Culture method for high-purity muse cells

Abstract

Provided is a culture method for high-purity Muse cells. The culture method comprises the following steps: S1, incubating mesenchymal stem cells with an SSEA3 primary antibody, performing centrifugation, removing a supernatant, adding immunomagnetic beads for incubation, and performing magnetic separation to obtain Muse cells; S2, culturing the Muse cells obtained in step S1 and a microcarrier in a culture medium under stirring at a rate of 40-60 r / min, performing digestion and passaging by means of using a pancreatic enzyme or protease, and replenishing the culture medium and the microcarrier; and S3, centrifugally washing the Muse cells cultured to third to sixth generations, detecting SSEA3 and CD105 phenotypes, performing cell counting, and performing cryopreservation. By means of combining the magnetic separation and microcarrier suspension culture, i.e. using the magnetic separation to improve the purity of Muse cells at the beginning, and then using the microcarrier to organically combine 3D spheroid culture and microsurface adherent culture, the present invention achieves amplification of 10 folds or more simple in about ten days, and the purity is higher than 80%. Therefore, the present invention can obtain high-purity Muse cells of clinical order of magnitudes in short time.

Description

A method for culturing high-purity Muse cells Technical Field

[0001] The present invention belongs to the field of cell culture, and particularly relates to a method for culturing high-purity Muse cells. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Muse cells (multilineage differentiating stress enduring cells) are a new type of human pluripotent stem cell discovered in 2010 by Professor Mari Dezawa and others at Tohoku University in Japan. They are found in the blood, bone marrow, and connective tissues of various organs. They express SSEA3 and comprise 0.03% of bone marrow mononuclear cells and 1-5% of mesenchymal stem cells. Muse cells have the ability to differentiate into three germ layers: endoderm (such as the lung, liver, and pancreas), mesoderm (such as the heart, kidney, bone, and blood vessels), and ectoderm (such as neural tissue and epidermis). Muse cells express receptors for sphingosine-1-phosphate (S1P), which is produced in large quantities by damaged cells, prompting them to selectively home to damaged tissues. Once homed, Muse cells spontaneously differentiate into multiple tissue components in situ, replacing damaged or apoptotic cells and promoting tissue repair. Muse cells possess a unique immune regulatory system and highly express HLA-G. This property allows allogeneic Muse cells to be administered directly without the need for HLA matching or immunosuppressive treatment. Currently, allogeneic Muse cells have been used in clinical trials via intravenous injection for the following diseases: myocardial infarction, stroke, epidermolysis bullosa, spinal cord injury, perinatal hypoxic-ischemic encephalopathy, and amyotrophic lateral sclerosis. Muse cells hold promise for overcoming the limitations of existing cell-based treatments for neurological diseases such as amyotrophic lateral sclerosis and spinal cord injury.

[0004] Muse cells are present in extremely low concentrations in normal tissues, constituting only 0.03% of bone marrow mononuclear cells and 1-5% of mesenchymal stem cells (MSCs). Furthermore, Muse cells readily differentiate into other cell types. Therefore, large-scale expansion and culture are essential for the industrialization of Muse cells. Classic Muse cell culture involves long-time trypsinization (LTT) of MSCs, followed by killing of non-Muse cells. The number of Muse cells is then increased through repeated sphere formation and adherence culture. During sphere formation, Muse cells barely proliferate. While they proliferate during adherence, most cells differentiate, resulting in very low purity. Repeating the LTT-sphere formation-adhesion process theoretically allows for the production of high-purity Muse cells. However, this method is complex and time-consuming, making it difficult to obtain large quantities of highly pure Muse cells in a short period of time. Each culture cycle requires nine days, with each cycle increasing purity by approximately 5%. Theoretically, starting with MSCs with 1% purity, it would take 144 days to reach 80% purity. There is also a method to obtain high-purity Muse cells through flow sorting and then culture them according to the classic method. However, even in MSCs with a high initial Muse cell content, only about 2% is present. This results in a long flow sorting time and increased voltage damage to the cells. Therefore, the viability of Muse cells sorted by flow cytometry is too low, less than 30%, making it difficult to carry out subsequent expansion.

[0005] Summary of the Invention

[0006] To address the deficiencies of the prior art, the present invention aims to provide a method for culturing high-purity Muse cells. This invention combines magnetic sorting with microcarrier suspension culture. This method, which improves the purity of Muse cells from the outset through a very gentle sorting method called magnetic sorting, organically combines 3D spheroid culture with microsurface adherent culture through microcarriers. This method achieves a 10-fold or greater expansion in just ten days, with a purity greater than 80%, enabling the acquisition of clinical-grade, high-purity Muse cells in a short period of time.

