A method for large-scale production of mesenchymal stem cells using induced pluripotent stem cells (iPS) and its application

Abstract

The present invention provides a method and application for large-scale production of mesenchymal stem cells using induced pluripotent stem cells (iPS), belonging to the field of stem cell technology. It includes: S1. Inoculating a human iPS cell line into a culture dish coated with Matrigel, culturing, changing the culture medium daily, and subculturing once every 6-7 days for 4-6 times to obtain subcultured cells; S2. Adding silicon dioxide nanospheres into water, adding ferric chloride and ferrous chloride, dropping ammonia water under the protection of an inert gas, and heating and stirring for reaction to prepare magnetic nanospheres; S3. Inoculating the subcultured cells into a culture medium, adding the magnetic nanospheres, stirring and mixing evenly, and culturing to obtain nanospheres adsorbed with mesenchymal stem cells. The present invention uses iPS to produce mesenchymal stem cells on a large scale. Under the induction of magnetic iron oxide, iPS is prone to transform into mesenchymal stem cells, affecting the directed differentiation and paracrine function of stem cells and promoting the repair of injured tendons.

Description

Technical Field

[0001] The present invention relates to the technical field of stem cells, and particularly relates to a method for large-scale production of mesenchymal stem cells using induced pluripotent stem cells (iPS) and its application. Background Art

[0002] Stem cells are the initial source of human body and various tissue cells. Their most significant biological characteristics are the ability of self-renewal and continuous proliferation, as well as the potential of multi-directional differentiation. Stem cells are classified into adult stem cells and embryonic stem cells (ES cells) according to different sources. Adult stem cells include bone marrow mesenchymal stem cells, pancreatic stem cells, neural stem cells, etc., and exist in adult tissues.

[0003] Mesenchymal stem cells are widely present in the connective tissues and organ interstitium throughout the body, with the highest content in bone marrow tissue, and can also be isolated from fetal cord blood. In addition, they also exist in placenta, amniotic fluid, subendothelial layer of umbilical vein, peripheral blood, and various tissues such as liver, fat, muscle, and skin. Mesenchymal stem cells have the characteristics of high proliferation, self-renewal, and multi-directional differentiation potential. They can not only differentiate into various tissues and cells, but also be easily isolated, cultured, and amplified. They are also easy to introduce and express foreign genes, and always maintain the potential of multi-directional differentiation during long-term in vitro culture, with a relatively stable genetic background.

[0004] In 1995, mesenchymal stem cells were first applied to clinical practice. In 2012, Osiris Company applied for the listing of mesenchymal stem cells as a drug and obtained the approval of the Canadian FDA, reaching a historical height in the clinical application of mesenchymal stem cells. Mesenchymal stem cells have now replaced placental stem cells and become the preferred seed cells in biomedical research, and are widely used in basic and clinical research of various diseases. Therefore, it is very necessary to provide a method for preparing mesenchymal stem cells, which is also a research hotspot in the current study of mesenchymal stem cells. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for large-scale production of mesenchymal stem cells using induced pluripotent stem cells (iPS) and its application. By using induced pluripotent stem cells (iPS) for large-scale production of mesenchymal stem cells, under the induction of magnetic iron oxide, induced pluripotent stem cells (iPS) are easily transformed into mesenchymal stem cells, which affects the directional differentiation and paracrine function of stem cells and promotes the repair of damaged tendons.

[0006] The technical solution of the present invention is realized as follows:

[0007] The present invention provides a method for large-scale production of mesenchymal stem cells using induced pluripotent stem cells (iPS), comprising the following steps:

[0008] S1. Inoculate a human iPS cell line into a culture dish coated with Matrigel, culture it, change the culture medium every day, passage once every 6 - 7 days, and passage 4 - 6 times to obtain passage cells.

[0009] S2. Add silica nanospheres into water, add ferric chloride and ferrous chloride, under the protection of inert gas, dropwise add ammonia water, heat and stir to react to obtain magnetic nanospheres.

[0010] S3. Inoculate the passage cells into a culture medium, add magnetic nanospheres, stir and mix evenly, culture to obtain nanospheres adsorbed with mesenchymal stem cells.

[0011] As a further improvement of the present invention, the inoculation amount of the human iPS cell line in step S1 is 2 - 3×10 4 cells / cm 2 .

[0012] As a further improvement of the present invention, the culture conditions in step S1 are 36 - 38°C, 5% CO2 cell culture incubator.

[0013] As a further improvement of the present invention, the culture medium in step S1 is mTeSRTM1 complete stem cell medium.

