CN117327647B - A method for culturing high-purity Muse cells - Google Patents

Abstract

The invention belongs to the field of cell culture, and particularly relates to a culture method of high-purity Muse cells. The culture method comprises the following steps: s1, incubating mesenchymal stem cells with SSEA3, centrifuging to remove supernatant, adding immunomagnetic beads for incubation, and magnetically separating to obtain Muse cells; s2, culturing the Muse cells obtained in the step S1 and the microcarriers in a culture medium at a speed of 40-60 rpm, digesting and passaging by using pancreatin or protease, and adding the culture medium and the microcarriers; s3, centrifugally washing Muse cells cultured to 3-6 generations, detecting SSEA3 and CD105 phenotypes, counting cells and freezing. The invention combines magnetic separation and microcarrier suspension culture, namely improves the purity of the Muse cells from the beginning through the magnetic separation, organically combines 3D balling culture and micro surface adherence culture through the microcarrier, can obtain more than 10 times of amplification only by about ten days, has the purity of more than 80 percent, and can obtain the high-purity Muse cells with clinical orders of magnitude in a short time.

Description

A method for culturing high-purity Muse cells

Technical field

The invention belongs to the field of cell culture, and specifically relates to a method for cultivating high-purity Muse cells.

Background technique

The information in this Background section is disclosed solely for the purpose of increasing understanding of the general background of the invention and is not necessarily considered to be an admission or in any way implying that the information constitutes prior art that is already known to a person of ordinary skill in the art.

Muse cells (multilineage differentiating stress enduring cells) are a new type of human pluripotent stem cells discovered in 2010 by Professor Mari Dezawa of Tohoku University and others. It is present in the blood, bone marrow and connective tissues of various organs. It expresses SSEA3 and accounts for 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: they can differentiate into various cells of endoderm (such as lung, liver, pancreas), mesoderm (such as heart, kidney, bone, blood vessels) and ectoderm (such as nervous tissue and epidermis). Muse cells express sphingosine-1-phosphate (S1P) receptors, which are abundantly produced by damaged cells, prompting Muse cells to selectively home to damaged tissue. Homing Muse cells spontaneously differentiate into constituent cells of multiple tissues in situ, replacing damaged or apoptotic cells, thus promoting tissue repair. Muse cells have a special immunomodulatory system and highly express HLA-G. This feature allows allogeneic Muse cells to be administered directly without HLA matching or immunosuppressive treatment. At present, allogeneic Muse cells have been used in clinical trials through intravenous injection in the following diseases: myocardial infarction, stroke, epidermolysis bullosa, spinal cord injury, perinatal hypoxic-ischemic encephalopathy and amyotrophic disease. Lateral sclerosis. Muse cells are expected to break through the limitations of existing cells in the treatment of neurological diseases, such as amyotrophic lateral sclerosis and spinal cord injury.

The content of Muse cells in normal tissues is extremely low, accounting for only 0.03% of bone marrow mononuclear cells and 1-5% of mesenchymal stem cells (MSC). Muse cells can easily differentiate into other cells. Therefore, in order to realize the industrialization of Muse cells, large-scale expansion and culture must be achieved. Classic Muse cell culture uses long-term trypsinization (LTT) to digest MSCs, then kills the non-Muse cells, and then increases the number of Muse cells through repeated spheroid culture and adherent culture. Muse cells almost do not proliferate. Although Muse cells proliferate during adhesion, most cells differentiate, resulting in very low purity. Repeating the LTT-spheroidization-adhesion process repeatedly can theoretically achieve the purpose of obtaining high-purity Muse. However, this method is complex to operate, has a long cycle, and cannot obtain a large number of high-purity Muse cells in a short period of time. Each culture cycle of this method takes 9 days, and each cycle can increase the purity by approximately 5%. According to theoretical calculation, starting from MSC culture with 1% purity, it will take 144 days to reach 80% purity. There are also methods to first obtain high-purity Muse cells through flow sorting, and then culture them according to the classic method. However, even the MSCs with a higher initial Muse cell content are only about 2%, which results in a longer flow sorting time. Too long increases voltage damage to cells. Therefore, the viability of Muse cells passed through flow cytometry is too low, less than 30%, making subsequent amplification difficult.

