cell strain into a 6-well plate coated with 10 mu g/ml human pluripotent stem cell culture adherent matrix, and culturing the human iPSC cell strain in an iPSC culture medium at 37 ℃ and 5% CO₂Culturing for 5-7 days under the saturated humidity condition, and replacing a fresh culture medium every day.

Preferably, said human pluripotent stem cell culture adherent matrix comprises 960. mu.l CellAdhere per 1000. mu.l of said human pluripotent stem cell culture adherent matrix^TMDiluetionBuffer and 40ul of Vitronectin XF of 250 mug/ml^TM。

Preferably, the iPSC culture medium is TeSR^TM-E8^TMBasic Medium and 25 × TeSR^TM-E8^TMSupplement, the volume ratio of the two is 24: 1.

Preferably, the iPSC-MSC is characterised prior to being aliquoted.

The identification indexes of the iPSC-MSC characteristics comprise stem cell specific antigen, osteogenic capacity, chondrogenic capacity and adipogenic differentiation capacity.

Preferably, the identification method of the stem cell specific antigen comprises determining an iPSC-MSC surface marker; the iPSC-MSC surface marker comprises CD73⁺、CD90⁺、CD105⁺、CD34^-、CD45^-And HLA-DR^-；CD73⁺、CD90⁺And CD105⁺The expression is more than 95 percent positive, CD34^-、CD45^-And HLA-DR^-Expression less than 1% was negative.

The identification of the osteogenic, chondrogenic and adipogenic differentiation capacity is that the commercial osteogenic, chondrogenic and adipogenic differentiation induction kit is used for carrying out iPSC-MSC osteogenic, chondrogenic and adipogenic induction, and the iPSC-MSC in-vitro osteogenic, chondrogenic and adipogenic differentiation capacity is evaluated by adopting alizarin red, toluidine blue and oil red O staining respectively; alizarin red, toluidine blue and oil red O staining are all positive, and the iPSC-MSC has the capability of multidirectional differentiation of in vitro osteogenesis, chondrogenesis and adipogenesis.

Preferably, the quality control of the mesenchymal stem cell preparation is also included.

According to the international cell therapy society and/or partial reference to the national food and drug administration for the partial standard of adult stem cells, the cell product is verified and evaluated, and the related procedures, technologies and specifications are provided, and the cell product is qualified as a finished product;

the detection and evaluation indexes of the cell product comprise cell activity, cell quality and additive quality.

The method for preparing the clinical-grade mesenchymal stem cell preparation by utilizing the human induced pluripotent stem cells, provided by the invention, is characterized in that the mesenchymal stem cells are differentiated and amplified from the human induced pluripotent stem cells through a system for abstaining heterotrophic layer-free cells of a xenogenic serum product (so as to avoid the pollution of heterologous germs and cells), and the mesenchymal stem cells are cultured.

Drawings

FIG. 1 shows the morphology of the induced pluripotent stem cell derived mesenchymal stem cell under a light microscope at different time points;

FIG. 2 is a flow identification diagram of surface markers of mesenchymal stem cells derived from pluripotent stem cells induced by the present inventors;

FIG. 3 is a diagram of directional induced differentiation of mesenchymal stem cells induced by pluripotent stem cells into bone, cartilage and fat in vitro.

Detailed Description

The invention provides a method for preparing a clinical-grade mesenchymal stem cell preparation by utilizing human induced pluripotent stem cells, which comprises the following steps:

1) when the human iPSC grows to 80-90% in the 6-well plate, replacing the iPSC-MSC induction culture medium for culturing for 8-12 days, and replacing the culture medium every 2-3 days to obtain adherent cells, and digesting to obtain iPSC-MSC single cells;

