CN112048470B - Method for preparing clinical grade mesenchymal stem cell preparation by using human induced pluripotent stem cells - Google Patents

Abstract

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

Description

Method for preparing clinical grade mesenchymal stem cell preparation by using 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, which is not only the basis for the multi-functional activity, but also the limiting factor affecting the experimental repeatability of MSCs, the quality of MSCs and even the clinical efficacy, and creates more variables and difficulties for standardized and normalized evaluation of the safety and effectiveness of MSC treatment. Human induced pluripotent stem cells (inducedpluripotent stem cell, iPSC) and related studies based on cell reprogramming technology significantly improve the understanding of stem cell maintenance pluripotency and stem property and their molecular regulatory mechanisms, while also providing an unlimited source of patient-specific personalized stem cells. With the improvement and optimization of non-integrated iPSC induction techniques, ipscs free of viral vectors and tumorigenic gene 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 passaged infinitely in principle, so that the source of MSC can be endless; MSCs derived from a single iPSC cell clone were theoretically more homogeneous. Based on the characteristics, the iPSC can provide stable and reliable infinite MSC sources suitable for individual demands 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 demands.

However, the defects and clinical needs of the prior art at present, the problems of limited sources, unstable quality and low homogeneity of the mesenchymal stem cells in the middle of cell transplantation treatment and tissue engineering of articular cartilage injury and degenerative diseases still exist.

Disclosure of Invention

In view of the above, the invention aims to provide a method for preparing clinical grade mesenchymal stem cell preparations by using human induced pluripotent stem cells, which meets the requirements of the market on mesenchymal stem cell sources with stable and reliable quality and suitability for individual requirements of various patients, and establishes in-vitro infinite expansion large-scale preparation and production technology.

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

1) When the human iPSC grows to 80-90% in a 6-hole plate, replacing the iPSC-MSC induction culture medium for culturing for 8-12 d, replacing liquid once every 2-3 d to obtain adherent cells, and digesting to obtain iPSC-MSC single cells;

2) The iPSC-MSC single cells were cultured according to 8X 10 ⁴ cell/cm ² Inoculating to 25cm ² In the cell culture flask, defined as iPSC-MSC-P0, the iPSC-MSC growth medium is changed to culture for 4-7 d, the liquid is changed every 2-3 d, and the mixture is digested to obtain 4X 10 ⁴ cell/cm ² Inoculating to 25cm ² In the cell culture flask, defined as iPSC-MSC-P1, the culture is continued for 4-7 d in iPSC-MSC growth medium, every 2-3 d liquid is changed, digested, and used for 2X 10 ⁴ cell/cm ² Inoculating to 75cm ² In the cell culture flask, when the cell grows to 80-90% every 4-7 d later, the cell is cultured in a culture flask at a ratio of 1X 10 ⁴ cell/cm ² Inoculating to 175cm ² The cell culture flask is subjected to passage at a ratio of 1:3-1:4 to obtain a large number of iPSC-MSC-P3 cells;

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

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

3) Clinical grade serum-free Recovery will be used ^TM Cell Culture Freezing Medium the content of the frozen stock solution is 1 multiplied by 10 ⁶ ～1×10 ¹⁰ And packaging the iPSC-MSC-P3 cells at a concentration of one mL, and storing the prepared mesenchymal stem cell preparation in a cell bank.

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

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

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

Preferably, the method of culturing human iPSC in step 1) comprises inoculating human iPSC cell lines in 6-well plates coated with 10 μg/ml human pluripotent stem cell culture adherent matrix in iPSC medium at 37deg.C, 5% CO ₂ Culturing for 5-7 d under saturated humidity, and changing fresh culture medium every day.

Preferably, the human pluripotent stem cell culture adherent matrix comprises 960 μl CellAdhere per 1000 μl ^TM DiluionBuffer and 40ul of Vitronnectin XF at 250 μg/ml ^TM 。

Preferably, the iPSC medium is TeSR ^TM -E8 ^TM Basal Medium and 25×TeSR ^TM -E8 ^TM And supporting, wherein the volume ratio of the supporting agent to the supporting agent is 24:1.

Preferably, the iPSC-MSC characteristics are identified prior to the dispensing.