[0007] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0008] In a first aspect, the present invention provides a method for culturing high-purity Muse cells, comprising the following steps:

[0009] S1. Incubate mesenchymal stem cells with SSEA3 primary antibody, remove supernatant by centrifugation, add immunomagnetic beads for incubation, and magnetically separate to obtain Muse cells;

[0010] S2. The Muse cells obtained in step S1 and the microcarriers are cultured in a culture medium at a stirring rate of 40-60 rpm, digested and passaged using trypsin or protease, and culture medium and microcarriers are added;

[0011] S3. The Muse cells cultured to passages 3-6 were centrifuged and washed, and their SSEA3 and CD105 phenotypes were detected. The cells were counted and frozen.

[0012] Preferably, in step S1, the mesenchymal stem cells are incubated with the SSEA3 primary antibody for 25-35 minutes.

[0013] Preferably, in step S1, immunomagnetic beads are added and incubated for 10-20 minutes.

[0014] Preferably, in step S1, the Muse cells are sorted using a magnetic rack and detected by flow cytometry to be positive for SSEA3 and CD105.

[0015] Preferably, the microcarrier includes but is not limited to at least one of polystyrene microcarriers, PHEMA microcarriers, gelatin microcarriers, chitin microcarriers, polyurethane foam microcarriers and alginate gel microcarriers.

[0016] Preferably, the initial concentration of Muse cells is 10,000-20,000 cells / mL, and the ratio of Muse cells to microcarriers is 4,000-6,000 cells:1 mg.

[0017] Preferably, cells are passaged every 3-4 days using trypsin or protease digestion.

[0018] Preferably, the purity of the cultured Muse cells is greater than 80%.

[0019] In a second aspect, the present invention provides a high-purity Muse cell obtained by the preparation method as described in the first aspect.

[0020] The beneficial effects achieved by one or more technical solutions of the present invention are as follows:

[0021] 1. Through magnetic separation, Muse cells with a purity exceeding 90% were obtained from MSCs, avoiding the damage to cells caused by flow cytometry. This also solved the difficulty of obtaining high-purity Muse cells by the traditional LTT method. The magnetic separation method has obvious advantages, such as high recovery efficiency, and only takes about 30 minutes to achieve a purity of over 90% and a viability of over 90%. The classic LTT method requires 4 hours and can only increase the purity of Muse cells to about 5%, and it also causes greater damage to the cells. Although the flow cytometry method can achieve a purity of over 90%, due to the low initial concentration of Muse, the separation time is long, and the damage to the cells is too great, the viability of the recovered cells is less than 30%.

[0022] 2. Through microcarrier suspension culture, an organic combination of 3D spheroid culture and microsurface adherent culture is achieved, solving the problem of Muse spheroid culture not proliferating and adherent culture mostly differentiating. Through 3D stirred suspension culture, the nutrient exchange efficiency of the cell spheroids is greatly improved, thereby greatly shortening the culture time of each cycle from 9 days to 3-4 days. Ultimately, it is possible to obtain 80% purity in about 10 days, which is achievable with the classic method of 144 days. Due to the reduction in the total culture volume, the culture cost is greatly reduced. The microcarrier-based suspension culture method is significantly superior to the LTT-spheroid-adherent method. a) The process is simple and the operation is easy. When the culture is scaled up, the work efficiency can be increased by at least 10 times. b) The time is short. The classic method takes 9 days per cycle, while this method takes 3-4 days per cycle. The theoretical time required to achieve 80% purity with the classic method is about 12 times that of this method. In actual production, cell viability may not be able to support such a long culture period. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0024] Figure 1 is a graph showing changes in the number of Muse cells over culture time in Example 1 and Comparative Examples 1-4;

[0025] FIG2 is a graph showing changes in the purity of Muse cells over culture time in Example 1 and Comparative Examples 1-4. DETAILED DESCRIPTION

[0026] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples.

[0027] Example 1

[0028] 1) Magnetic separation

[0029] MSC cells were incubated with SSEA3 primary antibody for 30 minutes, centrifuged to remove the supernatant, unbound primary antibody was removed, immunomagnetic beads were added and incubated for 15 minutes, SSEA3-positive cells (i.e., Muse cells) were sorted on a magnetic stand, and SSEA3 and CD105 were detected by flow cytometry. The cells were counted.