2) the iPSC-MSC single cells are arranged according to 8 multiplied by 10⁴cell/cm²Inoculating to 25cm²In a cell culture bottle, defined as iPSC-MSC-P0, replacing the iPSC-MSC growth culture medium for culture for 4-7 days, replacing the culture medium once every 2-3 days, digesting, and replacing by 4 multiplied by 10⁴cell/cm²Inoculating to 25cm²In a cell culture bottle, defined as iPSC-MSC-P1, continuously culturing for 4-7 d in iPSC-MSC growth culture medium, changing liquid every 2-3 d, digesting, and performing 2 × 10⁴cell/cm²Inoculating to 75cm²In the cell culture bottle, when the cells grow to 80-90% every 4-7 days later, the number is 1 multiplied by 10⁴cell/cm²Inoculating to 175cm²Carrying out passage culture in a cell culture bottle at a ratio of 1: 3-1: 4 to obtain a large amount of iPSC-MSC-P3 cells;

the iPSC-MSC induction culture medium is a DMEM culture medium containing 2-5% of human platelet lysate in volume concentration, 1% of 100 multiplied by trace nonessential amino acid solution in volume concentration, 1% of 100 multiplied by glutamine additive in volume concentration and 1.0 g/LD-glucose;

the iPSC-MSC growth medium is a DMEM medium containing 2-5% of human platelet lysate in volume concentration, 1% of 100 multiplied by trace nonessential amino acid solution in volume concentration, 1% of 100 multiplied by glutamine additive in volume concentration and 4.5 g/LD-glucose;

3) clinical grade serum-free Recovery will be used^TMThe frozen stock solution of Cell Culture Freezing Medium is 1 × 10⁶～1×10¹⁰The iPSC-MSC-P3 cells were aliquoted at individual/mL concentrations and stored in cell banks.

When the human iPSC grows to 80-90% in the 6-well plate, the iPSC-MSC induction culture medium is replaced to culture for 8-12 days, and the liquid is replaced once every 2-3 days to obtain adherent cells, and the adherent cells are digested to obtain the iPSC-MSC single cells.

In the invention, the culture method of the human iPSC is preferably to inoculate the human iPSC cell strain in a 6-well plate coated with 10 mu g/ml human pluripotent stem cell culture adherent matrix, and to culture the human iPSC in an iPSC culture medium at 37 ℃ and 5% CO₂Culturing for 5-7 days under the saturated humidity condition, and replacing a fresh culture medium every day. The iPSC culture medium is preferably TeSR^TM-E8^TMBasic Medium and 25 × TeSR^TM-E8^TMSupplement, the volume ratio of the two is 24: 1. TeSR^TM-E8^TMBasic Medium and TeSR^TM-E8^TMSupplement together forms a low-protein and feeder-layer-free TeSR^TM-E8^TMCulture medium capable of maintaining human polyploidyCapable of stem cell growth, all purchased from STEMCELL^TMTECHNOLOGIES Inc. The source of the human iPSC cell line is not particularly limited in the present invention, and a human iPSC cell line known in the art may be used. The human iPSC cell strain is purchased from Nuwacell company and is Nuwacell^TMScientific grade hiPSC cell line (RC 01001-B). Preferably, said human pluripotent stem cell culture adherent matrix comprises 960. mu.l CellAdhere per 1000. mu.l^TMDiluetionBuffer and 40. mu.l of 250. mu.g/ml Vitronectin XF^TM。

In the invention, the iPSC-MSC induction culture medium is preferably a DMEM culture medium containing 2-5% of clinical-grade human platelet lysate by volume concentration, 1% of 100 multiplied by trace nonessential amino acid solution by volume concentration, 1% of 100 multiplied by glutamine additive by volume concentration and 1.0g/L D-glucose. Advantages over other formulations of induction media in the art: clinical grade platelet lysate, which contains all the growth factors and proteins necessary for cell growth, is an efficient, high protein supplement to human cell culture and expansion, and can reduce the risk of xenogeneic immune responses or the spread of prions and zoonosis. The present invention has no particular limitation on the source of the components of the induction medium, and clinical, trace amount of non-essential amino acid solution, glutamine additive, D-glucose and DMEM medium well known in the art may be used.

In the invention, when the human iPSC grows to 80-90% in the 6-well plate, the cell edge is smooth and clear, the cell gap is compact, no obvious differentiation is generated, the human iPSC is cloned in a typical cobblestone shape, and the human iPSC can be digested and induced at the stage.