The identification index of the characteristics of the iPSC-MSC comprises stem cell specific antigen, osteogenesis, chondrogenesis and adipogenic differentiation capacity.

Preferably, the method of identifying a 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 ⁺ Positive expression, CD34, of over 95% ^- 、CD45 ^- And HLA-DR ^- Less than 1% of expression is negative.

The identification of the osteogenesis, chondrogenesis and adipogenesis differentiation capacity is to utilize a commercial osteogenesis, chondrogenesis and adipogenesis differentiation induction kit to perform iPSC-MSC osteogenesis, chondrogenesis and adipogenesis induction, and to evaluate the in vitro osteogenesis, chondrogenesis and adipogenesis differentiation capacity of the iPSC-MSC by respectively adopting alizarin red, toluidine blue and oil red O staining; alizarin red, toluidine blue and oil red O staining are positive, and the iPSC-MSC has the capacity of in vitro osteogenesis, cartilage formation and adipogenesis multidirectional differentiation.

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

According to the international cell therapy association and/or partially referring to the partial standard of the national food and drug administration on adult stem cells, the cell products are detected and evaluated, and related processes, technologies and specifications are provided, and the finished products are qualified;

the assay and evaluation indicators of cellular products include cellular activity, cellular 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 to induce the pluripotent stem cells to differentiate and expand and culture the mesenchymal stem cells from human through a system without the trophoblast cells of a heterogeneous serum product (so as to avoid the pollution of heterogeneous bacteria and cells), has the advantages of infinite source, stable quality, high homogeneity, relatively controllable property and the like compared with the mesenchymal stem cells from other sources, can provide reliable mesenchymal stem cells for the individual demands of various patients, can provide an important cell model for clinical research of related diseases and targeted drugs, and has very wide scale industrialization and clinical application market prospect.

Drawings

FIG. 1 shows the morphology of the present inventors under different time points during the induction of pluripotent stem cell-derived mesenchymal stem cells;

FIG. 2 is a flow chart of the identification of surface markers of the present inventors induced pluripotent stem cell-derived mesenchymal stem cells;

FIG. 3 is a diagram showing the in vitro osteogenic, chondrogenic, adipogenic directed induction differentiation of the mesenchymal stem cells derived from the induced pluripotent stem cells of the present invention.

Detailed Description

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

1) When the human iPSC grows to 80-90% in a 6-hole plate, replacing the iPSC-MSC induction culture medium for culturing for 8-12 d, replacing liquid once every 2-3 d to obtain adherent cells, and digesting to obtain iPSC-MSC single cells;

2) The iPSC-MSC single cells were cultured according to 8X 10 ⁴ cell/cm ² Inoculating to 25cm ² In the cell culture flask, defined as iPSC-MSC-P0, the iPSC-MSC growth medium is changed to culture for 4-7 d, the liquid is changed every 2-3 d, and the mixture is digested to obtain 4X 10 ⁴ cell/cm ² Inoculating to 25cm ² In the cell culture flask, defined as iPSC-MSC-P1, the culture is continued for 4-7 d in iPSC-MSC growth medium, every 2-3 d liquid is changed, digested, and used for 2X 10 ⁴ cell/cm ² Inoculating to 75cm ² In the cell culture flask, when the cell grows to 80-90% every 4-7 d later, the cell is cultured in a culture flask at a ratio of 1X 10 ⁴ cell/cm ² Inoculating to 175cm ² The cell culture flask is subjected to passage at a ratio of 1:3-1:4 to obtain a large number of iPSC-MSC-P3 cells;

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

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

3) Clinical grade serum-free Recovery will be used ^TM Cell Culture Freezing Medium the content of the frozen stock solution is 1 multiplied by 10 ⁶ ～1×10 ¹⁰ The iPSC-MSC-P3 cells were split at a concentration of individual/mL and stored in a cell bank.

When the human iPSC grows to 80-90% in a 6-hole plate, the invention replaces the iPSC-MSC induction culture medium to culture for 8-12 d, and the liquid is replaced once every 2-3 d to obtain adherent cells, and digestion is carried out to obtain iPSC-MSC single cells.