[0014] As a further improvement of the present invention, the average particle size of the silica nanospheres in step S2 is 500 - 700 nm.

[0015] As a further improvement of the present invention, the mass ratio of the silica nanospheres, ferric chloride, ferrous chloride, and ammonia water in step S2 is 10:3.24:1.26:2 - 4.

[0016] As a further improvement of the present invention, the temperature of the heating and stirring reaction in step S2 is 70 - 80°C, and the time is 3 - 5 h.

[0017] As a further improvement of the present invention, the inoculation amount in step S3 is 1 - 2×10 6 cells / mL.

[0018] As a further improvement of the present invention, the culture conditions in step S3 are 36 - 38°C, 5% CO2 cell culture incubator, and the time is 5 - 7 d.

[0019] The present invention further protects the application of the mesenchymal stem cells prepared by the above - mentioned method in large - scale production in the preparation of drugs for repairing chronic tendinopathy.

[0020] The present invention has the following beneficial effects:

[0021] The present invention uses induced pluripotent stem cells (iPS) to mass-produce mesenchymal stem cells. Under the induction of magnetic iron tetroxide, iPS are prone to transform into mesenchymal stem cells and adsorb and fix on the prepared magnetic nanospheres, accelerating the transformation from two-dimensional cell culture to three-dimensional cell culture. While promoting the "industrialized" culture of cells, it can also increase the communication and connection between cells, further affecting the directional differentiation and paracrine function of stem cells. At the same time, magnetic iron tetroxide can also release far-infrared rays, further synergistically promoting the repair of injured tendons. Detailed implementation mode

[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0023] Example 1

[0024] This example provides a method for mass-producing mesenchymal stem cells using induced pluripotent stem cells (iPS), including the following steps:

[0025] S1. Inoculate a human iPS cell line into a culture dish coated with Matrigel, with an inoculation amount of 2×10 4 cells / cm 2 . Culture in a 36°C, 5% CO2 cell incubator, with the culture medium being mTeSRTM1 complete stem cell medium. Change the medium every day and passage once every 6 - 7 days. Passage 4 - 6 times to obtain passage cells;

[0026] S2. Add 10 g of silica nanospheres with an average particle size of 500 nm to water, add 3.24 g of ferric chloride and 1.26 g of ferrous chloride. Under nitrogen protection, dropwise add 2 g of ammonia water, heat to 70°C, and stir and react for 3 h to prepare magnetic nanospheres;

[0027] S3. Inoculate the passage cells into mTeSRTM1 complete stem cell medium, with an inoculation amount of 1×10 6 cells / mL, add 10 wt% of the magnetic nanospheres based on the total mass of the system, stir and mix evenly, and culture in a 36°C, 5% CO2 cell incubator for 5 d to obtain nanospheres adsorbed with mesenchymal stem cells.

[0028] Example 2

[0029] This example provides a method for mass-producing mesenchymal stem cells using induced pluripotent stem cells (iPS), including the following steps:

[0030] S1. Seed the human iPS cell line in a culture dish coated with Matrigel at an inoculation density of 3×10 4 cells / cm 2 , culture in a 38°C, 5% CO2 cell incubator. The culture medium is mTeSR™1 complete stem cell medium. Change the medium daily and passage every 6 - 7 days for 4 - 6 passages to obtain passaged cells;

[0031] S2. Add 10 g of silica nanospheres with an average particle size of 700 nm to water, add 3.24 g of ferric chloride and 1.26 g of ferrous chloride. Under nitrogen protection, dropwise add 4 g of ammonia water, heat to 80°C, and stir and react for 5 h to prepare magnetic nanospheres;

[0032] S3. Seed the passaged cells in mTeSR™1 complete stem cell medium at an inoculation density of 2×10 6 cells / mL, add 10 wt% of the magnetic nanospheres based on the total mass of the system, stir and mix evenly, and culture in a 38°C, 5% CO2 cell incubator for 7 d to prepare nanospheres adsorbed with mesenchymal stem cells.