In the present invention, the enzyme for digestion is preferably TrypLE^TMSelecting; the time for digestion is preferably 5-8 min, and more preferably 6-7 min. Centrifuging and collecting single cells after digestion; the rotation speed of the centrifugation is preferably 1000-1500 rpm; the time for centrifugation is preferably 3-7 min. The iPSC-MSC single cell represents an MSC single cell induced by human iPSC differentiation.

After obtaining the iPSC-MSC single cell, the invention makes the iPSC-MSC single cell according to 8 multiplied by 10⁴cell/cm²Inoculating to 25cm²In a cell culture flask, fixingThe iPSC-MSC-P0 is defined as the iPSC-MSC growth culture medium, the iPSC-MSC growth culture medium is replaced to culture for 4-7 days, the liquid is replaced once every 2-3 days, digestion is carried out, and the culture medium is digested by 4 multiplied by 10⁴cell/cm²Inoculating to 25cm²In a cell culture bottle, defined as iPSC-MSC-P1, continuously culturing for 4-7 d in iPSC-MSC growth culture medium, changing liquid every 2-3 d, digesting, and performing 2 × 10⁴cell/cm²Inoculating to 75cm²In the cell culture bottle, when the cells grow to 80-90% every 4-7 days later, the number is 1 multiplied by 10⁴cell/cm²Inoculating to 175cm²And (4) carrying out passage culture in a cell culture bottle at a ratio of 1: 3-1: 4 to obtain a large amount of iPSC-MSC-P3 cells.

In the invention, the growth medium of the iPSC-MSC is preferably a DMEM medium containing 2-5% of human platelet lysate by volume concentration, 1% of 100 multiplied by trace nonessential amino acid solution by volume concentration, 1% of 100 multiplied by glutamine additive by volume concentration and 4.5g/L D-glucose. The temperature of the culture is preferably 36-38 ℃, and more preferably 37 ℃.

In the invention, after the iPSC-MSC-P3 cells are prepared, the characteristics of the iPSC-MSC are preferably identified; the identification indexes of the iPSC-MSC characteristics preferably comprise stem cell specific antigens, osteogenic capacity, chondrogenic capacity and adipogenic differentiation capacity. The identification method of the stem cell specific antigen preferably comprises measuring an iPSC-MSC surface marker by using a flow cytometer; the iPSC-MSC surface marker comprises CD73⁺、CD90⁺、CD105⁺、CD34^-、CD45^-And HLA-DR^-；CD73⁺、CD90⁺And CD105⁺The expression is more than 95 percent positive, CD34^-、CD45^-And HLA-DR^-Expression less than 1% was negative.

Preferably, the identification of osteogenic, chondrogenic and adipogenic differentiation capacity is carried out by using a commercial osteogenic, chondrogenic and adipogenic differentiation induction kit for iPSC-MSC osteogenic, chondrogenic and adipogenic induction, and the iPSC-MSC in vitro osteogenic, chondrogenic and adipogenic differentiation capacity is evaluated by adopting alizarin red, toluidine blue and oil red O staining respectively. Alizarin red, toluidine blue and oil red O staining are all positive, which indicates that the iPSC-MSC has the capability of multidirectional differentiation of in vitro osteogenesis, chondrogenesis and adipogenesis.

After the iPSC-MSC-P3 cells with qualified screening characteristics are obtained, the invention adopts clinical-grade serum-free Recovery^TMThe frozen stock solution of Cell Culture Freezing Medium is 1 × 10⁶～1×10¹⁰The iPSC-MSC-P3 cells are subpackaged at the concentration of each mL, and the prepared mesenchymal stem cell preparation is stored in a cell bank.

In the present invention, the concentration of the aliquot is preferably 1X 10⁷～1×10⁹one/mL, more preferably 1X 10⁸one/mL.