In the present invention, the method for culturing human iPSC is preferably to inoculate a human iPSC cell strain in a 6-well plate coated with a 10. Mu.g/ml human pluripotent stem cell culture adherent substrate in iPSC medium at 37℃and 5% CO ₂ Culturing for 5-7 d under saturated humidity, and changing fresh culture medium every day. The iPSC medium is preferably TeSR ^TM -E8 ^TM Basal Medium and 25×TeSR ^TM -E8 ^TM And supporting, wherein the volume ratio of the supporting to the supporting is 24:1.TeSR (Tesr) ^TM -E8 ^TM Basal Medium and TeSR ^TM -E8 ^TM Supplement together form a low protein, feeder-free TeSR ^TM -E8 ^TM Culture medium capable of maintaining human pluripotent stem cell growth, all available from STEMCELL ^TM Techenologies inc. The source of the human iPSC cell strain is not particularly limited, and the human iPSC cell strain well known in the art can be adopted. The human iPSC cell strain is purchased from Nuwacell company and is Nuwacell ^TM Scientific grade hiPSC cell line (RC 01001-B). The human pluripotent stem cell culture adherent matrix preferably comprises 960 μl CellAdhere per 1000 μl ^TM DiluionBuffer and 40 μl of Vitronnectin XF of 250 μg/ml ^TM 。

In the invention, the iPSC-MSC induction culture medium is preferably a DMEM culture medium containing clinical-grade human platelet lysate with the volume concentration of 2-5%, a 1% 100X trace non-essential amino acid solution with the volume concentration, a 1% 100X glutamine additive with the volume concentration and 1.0g/L D-glucose. Advantages of induction medium over other formulations in the art: clinical grade human platelet lysate contains all growth factors and proteins necessary for cell growth, is an effective, high protein supplement for human cell culture and expansion, and can reduce the risk of xenogenic immune responses or transmission of prion and zoonotic diseases. The source of the components of the induction medium is not particularly limited, and clinical, trace amounts of non-essential amino acid solution, glutamine additive, D-glucose and DMEM medium, which are well known in the art, may be used.

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

In the present invention, the enzyme for digestion is preferably TrypLE ^TM Select; the digestion time is preferably 5 to 8 minutes, more preferably 6 to 7 minutes. Centrifuging to collect single cells after the digestion; the rotation speed of the centrifugation is preferably 1000-1500 rpm; the centrifugation time is preferably 3 to 7 minutes. iPSC-MSC single cells represent MSC single cells derived from induced differentiation of human iPSC.

After iPSC-MSC single cells are obtained, the invention uses the iPSC-MSC single cells according to the ratio of 8 multiplied by 10 ⁴ cell/cm ² Inoculating to 25cm ² In the cell culture flask, defined as iPSC-MSC-P0, the iPSC-MSC growth medium is changed to culture for 4-7 d, the liquid is changed every 2-3 d, and the mixture is digested to obtain 4X 10 ⁴ cell/cm ² Inoculating to 25cm ² In the cell culture flask, defined as iPSC-MSC-P1, the culture is continued for 4-7 d in iPSC-MSC growth medium, every 2-3 d liquid is changed, digested, and used for 2X 10 ⁴ cell/cm ² Inoculating to 75cm ² In the cell culture flask, when the cell grows to 80-90% every 4-7 d later, the cell is cultured in a culture flask at a ratio of 1X 10 ⁴ cell/cm ² Inoculating to 175cm ² And (3) culturing the cells in a bottle at a ratio of 1:3-1:4 to obtain a large number of iPSC-MSC-P3 cells.

In the invention, the iPSC-MSC growth medium is preferably a DMEM medium containing 2-5% by volume of human platelet lysate, 1% by volume of 100 Xtrace non-essential amino acid solution, 1% by volume of 100 Xglutamine additive and 4.5g/L D-glucose. The temperature of the culture is preferably 36 to 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 of the iPSC-MSC characteristics preferably includes stem cell specific antigens, osteogenesis, chondrogenesis and adipogenic differentiation capacity. The method for identifying the stem cell specific antigen preferably comprises measuring the 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 ⁺ Positive expression, CD34, of over 95% ^- 、CD45 ^- And HLA-DR ^- Less than 1% of expression is negative.