[0033] Example 3

[0034] This example provides a method for large-scale production of mesenchymal stem cells using pluripotent stem cell iPS, including the following steps:

[0035] S1. Seed the human iPS cell line in a culture dish coated with Matrigel at an inoculation density of 2.5×10 4 cells / cm 2 , culture in a 37°C, 5% CO2 cell incubator. The culture medium is mTeSR™1 complete stem cell medium. Change the medium daily and passage every 6 - 7 days for 4 - 6 passages to obtain passaged cells;

[0036] S2. Add 10 g of silica nanospheres with an average particle size of 600 nm to water, add 3.24 g of ferric chloride and 1.26 g of ferrous chloride. Under nitrogen protection, dropwise add 3 g of ammonia water, heat to 75°C, and stir and react for 3 - 5 h to prepare magnetic nanospheres;

[0037] S3. Seed the passaged cells in mTeSR™1 complete stem cell medium at an inoculation density of 1.5×10 6 cells / mL, add 10 wt% of the magnetic nanospheres based on the total mass of the system, stir and mix evenly, and culture in a 37°C, 5% CO2 cell incubator for 6 d to prepare nanospheres adsorbed with mesenchymal stem cells.

[0038] Comparative Example 1

[0039] Compared with Example 3, the difference lies in that step S2 is not carried out, and in step S3, the magnetic nanospheres are replaced by an equal amount of silica nanospheres.

[0040] Comparative Example 2

[0041] Compared with Example 3, the difference lies in that steps S2 and S3 are not carried out.

[0042] Test Example 1

[0043] A rat tendon disease model was constructed by injecting type I collagenase. Before modeling, the rats were separately caged and adaptively raised for 1 week, fed freely, and randomly divided into 7 groups with 10 rats in each group. Normal group: injected with an equal amount of normal saline; Tendon disease group: a tendon disease model was established by injecting type I collagenase; Example 1 - 3 and Comparative Example 1 - 2 groups: a tendon disease model was established by injecting type I collagenase, and the corresponding prepared products (50 mg / L) were injected. The rats were anesthetized with 3% sodium pentobarbital (1 mL / kg), the Achilles tendon of the right hind limb of SD rats was located, and 50 μL of type I collagenase (2 mg / mL, injected once every 3 days for a total of 5 times) was injected with a microinjector. Two weeks later, the tendon disease model was successfully established, and then stem cell transplantation treatment was given with a microinjector, with a total of 1 injection.

[0044] After 4 weeks of treatment, a small animal gait analyzer (CatWalk) was used to detect the functional recovery of the rats. Under light - avoiding conditions, the rats adapted to the surrounding environment. The rats were placed on the runway of the small animal gait analyzer with pre - set gait parameters. When the rats passed through the fixed detection area runway at a uniform speed, the gait characteristics during the process of passing through the runway could be detected based on the pressure generated by the four limbs of the rats. The results are shown in Table 1.

[0045] Table 1

[0046] Group Standing time (s) <![CDATA[Contact area (mm 2 ) <!-- 3 -->]]> Normal group 0.43±0.19 221±14 Model group 0.25±0.12 134±5 Example 1 0.35±0.14 178±7 Example 2 0.37±0.15 181±9 Example 3 0.38±0.11 184±10 Comparative example 1 0.30±0.10 167±8 Comparative example 2 0.28±0.09 159±6

[0047] As can be seen from the above table, the nanospheres for adsorbing mesenchymal stem cells prepared in Examples 1 - 3 of the present invention have a good effect on repairing chronic tendon disease.

[0048] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for large-scale production of mesenchymal stem cells using pluripotent stem cells (iPS), characterized in that: The following steps are involved: S1. The human iPS cell line was inoculated in a culture dish covered with matrix gel, cultured, and the medium was changed every day. The cells were subcultured once every 6-7 days, and the subcultured cells were obtained after 4-6 subcultures. The inoculation amount of the human iPS cell line was 2-3×10 4 Cells / cm 2 ; The culture conditions are 36-38°C, 5% CO2 cell culture incubator; the culture medium is mTeSRTM1 stem cell complete culture medium; S2. Add silica nanospheres to water, add ferric chloride and ferrous chloride, add ammonia water dropwise under inert gas protection, heat and stir to react, and obtain magnetic nanospheres; the mass ratio of the silica nanospheres, ferric chloride, ferrous chloride, and ammonia water is 10:3.24:1.26:(2-4); the average particle size of the silica nanospheres is 500-700nm; the temperature of the heating and stirring reaction is 70-80°C, and the time is 3-5h; S3. The passaged cells are inoculated into the culture medium, and the magnetic nanospheres are added, stirred and mixed evenly, and cultured to obtain nanospheres adsorbing mesenchymal stem cells; the inoculation amount is 1-2×10 6 cells / mL; the culture conditions are 36-38°C, 5% CO2 cell culture incubator, and the culture time is 5-7 days.

2. Use of the large-scale production of mesenchymal stem cells obtained by the method according to claim 1 in the preparation of drugs for repairing chronic tendinopathy.