In the present invention, it is preferable to further include quality control of the mesenchymal stem cell preparation: standard operation procedures of the stem cell preparation process and Standard Operation Procedures (SOP) of each process are established according to the International society for cell therapy and/or partial reference to the national food and drug administration for the partial standards of adult stem cells, and are regularly reviewed and revised. The preparation process of the stem cell preparation comprises the steps of induction preparation, purification, amplification and passage of stem cells, selection standard and use of culture medium, auxiliary materials and packing materials, freezing and storing of the cells, recovery, subpackaging and marking, residue removal and the like. The whole preparation process is subjected to comprehensive process research and verification, appropriate process parameters and quality standards are formulated, and effective control of each process is ensured. And (4) verifying and evaluating the cell product, and providing related procedures, technologies and specifications to obtain a qualified finished product.

The assay and evaluation criteria for the cell product preferably include cell viability, cell mass and additive mass. The cell activity detection adopts different cell biological activity detection methods to judge the cell activity and growth condition. The basic quality requirement of the cells is that the cells have definite cell identification characteristics and no infection of exogenous microorganisms. The quality requirement of the additive is to make clear the source, batch number and quality certification qualification report, and to adopt the clinically applicable products approved by the state as much as possible.

The relevant data of the cell preparation needs to be filed and stored for a long time.

The method for preparing clinical-grade mesenchymal stem cell preparation by using human induced pluripotent stem cells according to the present invention will be described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.

Example 1

A method for preparing clinical-grade mesenchymal stem cell preparation by utilizing human induced pluripotent stem cells comprises the following steps:

i, conventional culture of human iPSCs: the human iPSC cell strain is purchased from Nuwacell company, is a Nuwacell TM research grade hipSC cell strain (RC01001-B), and is inoculated to 10 mu g/ml Vitronectin XF^TM(composition includes 960. mu.l CellAdhere^TMDilutionBuffer+40μl 250μg/ml Vitronectin XF^TM) In the coated 6-well plate, iPSC conventional medium (1920. mu.l TeSR) was used^TM-E8^TMBasal Medium+80μl 25×TeSR^TM-E8^TMSupplement) at 37 ℃ with 5% CO₂And (5) culturing for 6d under saturated humidity conditions. The culture was continued by replacing the above conventional medium with fresh one every day. During the period, the morphology of human ipscs at different culture times was photographed using an optical microscope. The results are shown in FIG. 1. Before differentiation, Day-2 of undifferentiated human iPSC grows in a 6-well plate to 70% of compact cobblestone-shaped multicellular clonic masses with high nucleo-cytoplasmic ratio, prominent nucleolus and clear boundaries; the 90% compact, high nucleo-cytoplasmic ratio and with prominent nucleoli, well-defined cobblestone-like multicellular colonies were grown in Day 0 in 6-well plates.

And II, inducing and differentiating iPSC into iPSC-MSC and amplifying the iPSC-MSC: when Human iPSC grew to 90% in Day 0 in 6-well plates (smooth and clear cell edges, compact intercellular spaces, no apparent differentiation, typical cobblestone cloning) it was replaced with iPSC-MSC induction medium (containing 5% PLT GOLD Human Platelet Lysate (Clinical Grade) + 1% MEM Non-Essential Amino Acids Solution (100 ×) + 1% GultaMAX^TMDMEM basic (1X) [ + (100X)]1.0g/L D-Glucose medium), changing the culture solution every 2 days, culturing for 4 days to obtain adherent cells, and culturing with TrypLE^TMSelect (1X) was digested for 7min, then centrifuged at 1500rpm for 5min, and the pellet was collected. At 8X 10⁴cell/cm²Inoculating to 25cm²In a cell culture flask, defined as iPSC-MSC-P0, and replaced with iPSC-MSC growth medium (containing 5% PLT GOLD Human Platlet Lysate (Clinica)l Grade)+1％MEM Non-Essential Amino Acids Solution(100×)+1％GultaMAX^TMDMEM basic (1X) [ + (100X)]4.5g/L D-Glucose medium), changing the medium every 2 days, culturing for 4 days, and using TrypLE^TMSelect (1X) digest, digest single cells at 4X 10⁴cell/cm²Inoculating to 25cm²And (3) continuously culturing the cells for 4 days in a cell culture flask defined as iPSC-MSC-P1, wherein the cells grow in a spindle shape and grow at a high speed, and changing the culture solution every 2 days. Using TrypLE^TMSelect (1X) was digested for 7min, then centrifuged at 1200rpm for 5min and the pellet collected. At 2X 10⁴cell/cm²Inoculating to 75cm²Cell culture flasks, defined as iPSC-MSC-P2, then 1X 10 cells every 4d when the cells were 90% long⁴cell/cm²Inoculating to 175cm²The cells were cultured in flasks and passaged at 1:3 to give iPSC-MSC-P3. The iPSC-MSC-P3 generation cells were used for subsequent iPSC-MSC identification and preparation. During the period, the forms of human iPSC-MSC at different culture times were photographed by an optical microscope. The results are shown in FIG. 1. Day10 shows fusiform IPSC-MSC P0, Day 14 shows fusiform IPSC-MSC P1 with obviously increased number, Day 18 shows dense fibrous IPSC-MSC P2, and Day 22 shows homogeneous cell group iPSC-MSC-P3 with fusiform and MSC-like morphology.