The identification of the osteogenic, chondrogenic and adipogenic differentiation capacity is preferably to utilize a commercial osteogenic, chondrogenic and adipogenic differentiation induction kit to perform iPSC-MSC osteogenesis, chondrogenic and adipogenic induction, and to evaluate the in vitro osteogenic, chondrogenic and adipogenic differentiation capacity of the iPSC-MSC by respectively adopting alizarin red, toluidine blue and oil red O staining. Alizarin red, toluidine blue and oil red O staining are positive, which shows that the iPSC-MSC has the capacity of in vitro osteogenesis, cartilage formation and adipogenesis multidirectional differentiation.

After the iPSC-MSC-P3 cells with qualified screening characteristics are obtained, the invention adopts clinical grade serum-free Recovery ^TM Cell Culture Freezing Medium the content of the frozen stock solution is 1 multiplied by 10 ⁶ ～1×10 ¹⁰ And packaging the iPSC-MSC-P3 cells at a concentration of one mL, and storing the prepared mesenchymal stem cell preparation in a cell bank.

In the present invention, the concentration of the split charge is preferably 1X 10 ⁷ ～1×10 ⁹ And more preferably 1X 10 per mL ⁸ And each mL.

In the present invention, it is preferable to further include quality control of the mesenchymal stem cell preparation: standard procedures for stem cell preparation processes and standard procedures for each process (SOP) are formulated and periodically reviewed and revised in accordance with the international cytotherapeutic association and/or with reference in part to the national food and drug administration's standards regarding parts of adult stem cells. The preparation process of the stem cell preparation comprises the steps of induction preparation, purification, amplification and passage of stem cells, selection criteria and use of culture medium, auxiliary materials and packing materials, freezing storage, resuscitating, split charging and marking of cells, residue removal and the like. The whole preparation process is subjected to comprehensive process research and verification, and proper process parameters and quality standards are formulated to ensure effective control of each process. And (3) detecting and evaluating the cell products, and providing related processes, technologies and specifications, wherein the cell products are qualified to obtain finished products.

The assay and evaluation indicators of the cell product preferably include cell activity, cell mass, and additive mass. The cell activity detection adopts different cell biological activity detection methods to judge the cell activity and the growth condition. The basic quality requirement of the cell is that the cell has definite cell identification characteristics and no infection of exogenous microorganisms. The quality requirements of the additive are that the source, the batch number and the quality verification qualification report should be defined, and the clinically applicable products approved by the country are adopted as much as possible.

Relevant information of the cell preparation needs to be established and stored for a long time.

The following is a detailed description of a method for preparing clinical grade mesenchymal stem cell preparations using human induced pluripotent stem cells according to the present invention, but is not to be construed as limiting the scope of the present invention.

Example 1

A method for preparing a clinical grade mesenchymal stem cell preparation by using human induced pluripotent stem cells, comprising the following steps:

1. routine culture of human iPSC: the human iPSC cell line was purchased from Nuwacell corporation as Nuwacell TM scientific grade hiPSC cell line (RC 01001-B) and inoculated into 10. Mu.g/ml Vitronectin XF ^TM (composition comprising 960. Mu.l CellAdhere) ^TM DilutionBuffer+40μl 250μg/ml Vitronectin XF ^TM ) In coated 6-well plates, iPSC conventional medium (1920 μl TeSR was used ^TM -E8 ^TM Basal Medium+80μl 25×TeSR ^TM -E8 ^TM Support) at 37℃with 5% CO ₂ Culturing under saturated humidity for 6d. The culture was continued with daily replacement of fresh conventional medium as described above. During the period, the optical microscope is adopted to shoot the human iPSC shape with different culture timeA state. The results are shown in FIG. 1. Undifferentiated human iPSC were grown in 6 well plates to 70% compact, higher nuclear to cytoplasmic ratio with prominent nucleoli, well-defined cobblestone-like multicellular clusters in Day-2 plates prior to differentiation; cobblestone-like multicellular clusters with high nuclear-to-mass ratios and with prominent nucleoli were grown to 90% compactness in Day 0 in 6 well plates.