[0030] 2) Suspension culture of Muse cells:

[0031] Add the magnetically separated Muse cells and gelatin microcarriers to a ventilated culture flask containing culture medium and gently stir with an impeller. Culture at a starting concentration of 10,000 Muse cells / ml and 2 g / L microcarriers at 50 rpm. Subculture with trypsin or protease every 3-4 days, increasing the culture medium and microcarrier volume proportionally.

[0032] 3) Muse cell harvest

[0033] Muse cells cultured to passage 3-6 were digested with trypsin or protease, washed by centrifugation at 200 g, and the phenotypes of SSEA3 and CD105 were detected by flow cytometry. Cell count was performed according to 5*10 6 / ml frozen.

[0034] Comparative Example 1

[0035] 1) Magnetic separation is the same as in Example 1.

[0036] 2) Cultivation of Muse cells (muse-3D-static):

[0037] Add the magnetically separated Muse cells and gelatin microcarriers to a low-adsorption culture flask for culture. The starting concentration is 10,000 Muse cells / ml and 2 g / L microcarriers. Digest and passage the cells every 3-4 days using trypsin or protease, increasing the culture medium and microcarriers proportionally.

[0038] 3) Muse cell harvesting was the same as in Example 1.

[0039] Comparative Example 2

[0040] 1) Culture of Muse cells (msc-LTT-classical):

[0041] Unsorted MSC cells (Muse cell purity of 1-5%) were trypsinized for 4 hours and then added to low-adsorption culture flasks for 6 days. The cells aggregated into small balls, then trypsinized into single cells and inoculated into ordinary culture flasks for adherent culture for 3 days. Then, LTT, re-balling, and re-adherence were repeated for multiple cycles.

[0042] 2) Muse cell harvesting was the same as in Example 1.

[0043] Comparative Example 3

[0044] 1) Culture of Muse cells (msc-no-LTT-classical):

[0045] Unsorted MSC cells (Muse cell purity of 1-5%) were directly added to low-adsorption culture flasks and cultured for 6 days without LTT. The cells aggregated into small balls, then digested with trypsin into single cells and inoculated into ordinary culture flasks for adherent culture for 3 days, then formed into balls and adhered again, and this cycle was repeated multiple times.

[0046] 2) Muse cell harvesting was the same as in Example 1.

[0047] Comparative Example 4

[0048] 1) Magnetic separation is the same as in Example 1.

[0049] 2) Culture of Muse cells (Muse-2D):

[0050] The magnetically separated Muse cells were directly inoculated into ordinary culture bottles for adherent culture. After they were fully grown, the cells were cultured at a rate of 10,000 cells / cm 2 Repeat this process for multiple cycles.

[0051] 3) Muse cell harvesting was the same as in Example 1.

[0052] Comparative Example 5

[0053] 1) Magnetic separation is the same as in Example 1

[0054] 2) Cultivation of Muse Cells (Muse-classical)

[0055] The sorted muse cells (high purity after magnetic separation, no LTT is required in the first cycle) are added to low-adsorption culture flasks and cultured for 6 days. The cells aggregate into small balls, then digested with trypsin into single cells and inoculated into ordinary culture flasks for adherent culture for 3 days. Then LTT, re-balling, and re-adherence are repeated for multiple cycles.

[0056] 3) Muse cell harvesting was the same as in Example 1.

[0057] As shown in FIG1 , when the initial number of Muse cells is the same, compared with Comparative Examples 1-4, the culture method of Example 1 can culture Muse cells to 5×10 6 The culture efficiency is much higher than other methods. Although microcarriers were used for culture in Comparative Example 1, the state of Muse cells in a static environment without stirring was close to that of cells in spherical culture, and Muse cells did not proliferate during this process. In Comparative Example 4, traditional adherent culture was used after magnetic separation, and the culture efficiency was low. Only 2-3×10 6 Although the cells in Comparative Example 5 were first subjected to magnetic separation, the culture efficiency of the repeated LTT-sphere formation-adhesion process at the same initial Muse level was still not as good as that of Example 1.

[0058] As shown in Figure 2, in Example 1, Muse with a purity close to 100% can be obtained by magnetic sorting, and the use of microcarrier suspension culture avoids the large-scale differentiation of Muse, which causes a significant decrease in its purity, so that the purity of Muse cells can be maintained at above 90%, which is much higher than the traditional 2D culture method and LTT-spheroidization-adhesion method. In Comparative Example 1, Muse cells with a purity close to 100% were obtained after magnetic sorting. Although microcarrier culture was used, the cells did not proliferate or differentiate in the absence of stirring, and the purity did not change significantly. In Comparative Example 4, the initial purity of Muse after magnetic sorting was close to 100%, but the differentiation of Muse during the adherent culture process caused a significant decrease in purification. Although Comparative Example 5 was first subjected to magnetic sorting, the repeated LTT-spheroidization-adhesion process caused the Muse cells to differentiate in large quantities, resulting in a decrease in the purity of Muse cells.