Thirdly, identifying the characteristics of iPSC-MSC: iPSC-MSC surface marker (CD 73) using flow cytometry⁺、CD90⁺、CD105⁺、CD34^-、CD45^-、HLA-DR^-) An assay to identify stem cell specific antigens; the results are shown in FIG. 2. CD73⁺、CD90⁺And CD105⁺The expression is more than 95 percent positive, CD34^-、CD45^-And HLA-DR^-Expression less than 1% was negative. The iPSC-MSC is shown to have mesenchymal stem cell specific antigen.

And carrying out osteogenic, chondrogenic and adipogenic induction on the iPSC-MSC by respectively utilizing commercial osteogenic, chondrogenic and adipogenic differentiation induction kits, and evaluating the multidirectional differentiation capacities of the iPSC-MSC in vitro such as osteogenic, chondrogenic and adipogenic capacities through alizarin red, toluidine blue and oil red O staining. The results are shown in FIG. 3. Alizarin red, toluidine blue and oil red O staining are all positive, which indicates that the iPSC-MSC has the capability of multidirectional differentiation of in vitro osteogenesis, chondrogenesis and adipogenesis.

Fourthly, preparing a mesenchymal stem cell preparation: serum-free Recovery of the obtained mesenchymal stem cells at clinical level^TMThe frozen stock solution of Cell Culture Freezing Medium is 1 × 10⁸The concentration of each/mL is stored in separate aliquots in a cell bank.

Fifthly, quality control of the mesenchymal stem cell preparation: standard operation procedures of the stem cell preparation process and Standard Operation Procedures (SOP) of each process are established according to the International society for cell therapy and/or partial reference to the national food and drug administration for the partial standards of adult stem cells, and are regularly reviewed and revised. The preparation process of the stem cell preparation comprises the steps of induction preparation, purification, amplification and passage of stem cells, selection standard and use of culture medium, auxiliary materials and packing materials, freezing and storing of the cells, recovery, subpackaging and marking, residue removal and the like. The whole preparation process is subjected to comprehensive process research and verification, appropriate process parameters and quality standards are formulated, and effective control of each process is ensured. And (4) verifying and evaluating the cell product, and providing related procedures, technologies and specifications to obtain a qualified finished product.

The assay and evaluation criteria for the cell product preferably include cell viability, cell mass and additive mass. The cell activity detection adopts different cell biological activity detection methods. The basic quality of the cells is that the iPSC-MSC prepared by the invention has clear cell characteristics and has no infection of exogenous microorganisms. The quality requirement of the additive is to make clear the source, batch number and quality certification qualification report, and to adopt the clinically applicable products approved by the state as much as possible.

The relevant data of the cell preparation needs to be filed and stored for a long time.