2. Inducing differentiation of iPSC into iPSC-MSC and amplifying iPSC-MSC: when human iPSC grew to 90% in Day 0 in 6 well plates (smooth and clear cell edges, tight cell gaps, no apparent differentiation, and typical cobblestone-like clones) 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) ^TM DMEM basic (1×) [ + of (100×)]1.0g/L D-Glucose medium), changing the liquid every 2d, culturing for 4d to obtain adherent cells, and culturing with TrypLE ^TM Select (1×) was digested for 7min and then centrifuged at 1500rpm for 5min and the pellet was collected. At 8×10 ⁴ cell/cm ² Inoculum size of 25cm ² In the cell culture flask, defined as iPSC-MSC-P0, and replaced with iPSC-MSC growth medium (containing 5%PLT GOLD Human Platelet Lysate (Clinical Grade) +1% MEM Non-Essential Amino Acids Solution (100×) +1% GultaMAX) ^TM DMEM basic (1×) [ + of (100×)]4.5g/L D-Glucose medium), liquid was changed every 2d, and after 4d of incubation, trypLE was used ^TM Select (1×) digested single cells at 4×10 ⁴ cell/cm ² Inoculating to 25cm ² Cell culture bottle, defined as iPSC-MSC-P1, continues to cultivate for 4d, observes cell growth state during which time, the cell should be shuttle growth, growth rate is fast, trades liquid every 2 d. By TrypLE ^TM Select (1×) was digested for 7min and then centrifuged at 1200rpm for 5min and the pellet was collected. At 2X 10 ⁴ cell/cm ² Inoculum size of 75cm ² Cell culture flasks, defined as iPSC-MSC-P2, were then grown at 1X 10 every 4d when the cells were 90% ⁴ cell/cm ² Inoculum size of 175cm ² Cell culture flasks were passaged 1:3 to give iPSC-MSC-P3. Subsequent iPSC-MSC identification and preparation of formulations were performed using iPSC-MSC-P3 generation cells. During which the optical microscope is used for shootingHuman iPSC-MSC morphology at different culture times. The results are shown in FIG. 1.Day10 shows up as a fusiform IPSC-MSC P0, day 14 shows up as a significantly increased number of fusiform IPSC-MSCs P1, day 18 shows up as a densely fibrous IPSC-MSC P2, and Day 22 shows up as a homogeneous population IPSC-MSC-P3 with spindle and MSC-like morphology.

3. identification of iPSC-MSC characteristics: iPSC-MSC surface marker (CD 73) using flow cytometry ⁺ 、CD90 ⁺ 、CD105 ⁺ 、CD34 ^- 、CD45 ^- 、HLA-DR ^- ) Determining, thereby identifying stem cell specific antigens; the results are shown in FIG. 2.CD73 ⁺ 、CD90 ⁺ And CD105 ⁺ Positive expression, CD34, of over 95% ^- 、CD45 ^- And HLA-DR ^- Less than 1% of expression is negative. Indicating that the iPSC-MSC has the mesenchymal stem cell specific antigen.

The commercial osteogenesis, chondrogenesis and adipogenesis differentiation induction kit is used for carrying out the osteogenesis, the chondrogenesis and the adipogenesis induction of the iPSC-MSC, and the multidirectional differentiation capacities of the iPSC-MSC such as in-vitro osteogenesis, chondrogenesis, adipogenesis and the like are respectively evaluated 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 positive, which shows that the iPSC-MSC has the capacity of in vitro osteogenesis, cartilage formation and adipogenesis multidirectional differentiation.

4. Preparation of mesenchymal stem cell preparation: serum-free Recovery of the resulting mesenchymal stem cells at clinical level ^TM Cell Culture Freezing Medium the content of the frozen stock solution is 1 multiplied by 10 ⁸ The concentration of each mL is split and stored in a cell bank.

5. Quality control of mesenchymal stem cell preparation: standard procedures for stem cell preparation processes and standard procedures for each process (SOP) are formulated and periodically reviewed and revised in accordance with the international cytotherapeutic association and/or with reference in part to the national food and drug administration's standards regarding parts of adult stem cells. The preparation process of the stem cell preparation comprises the steps of induction preparation, purification, amplification and passage of stem cells, selection criteria and use of culture medium, auxiliary materials and packing materials, freezing storage, resuscitating, split charging and marking of cells, residue removal and the like. The whole preparation process is subjected to comprehensive process research and verification, and proper process parameters and quality standards are formulated to ensure effective control of each process. And (3) detecting and evaluating the cell products, and providing related processes, technologies and specifications, wherein the cell products are qualified to obtain finished products.