Contents of the invention

In order to solve the deficiencies of the existing technology, the purpose of the present invention is to provide a method for cultivating high-purity Muse cells. This invention combines magnetic sorting and microcarrier suspension culture. That is, through magnetic sorting, a very gentle sorting method, the purity of Muse cells is improved from the beginning, and through microcarriers, 3D spheroid culture and microsurface adhesion are achieved. By culturing organically, it only takes about ten days to obtain more than 10-fold amplification, with a purity greater than 80%. High-purity Muse cells of clinical magnitude can be obtained in a short time.

In order to achieve the above objects, the present invention is achieved through the following technical solutions:

In a first aspect, the present invention provides a method for cultivating high-purity Muse cells, which includes the following steps:

S1. Incubate mesenchymal stem cells with SSEA3 primary antibody, centrifuge to remove the supernatant, add immunomagnetic beads for incubation, and magnetically sort to obtain Muse cells;

S2. Cultivate the Muse cells and microcarriers obtained in step S1 in the culture medium with stirring at a rate of 40-60 rpm, use trypsin or protease to digest and passage, and add the culture medium and microcarriers;

S3. Centrifuge and wash the Muse cells cultured to passage 3-6, detect SSEA3 and CD105 phenotypes, count the cells, and freeze them.

Preferably, in step S1, the incubation time of mesenchymal stem cells and SSEA3 primary antibody is 25-35 minutes.

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

Preferably, in step S1, a magnetic frame is used for sorting and a flow cytometer is used to detect that Muse cells are positive for SSEA3 and CD105.

Preferably, the microcarriers include but are not limited to at least one of polystyrene microcarriers, PHEMA microcarriers, gelatin microcarriers, chitin microcarriers, polyurethane foam microcarriers and alginate gel microcarriers.

Preferably, the starting 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.

Preferably, trypsin or protease digestion is used for passage every 3-4 days.

Preferably, the purity of Muse cells obtained by culture is greater than 80%.

In a second aspect, the present invention provides a high-purity Muse cell, which is obtained by the preparation method described in the first aspect.

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

1. Through magnetic sorting, Muse cells with a purity higher than 90% are obtained from MSCs, which avoids damage to cells by flow sorting and also solves the difficulty of obtaining high-purity Muse using the traditional LTT method. The magnetic sorting method has obvious advantages and high recovery efficiency. It only takes about 30 minutes, the purity is more than 90%, and the activity rate is more than 90%. The LTT in the classic method takes 4 hours and can only increase the purity of Muse cells to about 5%, and It causes great damage to cells. Although the flow sorting method can achieve a purity of more than 90%, the initial concentration of Muse is too low and the sorting time is long, which causes too much damage to the cells. The cell viability after recovery is lower than 30%.

2. Through microcarrier suspension culture, the organic combination of 3D spheroid culture and micro-surface adherent culture is achieved, which solves the problem that Muse spheroid culture does not proliferate and adherent culture mostly differentiates. Through 3D stirring 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, and finally able to obtain the results in about 10 days that the classic method took 144 days to achieve. 80% purity, and due to the reduction of the total culture volume, the culture cost is greatly reduced. The suspension culture method based on microcarriers is significantly better than the LTT-spheroidization-adhesion method. a) The process is simple and the operation difficulty is small. When scaling up the culture, the work efficiency can be increased by at least 10 times; b) The time is short and the classic method Each cycle is 9 days, while this method has 3-4 days per cycle. The theoretical time required by the classic method to achieve 80% purity is about 12 times that of this method. In actual production, cell viability may not be able to support such a long period of culture.

Description of drawings

The description and drawings that constitute a part of the present invention are used to provide a further understanding of the present invention. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

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

Figure 2 is a graph showing changes in the purity of Muse cells with culture time in Example 1 and Comparative Examples 1-4.