Example 2

A method for preparing clinical-grade mesenchymal stem cell preparation by utilizing human induced pluripotent stem cells comprises the following steps:

i, conventional culture of human iPSCs: human iPSC cell lines (purchased from Nuwacell Co.) were inoculated to 10. mu.g/ml Vitronectin XF^TM(composition includes 960. mul CellAdhere^TMDilutionBuffer+40μl 250μg/ml Vitronectin XF^TM) In the coated 6-well plate, iPSC conventional medium (1920. mu.l TeSR) was used^TM-E8^TMBasal Medium+80μl 25×TeSR^TM-E8^TMSupplement) at 37 ℃ with 5% CO₂And (5) culturing for 6d under saturated humidity conditions. The culture was continued by replacing the above conventional medium with fresh one every day. During the period, the morphology of human ipscs at different culture times was photographed using an optical microscope. The results are shown in FIG. 1. Before differentiation, Day-2 of undifferentiated human iPSC grows in a 6-well plate to 70% of compact cobblestone-shaped multicellular clonic masses with high nucleo-cytoplasmic ratio, prominent nucleolus and clear boundaries; the 90% compact, high nucleo-cytoplasmic ratio and with prominent nucleoli, well-defined cobblestone-like multicellular colonies were grown in Day 0 in 6-well plates.

And II, inducing and differentiating iPSC into iPSC-MSC and amplifying the iPSC-MSC: when Human iPSC grew up to 80% in Day 0 in 6-well plates (smooth and clear cell edges, compact intercellular spaces, no apparent differentiation, typical cobblestone cloning) it was replaced with iPSC-MSC induction medium (containing 2% PLT GOLD Human Platelet Lysate (Clinical Grade) + 1% MEM Non-Essential Amino Acids Solution (100 ×) + 1% GultaMAX^TMDMEM basic (1X) [ + (100X)]1.0g/L D-Glucose medium), changing the culture solution every 3 days, culturing for 7 days to obtain adherent cells, and culturing with TrypLE^TMSelect (1X) was digested for 5min, then centrifuged at 1000rpm for 7min and the pellet collected. At 8X 10⁴cell/cm²Inoculating to 25cm²In cell culture flasks, defined as iPSC-MSC-P0, and replaced with iPSC-MSC growth medium (containing 2% PLT GOLD Human Platlet Lysate (Clinical Grade) + 1% MEM Non-Essential Amino Acids Solution (100X) + 1% GultaMAX^TMDMEM basic (1X) [ + (100X)]4.5g/L D-Glucose medium), changing the medium every 3 days, culturing for 7 days, and using TrypLE^TMSelect (1X) digest, digest single cells at 4X 10⁴cell/cm²Inoculating to 25cm²And (3) continuously culturing the cells for 7d in a cell culture flask defined as iPSC-MSC-P1, wherein the cells grow in a spindle shape and grow at a high speed, and changing the liquid every 2 d. Using TrypLE^TMDigest for 5min with Select (1X), howeverThen, the mixture was centrifuged at 1200rpm for 5min, and the precipitate was collected. At 2X 10⁴cell/cm²Inoculating to 75cm²Cell culture flasks, defined as iPSC-MSC-P2, then every 6d when the cells were 80% long at 1X 10⁴cell/cm²Inoculating to 175cm²The cells were cultured in flasks and passaged at 1:4 to give iPSC-MSC-P3. The iPSC-MSC-P3 generation cells were used for subsequent iPSC-MSC identification and preparation. During the period, the forms of human iPSC-MSC at different culture times were photographed by an optical microscope. Day9 shows fusiform IPSC-MSC P0, Day12 shows fusiform IPSC-MSC P1 with obviously increased number, Day17 shows dense fibrous IPSC-MSC P2, and Day 21 shows homogeneous cell group iPSC-MSC-P3 with fusiform and MSC-like morphology.

Thirdly, identifying the characteristics of iPSC-MSC: iPSC-MSC surface marker (CD 73) using flow cytometry⁺、CD90⁺、CD105⁺、CD34^-、CD45^-、HLA-DR^-) Assaying, thereby identifying a stem cell-specific antigen. The results show that CD73⁺、CD90⁺And CD105⁺The expression is more than 95 percent positive, CD34^-、CD45^-And HLA-DR^-Expression less than 1% was negative. The iPSC-MSC is shown to have mesenchymal stem cell specific antigen.