The assay and evaluation indicators of the cell product preferably include cell activity, 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 definite cell characteristics and no exogenous microorganism infection. The quality requirements of the additive are that the source, the batch number and the quality verification qualification report should be defined, and the clinically applicable products approved by the country are adopted as much as possible.

Relevant information of the cell preparation needs to be established and stored for a long time.

Example 2

A method for preparing a clinical grade mesenchymal stem cell preparation by using human induced pluripotent stem cells, comprising the following steps:

1. routine culture of human iPSC: human iPSC cell lines (from Nuwacell) were inoculated at 10. Mu.g/ml Vitronectin XF ^TM (composition comprising 960. Mu.l CellAdhere) ^TM DilutionBuffer+40μl 250μg/ml Vitronectin XF ^TM ) In coated 6-well plates, iPSC conventional medium (1920 μl TeSR was used ^TM -E8 ^TM Basal Medium+80μl 25×TeSR ^TM -E8 ^TM Support) at 37℃with 5% CO ₂ Culturing under saturated humidity for 6d. The culture was continued with daily replacement of fresh conventional medium as described above. During the period, the human iPSC forms with different culture times are photographed by an optical microscope. The results are shown in FIG. 1. Undifferentiated human iPSC were grown in 6 well plates to 70% compact, higher nuclear to cytoplasmic ratio with prominent nucleoli, well-defined cobblestone-like multicellular clusters in Day-2 plates prior to differentiation; cobblestone-like multicellular clusters with high nuclear-to-mass ratios and with prominent nucleoli were grown to 90% compactness in Day 0 in 6 well plates.

2. Inducing differentiation of iPSC into iPSC-MSC and amplifying iPSC-MSC: when human ipscs were grown to 80% in Day 0 in 6 well plates (smooth and clear cell edges,the cell gap was compact, no obvious differentiation was observed, and the typical cobblestone clone 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) ^TM DMEM basic (1×) [ + of (100×)]1.0g/L D-Glucose medium), changing the liquid every 3d, culturing for 7d to obtain adherent cells, and culturing with TrypLE ^TM Select (1×) was digested for 5min, then centrifuged at 1000rpm for 7min and the pellet was collected. At 8×10 ⁴ cell/cm ² Inoculum size of 25cm ² In the cell culture flask, defined as iPSC-MSC-P0, and replaced with iPSC-MSC growth medium (containing 2%PLT GOLD Human Platelet Lysate (Clinical Grade) +1% MEM Non-Essential Amino Acids Solution (100×) +1% GultaMAX) ^TM DMEM basic (1×) [ + of (100×)]4.5g/L D-Glucose medium), liquid was changed every 3d, and after 7d of incubation, trypLE was used ^TM Select (1×) digested single cells at 4×10 ⁴ cell/cm ² Inoculating to 25cm ² Cell culture bottle, defined as iPSC-MSC-P1, continues to cultivate for 7d, observes cell growth state during which time, the cell should be shuttle growth, growth rate is fast, trades liquid every 2 d. By TrypLE ^TM Select (1×) was digested for 5min, then centrifuged at 1200rpm for 5min and the pellet was collected. At 2X 10 ⁴ cell/cm ² Inoculum size of 75cm ² Cell culture flasks, defined as iPSC-MSC-P2, were then grown 1X 10 every 6d when the cells were 80% long ⁴ cell/cm ² Inoculum size of 175cm ² Cell culture flasks were passaged 1:4 to give iPSC-MSC-P3. Subsequent iPSC-MSC identification and preparation of formulations were performed using iPSC-MSC-P3 generation cells. During the period, the human iPSC-MSC forms with different culture times are photographed by an optical microscope. Day9 shows up as a fusiform IPSC-MSC P0, day12 shows up as a significantly increased number of fusiform IPSC-MSCs P1, day17 shows up as a densely fibrous IPSC-MSC P2, and Day 21 shows up as a homogeneous cell population IPSC-MSC-P3 with spindle and MSC-like morphology.