Detailed ways

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

Example 1

1) Magnetic sorting

Incubate MSC cells with SSEA3 primary antibody for 30 minutes, centrifuge to remove the supernatant, remove unbound primary antibody, add immunomagnetic beads and incubate for 15 minutes, put on a magnetic stand to sort SSEA3 positive cells (i.e. Muse cells), and detect with flow cytometry SSEA3 and CD105 were both positive, and the cells were counted.

2) Suspension culture of Muse cells:

Add the magnetically separated Muse cells and gelatin microcarriers into a breathable culture bottle with a built-in impeller containing culture medium and culture with gentle stirring. The starting concentration of Muse cells is 10,000 cells/ml, the microcarriers are 2g/L, and 50 rpm. Digest and passage with trypsin or protease every 3-4 days, and increase culture medium and microcarriers proportionally.

3) Muse cell harvesting

Muse cells cultured to passage 3-6 were digested with trypsin or protease, washed by centrifugation at 200g, and SSEA3 and CD105 phenotypes were detected by flow cytometry. The cells were counted and frozen at 5*10 ⁶ /ml.

Comparative example 1

1) Magnetic separation is the same as in Example 1.

2) Culture of Muse cells (muse-3D-static):

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

3) Muse cell harvesting is the same as in Example 1.

Comparative example 2

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

Unsorted MSC cells (the purity of Muse cells is 1-5%) are digested with trypsin for 4 hours, then added to a low-adsorption culture flask and cultured for 6 days. The cells aggregate into pellets, and then trypsinized After digestion into single cells, they are inoculated into ordinary culture bottles for adherent culture for 3 days, then LTT, then formed into balls, and then adhered again, repeating this for multiple cycles.

2) Muse cell harvesting is the same as in Example 1.

Comparative example 3

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

Unsorted MSC cells (the purity of Muse cells is 1-5%) are directly added to the low-adsorption culture flask without LTT and cultured for 6 days. The cells aggregate into pellets and are then digested into single cells with trypsin. Then inoculate into ordinary culture bottles for adhesion culture for 3 days, then form into balls, and then adhere to the wall. Repeat multiple cycles.

2) Muse cell harvesting is the same as in Example 1.

Comparative example 4

1) Magnetic separation is the same as in Example 1.

2) Culture of Muse cells (Muse-2D):

The Muse cells after magnetic separation are directly inoculated into ordinary culture bottles for adherent culture. After reaching full growth, they are passaged at 10,000/ ^cm2 and repeated for multiple cycles.

3) Muse cell harvesting is the same as in Example 1.

Comparative example 5

1) Magnetic separation is the same as in Example 1

2) Culture of Muse cells (Muse-classical)

Add the sorted muse cells (purity is higher after magnetic separation, no need for LTT in the first cycle) into a low-adsorption culture flask and culture for 6 days. The cells aggregate into small balls and are then digested into single cells with trypsin. Inoculate into ordinary culture bottles and adhere to the culture for 3 days, then LTT, then form into balls, and then adhere to the wall. Repeat for multiple cycles.

3) Muse cell harvesting is the same as in Example 1.

As shown in Figure 1, 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 in 12 days, and 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 spheroid culture, and Muse cells did not proliferate during this process. In Comparative Example 4, traditional adherent culture was used after magnetic sorting, but the culture efficiency was low, and only 2-3×10 ⁶ cells could be obtained in 12 days. Although Comparative Example 5 underwent magnetic sorting first, the culture efficiency of repeated LTT-spheroidization-adherence process under the same initial Muse level was still not as good as that of Example 1.