And carrying out osteogenic, chondrogenic and adipogenic induction on the iPSC-MSC by respectively utilizing commercial osteogenic, chondrogenic and adipogenic differentiation induction kits, and evaluating the multidirectional differentiation capacities of the iPSC-MSC in vitro such as osteogenic, chondrogenic and adipogenic capacities through alizarin red, toluidine blue and oil red O staining. Alizarin red, toluidine blue and oil red O staining are all positive, which indicates that the iPSC-MSC has the capability of multidirectional differentiation of in vitro osteogenesis, chondrogenesis and adipogenesis.

Fourthly, preparing a mesenchymal stem cell preparation: serum-free Recovery of the obtained mesenchymal stem cells at clinical level^TMThe frozen stock solution of Cell Culture Freezing Medium is 1 × 10¹⁰The concentration of each/mL is divided and stored in a cell bank;

fifthly, quality control of the mesenchymal stem cell preparation: standard operation procedures of the stem cell preparation process and Standard Operation Procedures (SOP) of each process are established according to the International society for cell therapy and/or partial reference to the national food and drug administration for the partial standards of adult stem cells, and are regularly reviewed and revised. The preparation process of the stem cell preparation comprises the steps of induction preparation, purification, amplification and passage of stem cells, selection standard and use of culture medium, auxiliary materials and packing materials, freezing and storing of the cells, recovery, subpackaging and marking, residue removal and the like. The whole preparation process is subjected to comprehensive process research and verification, appropriate process parameters and quality standards are formulated, and effective control of each process is ensured. And (4) verifying and evaluating the cell product, and providing related procedures, technologies and specifications to obtain a qualified finished product.

The assay and evaluation criteria for the cell product preferably include cell viability, cell mass and additive mass. The cell activity detection adopts different cell biological activity detection methods. The basic quality of the cells is that the iPSC-MSC prepared by the invention has clear cell characteristics and has no infection of exogenous microorganisms. The quality requirement of the additive is to make clear the source, batch number and quality certification qualification report, and to adopt the clinically applicable products approved by the state as much as possible.

The relevant data of the cell preparation needs to be filed and stored for a long time.

From the above examples, it can be seen that the method provided by the present invention only requires replacing the induction medium and the growth medium containing clinical-grade human platelet lysate, and then using non-porcine pancreatic digestive juice TrypLE^TMAnd (3) carrying out continuous gradient density single cell passage on the Select (1 x), and successfully optimizing the method for differentiating the human iPSC into the clinical iPSC-MSC with low cost, simplicity and high efficiency. The method overcomes the defects of the prior art, does not relate to heterogeneous fetal calf serum, does not need gelatin to coat a cell carrier, does not need to add a small molecular compound and a growth factor, does not need special culture conditions and flow cytometer sorting, and can simply and efficiently produce a large number of clinical iPSC-MSCs from human iPSCs.

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.

Claims (10)

1. A method for preparing a clinical-grade mesenchymal stem cell preparation by utilizing human induced pluripotent stem cells is characterized by comprising the following steps:

1) when the human iPSC grows to 80-90% in the 6-well plate, replacing the iPSC-MSC induction culture medium for culturing for 8-12 days, and replacing the culture medium every 2-3 days to obtain adherent cells, and digesting to obtain iPSC-MSC single cells;

2) the iPSC-MSC single cells are arranged according to 8 multiplied by 10⁴cell/cm²Inoculating to 25cm²In a cell culture bottle, defined as iPSC-MSC-P0, replacing the iPSC-MSC growth culture medium for culture for 4-7 days, replacing the culture medium once every 2-3 days, digesting, and replacing by 4 multiplied by 10⁴cell/cm²Inoculating to 25cm²In a cell culture bottle, defined as iPSC-MSC-P1, continuously culturing for 4-7 d in iPSC-MSC growth culture medium, changing liquid every 2-3 d, digesting, and performing 2 × 10⁴cell/cm²Inoculating to 75cm²In the cell culture bottle, when the cells grow to 80-90% every 4-7 days later, the number is 1 multiplied by 10⁴cell/cm²Inoculating to 175cm²Carrying out passage culture in a cell culture bottle at a ratio of 1: 3-1: 4 to obtain a large amount of iPSC-MSC-P3 cells;