3. identification of iPSC-MSC characteristics: iPSC-MSC surface marker (CD 73) using flow cytometry ⁺ 、CD90 ⁺ 、CD105 ⁺ 、CD34 ^- 、CD45 ^- 、HLA-DR ^- ) MeasurementThereby identifying stem cell specific antigens. The results showed that CD73 ⁺ 、CD90 ⁺ And CD105 ⁺ Positive expression, CD34, of over 95% ^- 、CD45 ^- And HLA-DR ^- Less than 1% of expression is negative. Indicating that the iPSC-MSC has the mesenchymal stem cell specific antigen.

The commercial osteogenesis, chondrogenesis and adipogenesis differentiation induction kit is used for carrying out the osteogenesis, the chondrogenesis and the adipogenesis induction of the iPSC-MSC, and the multidirectional differentiation capacities of the iPSC-MSC such as in-vitro osteogenesis, chondrogenesis, adipogenesis and the like are respectively evaluated through alizarin red, toluidine blue and oil red O staining. Alizarin red, toluidine blue and oil red O staining are positive, which shows that the iPSC-MSC has the capacity of in vitro osteogenesis, cartilage formation and adipogenesis multidirectional differentiation.

4. Preparation of mesenchymal stem cell preparation: serum-free Recovery of the resulting mesenchymal stem cells at clinical level ^TM Cell Culture Freezing Medium the content of the frozen stock solution is 1 multiplied by 10 ¹⁰ The concentration of each mL is split-packed and stored in a cell bank;

5. quality control of mesenchymal stem cell preparation: standard procedures for stem cell preparation processes and standard procedures for each process (SOP) are formulated and periodically reviewed and revised in accordance with the international cytotherapeutic association and/or with reference in part to the national food and drug administration's standards regarding parts of adult stem cells. The preparation process of the stem cell preparation comprises the steps of induction preparation, purification, amplification and passage of stem cells, selection criteria and use of culture medium, auxiliary materials and packing materials, freezing storage, resuscitating, split charging and marking of cells, residue removal and the like. The whole preparation process is subjected to comprehensive process research and verification, and proper process parameters and quality standards are formulated to ensure effective control of each process. And (3) detecting and evaluating the cell products, and providing related processes, technologies and specifications, wherein the cell products are qualified to obtain finished products.

The assay and evaluation indicators of the cell product preferably include cell activity, 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 definite cell characteristics and no exogenous microorganism infection. The quality requirements of the additive are that the source, the batch number and the quality verification qualification report should be defined, and the clinically applicable products approved by the country are adopted as much as possible.

Relevant information of the cell preparation needs to be established and stored for a long time.

As can be seen from the above examples, the method provided by the present invention requires only replacement of the induction medium and the growth medium containing clinical-grade human platelet lysate, and then uses non-porcine pancreatin digestive juice TrypLE ^TM Serial gradient density single cell passage is carried out by Select (1×), and a method for differentiating human iPSC into clinical-grade iPSC-MSC with low cost, simplicity and high efficiency is successfully optimized. The method overcomes the defects of the prior art, does not relate to the xenogeneic bovine serum, does not need gelatin to coat cell carriers, does not need small molecular compounds and growth factors to be added, does not need special culture conditions and flow cytometry sorting, and can simply and efficiently generate a large number of iPSC-MSCs which are suitable for clinical human iPSC sources.

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 (10)

1. A method for preparing a clinical grade mesenchymal stem cell preparation by using human induced pluripotent stem cells, comprising the steps of:

1) When the human iPSC grows to 80-90% in a 6-hole plate, replacing an iPSC-MSC induction culture medium for culturing for 8-12 d, replacing liquid once every 2-3 d to obtain adherent cells, and digesting to obtain iPSC-MSC single cells;