[0059] Example 2

[0060] 1) Magnetic separation

[0061] MSC cells were incubated with SSEA3 primary antibody for 25 minutes, centrifuged to remove the supernatant, unbound primary antibody was removed, immunomagnetic beads were added and incubated for 10 minutes, SSEA3-positive cells (i.e., Muse cells) were sorted on a magnetic stand, and SSEA3 and CD105 were detected by flow cytometry. The cells were counted.

[0062] 2) Suspension culture of Muse cells:

[0063] Add the magnetically separated Muse cells and microcarriers to a ventilated culture flask with a built-in impeller and gently stir and culture at a starting concentration of 15,000 Muse cells / ml and 3g / L microcarriers at 50 rpm. Subculture with trypsin or protease every 3-4 days, increasing the culture medium and microcarriers proportionally.

[0064] 3) Muse cell harvest

[0065] Muse cells cultured to passage 3-6 were digested with trypsin or protease, washed by centrifugation at 200 g, and the phenotypes of SSEA3 and CD105 were detected by flow cytometry. Cell count was performed according to 5*10 6 / ml frozen.

[0066] Example 3

[0067] 1) Magnetic separation

[0068] MSC cells were incubated with SSEA3 primary antibody for 35 minutes, centrifuged to remove the supernatant, unbound primary antibody was removed, immunomagnetic beads were added and incubated for 20 minutes, SSEA3-positive cells (i.e., Muse cells) were sorted on a magnetic stand, and SSEA3 and CD105 were detected by flow cytometry. The cells were counted.

[0069] 2) Suspension culture of Muse cells:

[0070] Add the magnetically separated Muse cells and microcarriers to a ventilated culture flask with a built-in impeller and gently stir and culture at a starting concentration of 20,000 Muse cells / ml and 4g / L microcarriers at 50 rpm. Subculture with trypsin or protease every 3-4 days, increasing the culture medium and microcarriers proportionally.

[0071] 3) Muse cell harvest

[0072] Muse cells cultured to passage 3-6 were digested with trypsin or protease, washed by centrifugation at 200 g, and the phenotypes of SSEA3 and CD105 were detected by flow cytometry. Cell count was performed according to 5*10 6 / ml frozen.

[0073] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for culturing high-purity Muse cells, characterized in that: The following steps are involved: S1. Incubate mesenchymal stem cells with SSEA3 primary antibody, remove supernatant by centrifugation, add immunomagnetic beads for incubation, and obtain Muse cells by magnetic sorting; S2, the Muse cells obtained in step S1 and the microcarriers are cultured in a culture medium at a stirring rate of 40-60 rpm, digested and passaged using trypsin or protease, and the culture medium and microcarriers are added; S3. The Muse cells cultured to passages 3-6 were centrifuged and washed, and the SSEA3 and CD105 phenotypes were detected. The cells were counted and frozen.

2. The culture method according to claim 1, characterized in that In step S1, the mesenchymal stem cells are incubated with the SSEA3 primary antibody for 25-35 minutes.

3. The culture method according to claim 1, characterized in that In step S1, immunomagnetic beads are added and incubated for 10-20 minutes.

4. The culture method according to claim 1, characterized in that In step S1, the Muse cells were sorted using a magnetic rack and detected by flow cytometry, and both SSEA3 and CD105 were positive.

5. The culture method according to claim 1, characterized in that The microcarrier comprises at least one of a gelatin microcarrier, a polystyrene microcarrier, a PHEMA microcarrier, a chitin microcarrier, a polyurethane foam microcarrier and an alginate gel microcarrier.

6. The culture method according to claim 1, characterized in that The starting concentration of Muse cells is 10000-20000 / mL, and the ratio of Muse cells to microcarriers is 4000-6000:1 mg.

7. The culture method according to claim 1, characterized in that Subculture every 3-4 days using trypsin or protease digestion.

8. The culture method according to claim 1, characterized in that The purity of the cultured Muse cells was greater than 80%.

9. A high-purity Muse cell, characterized in that The method is obtained by the preparation method according to any one of claims 1 to 8.

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