the iPSC-MSC induction culture medium is a DMEM culture medium containing 2-5% of human platelet lysate in volume concentration, 1% of 100 multiplied by trace nonessential amino acid solution in volume concentration, 1% of 100 multiplied by glutamine additive in volume concentration and 1.0 g/LD-glucose;

the iPSC-MSC growth medium is a DMEM medium containing 2-5% of human platelet lysate in volume concentration, 1% of 100 multiplied by trace nonessential amino acid solution in volume concentration, 1% of 100 multiplied by glutamine additive in volume concentration and 4.5 g/LD-glucose;

3) clinical grade serum-free Recovery will be used^TMThe frozen stock solution of Cell Culture Freezing Medium is 1 × 10⁶～1×10¹⁰The iPSC-MSC-P3 cells are subpackaged at the concentration of each mL, and the prepared mesenchymal stem cell preparation is stored in a cell bank.

2. The method according to claim 1, wherein the digestive enzyme in step 1) or step 2) is TrypLE^TMSelecting; the digestion time is 5-8 min.

3. The method of claim 2, wherein said digesting is followed by centrifugation; the rotating speed of the centrifugation is 1000-1500 rpm; the centrifugation time is 3-7 min.

4. The method according to claim 1, wherein the temperature of the culture in step 2) is 36 to 38 ℃.

5. The method according to claim 1, wherein the human iPSC is cultured in step 1) by inoculating a human iPSC cell line in a 6-well plate coated with 10 μ g/ml human pluripotent stem cell culture adherent substrate in an iPSC culture medium at 37 ℃ and 5% CO₂Culturing for 5-7 days under the saturated humidity condition, and replacing a fresh culture medium every day.

6. The method of claim 5, wherein the human pluripotent stem cell culture adherent matrix comprises 960 μ L CellAdhere per 1000 μ L^TMDiluetionBuffer and 40ul of Vitronectin XF of 250 mug/ml^TM。

7. The method of claim 5, wherein the iPSC medium is TeSR at a volume ratio of 24:1^TM-E8^TMBasic Medium and 25 × TeSR^TM-E8^TMSupplement。

8. The method according to any one of claims 1 to 7, wherein the iPSC-MSC is characterized prior to being aliquoted;

the identification indexes of the iPSC-MSC characteristics comprise stem cell specific antigen, osteogenic capacity, chondrogenic capacity and adipogenic differentiation capacity.

9. The method of claim 8, wherein the method of identifying stem cell specific antigens comprises determining iPSC-MSC surface markers; the iPSC-MSC surface marker comprises CD73⁺、CD90⁺、CD105⁺、CD34^-、CD45^-And HLA-DR^-；

CD73⁺、CD90⁺And CD105⁺The expression is more than 95 percent positive, CD34^-、CD45^-And HLA-DR^-Expression less than 1% was negative.

The identification of the osteogenic, chondrogenic and adipogenic differentiation capacity is that the commercial osteogenic, chondrogenic and adipogenic differentiation induction kit is used for carrying out iPSC-MSC osteogenic, chondrogenic and adipogenic induction, and the iPSC-MSC in-vitro osteogenic, chondrogenic and adipogenic differentiation capacity is evaluated by adopting alizarin red, toluidine blue and oil red O staining respectively;

alizarin red, toluidine blue and oil red O staining results are positive, and the iPSC-MSC has the capability of multidirectional differentiation of in vitro osteogenesis, chondrogenesis and adipogenesis.

10. The method of any one of claims 1 to 7, further comprising quality control of the mesenchymal stem cell preparation;

according to the international cell therapy society and/or partial reference to the national food and drug administration for the partial standard of adult stem cells, the cell product is verified and evaluated, and the related procedures, technologies and specifications are provided, and the cell product is qualified as a finished product;

the detection and evaluation indexes of the cell product comprise cell activity, cell quality and additive quality.

Images