2) The iPSC-MSC single cells were cultured according to 8X 10 ⁴ cell/cm ² Inoculating to 25cm ² In a cell culture flask, defined as iPSC-MSC-P0, the iPSC-MSC growth medium is replaced to perform culture for 4-7 d, the liquid is replaced once every 2-3 d, and digestion is performed to obtain the product with the concentration of 4 multiplied by 10 ⁴ cell/cm ² Inoculating to 25cm ² In a cell culture bottle, defined as iPSC-MSC-P1, culturing in iPSC-MSC growth medium for 4-7 d continuously, each timeChanging the liquid for 2-3 days, digesting, and obtaining 2X 10 ⁴ cell/cm ² Inoculating to 75cm ² In the cell culture flask, when the cell grows to 80-90% every 4-7 d later, the cell is cultured in a culture flask with a ratio of 1X 10 ⁴ Inoculating cell/cm2 to 175cm ² The cell culture flask is subjected to passage at a ratio of 1:3-1:4 to obtain a large number of iPSC-MSC-P3 cells;

the iPSC-MSC induction culture medium is a DMEM culture medium containing 2-5% by volume of human platelet lysate, 1% by volume of 100X trace non-essential amino acid solution, 1% by volume of 100X glutamine additive and 1.0g/L D-glucose;

the iPSC-MSC growth medium is a DMEM medium containing 2-5% by volume of human platelet lysate, 1% by volume of 100X trace non-essential amino acid solution, 1% by volume of 100X glutamine additive and 4.5g/L D-glucose;

3) Clinical grade serum-free Recovery ™ Cell Culture Freezing Medium frozen stock solution is adopted according to the ratio of 1 multiplied by 10 ⁶ ~1×10 ¹⁰ And packaging the iPSC-MSC-P3 cells at a concentration of one mL, and storing the prepared mesenchymal stem cell preparation in a cell bank.

2. The method of claim 1, wherein the digestive enzyme in step 1) or step 2) is TrypLE ™ Select; the digestion time is 5-8 min.

3. The method of claim 2, wherein the digestion is followed by centrifugation; the rotational speed of the centrifugation is 1000-1500 rpm; and the centrifugation time is 3-7 min.

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

5. The method according to claim 1, wherein the method of culturing human iPSC in step 1) comprises inoculating human iPSC cell lines in 6-well plates coated with 10 μg/ml human pluripotent stem cell culture adherent substrate in iPSC medium at 37deg.C, 5% CO ₂ SaturatedCulturing for 5-7 d under the humidity condition, and replacing fresh culture medium every day.

6. The method of claim 5, wherein each 1000 μl of the human pluripotent stem cell culture adherent matrix comprises 960 μl cellsphere ™ differential buffer and 40 μl 250 μg/ml Vitronectin XF ™.

7. The method of claim 5, wherein the iPSC Medium is a 24:1 volumetric ratio of TeSR ™ -E8 ™ Basal Medium and 25 x TeSR ™ -E8 ™ supply.

8. The method according to any one of claims 1 to 7, wherein the iPSC-MSC characteristics are identified prior to packaging;

the identification index of the characteristics of the iPSC-MSC comprises stem cell specific antigen, osteogenesis, chondrogenesis 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 markers include CD73+, CD90+, CD105+, CD34-, CD 45-and HLA-DR-;

cd73+, cd90+ and cd105+ were positive for more than 95% and negative for less than 1% of CD34-, CD 45-and HLA-DR-expression;

the identification of the osteogenesis, chondrogenesis and adipogenesis differentiation capacity is to utilize a commercial osteogenesis, chondrogenesis and adipogenesis differentiation induction kit to perform iPSC-MSC osteogenesis, chondrogenesis and adipogenesis induction, and to evaluate the in vitro osteogenesis, chondrogenesis and adipogenesis differentiation capacity of the iPSC-MSC by respectively adopting alizarin red, toluidine blue and oil red O staining;

alizarin red, toluidine blue and oil red O have positive staining results, and the iPSC-MSC has the capacity of in vitro osteogenesis, cartilage formation and adipogenesis multidirectional differentiation.

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

according to the international cell therapy association and/or partially referring to the partial standard of the national food and drug administration on adult stem cells, the cell products are detected and evaluated, and related processes, technologies and specifications are provided, and the finished products are qualified;

the assay and evaluation indicators of cellular products include cellular activity, cellular quality, and additive quality.

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