As shown in Figure 2, in Example 1, Muse with a purity close to 100% can be obtained through magnetic sorting, and the use of microcarrier suspension culture avoids large-scale differentiation of Muse, resulting in a significant decrease in its purity, allowing the purity of Muse cells to be maintained. At more than 90%, it is much higher than the traditional 2D culture method and LTT-spheroid-adhesion method. In Comparative Example 1, Muse cells with a purity close to 100% were obtained after magnetic sorting. Although microcarriers were used for culture, the cells did not proliferate or differentiate without stirring, and the purity did not change significantly. In Comparative Example 4, the initial purity of Muse after magnetic separation can be close to 100%, but the differentiation of Muse during the adherent culture process leads to a significant reduction in purification. Although Comparative Example 5 was first subjected to magnetic sorting, repeating the LTT-spheroidization-adhesion process caused a large number of Muse cells to differentiate, resulting in a decrease in the purity of the Muse cells.

Example 2

1) Magnetic sorting

Incubate MSC cells with SSEA3 primary antibody for 25 minutes, centrifuge to remove the supernatant, remove unbound primary antibody, add immunomagnetic beads and incubate for 10 minutes, put on a magnetic stand to sort SSEA3 positive cells (i.e. Muse cells), and detect with flow cytometry SSEA3 and CD105 were both positive, and the cells were counted.

2) Suspension culture of Muse cells:

Add the magnetically separated Muse cells and microcarriers to a culture bottle with a breathable built-in impeller and culture with gentle stirring. The initial concentration of Muse cells is 15,000 cells/ml, microcarriers are 3g/L, and 50 rpm is used. Digest and passage with trypsin or protease every 3-4 days, and increase culture medium and microcarriers proportionally.

3) Muse cell harvesting

Muse cells cultured to passage 3-6 were digested with trypsin or protease, washed by centrifugation at 200g, and SSEA3 and CD105 phenotypes were detected by flow cytometry. The cells were counted and frozen at 5*10 ⁶ /ml.

Example 3

1) Magnetic sorting

Incubate MSC cells with SSEA3 primary antibody for 35 minutes, centrifuge to remove the supernatant, remove unbound primary antibody, add immunomagnetic beads and incubate for 20 minutes, put on a magnetic stand to sort SSEA3 positive cells (i.e. Muse cells), and detect with flow cytometry SSEA3 and CD105 were both positive, and the cells were counted.

2) Suspension culture of Muse cells:

Add the magnetically separated Muse cells and microcarriers to a culture bottle with a breathable built-in impeller and culture with gentle stirring. The initial concentration of Muse cells is 20,000 cells/ml, microcarriers are 4g/L, and 50 rpm is used. Digest and passage with trypsin or protease every 3-4 days, and increase culture medium and microcarriers proportionally.

3) Muse cell harvesting

Muse cells cultured to passage 3-6 were digested with trypsin or protease, washed by centrifugation at 200g, and SSEA3 and CD105 phenotypes were detected by flow cytometry. The cells were counted and frozen at 5*10 ⁶ /ml.

The above descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims (5)

1. A method for culturing high purity Muse cells, comprising the steps of:

s1, incubating mesenchymal stem cells with SSEA3, centrifuging to remove supernatant, adding immunomagnetic beads for incubation, and magnetically separating to obtain Muse cells;

s2, culturing the Muse cells obtained in the step S1 and the gelatin microcarrier in a culture medium at a speed of 40-60 rpm, digesting and passaging by using pancreatin or protease, and adding the culture medium and the gelatin microcarrier;

s3, centrifugally washing Muse cells cultured to 3-6 generations, detecting SSEA3 and CD105 phenotypes, counting cells, and freezing;

the initial concentration of the Muse cells in the step S2 is 10000-20000/mL, and the ratio of the Muse cells to the gelatin microcarrier is 4000-6000:1 mg;

the purity of the Muse cells obtained by culture is more than 80 percent.

2. The method of claim 1, wherein in step S1, the mesenchymal stem cells are incubated with SSEA3 for a period of 25-35 minutes.

3. The method according to claim 1, wherein in step S1, the immunomagnetic beads are added and incubated for 10 to 20 minutes.

4. The culture method according to claim 1, wherein in step S1, both sse cells SSEA3 and CD105 are detected positive using a flow cytometer by sorting using a magnetic rack.

5. The culture method according to claim 1, wherein the passage is digested with pancreatin or protease every 3 to 4 days.