Juvenile pig-derived stem cells and preparation method thereof

Abstract

The present invention aims to provide stem cells with excellent proliferation and differentiation capabilities. The present invention relates to stem cells isolated from young pigs and a method for preparing the same.

Description

[Technical field]

[0001] The present invention relates to a young pig-derived stem cell and a preparation method thereof, and more particularly to a young pig-derived mesenchymal stem cell capable of differentiating into adipocytes, osteocytes, and chondrocytes and a preparation method thereof. [Background technology]

[0002] With recent advances in research on somatic stem cells, including mesenchymal stem cells, the clinical application of somatic stem cells has already moved from the basic research stage to the development stage. Somatic stem cells have three major functions (multilineage differentiation ability, immune regulation ability, and extracellular environment remodeling ability), and are expected to be used as therapeutic cells for intractable diseases.

[0003] The first, pluripotency, is the ability of somatic stem cells to directly differentiate into bone, cartilage, etc., and the administered somatic stem cells exert a therapeutic effect by supplementing lost cells or replacing cells with insufficient function. The second immunomodulatory function exerts its therapeutic effect by acting on the patient's immune cells through the secretion of anti-inflammatory cytokines, chemokines, exosomes, etc. from somatic stem cells, or through intercellular adhesion factors, thereby suppressing immune responses such as inflammation and graft-versus-host disease. The third ability, remodeling of the extracellular environment, exerts a therapeutic effect on areas such as infarcted areas in ischemic diseases and areas of fibrosis caused by inflammation by secreting vascular induction factors, growth factors, and anti-fibrotic factors from somatic stem cells.

[0004] Mesenchymal stem cells are present in mammalian bone marrow, fat, pancreatic islets, umbilical cord blood, etc., and are somatic stem cells derived from mesodermal tissue (mesenchyme) and have the ability to differentiate into cells belonging to the mesenchymal system. In recent years, clinical trials have been conducted for diseases such as graft-versus-host disease, cardiovascular disorders, autoimmune diseases, osteoarthritis, osteogenesis imperfecta, liver disorders, respiratory diseases, spinal cord injuries, cerebral infarction, and renal failure (Non-Patent Document 1).

[0005] Mesenchymal stem cells are expected to have various clinical applications, but there are issues such as securing donors, invasiveness to donors, and ensuring safety such as virus-negative testing for each donor. In addition, the efficacy of the obtained mesenchymal stem cells varies greatly depending on conditions such as the donor and their age, and ensuring a stable quality of therapeutic cells is also a major issue. A technology in which mesenchymal stem cells derived from a patient's bone marrow are proliferated outside the body and the same patient is treated using the cells could be an alternative treatment to tissue and organ transplants, which are problematic due to a lack of donors. However, there are individual differences in the proliferation and differentiation capabilities of cells, and cells derived from all patients do not behave in the same way (Non-Patent Document 2). As described above, in order to prepare sufficient stem cells for treatment, it is necessary to secure donors, confirm safety, and ensure excellent proliferation and differentiation capabilities of stem cells. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Lemos NE, de Almeida Brondani L, Dieter C, Rheinheimer J, Boucas AP, Bauermann Leitao C, Crispim D, Bauer AC. Islets. 2017 Jul 5:e1335842. doi: 10.1080 / 19382014.2017.1335842 [Non-Patent Document 2] Hajime Ohgushi, Biochemistry, Vol. 81, No. 2, pp. 99-104, February 2009 Summary of the Invention [Problem to be solved by the invention]

[0007] As described above, when using stem cells for regenerative therapy, it is necessary to secure donors, confirm safety, and ensure excellent proliferation and differentiation capabilities of stem cells, but since the proliferation and differentiation capabilities of stem cells decrease after several passages, there is a problem that it takes time and cost to prepare stem cells sufficient for therapy. Therefore, the object of the present invention is to provide stem cells with excellent proliferation and differentiation capabilities using young medical pigs as a donor source, which allows for a stable supply and control of pathogens. [Means for solving the problem]

[0008] The present inventors discovered that mesenchymal stem cells prepared from the bone marrow of young pigs have the excellent properties of a significantly faster proliferation rate, superior proliferation ability, and smaller cell size compared to conventional mesenchymal stem cells, and thus completed the present invention.

[0009] That is, the present invention relates to the following. 1. Stem cells isolated from young pigs. 2. The stem cell according to 1 above, having an average diameter of 17 μm or less. 3. The stem cell according to 1 or 2 above, having a doubling time during logarithmic growth phase of 36 hours or less. 4. The stem cell according to any one of 1 to 3 above, which is a mesenchymal stem cell. 5. The stem cell according to any one of 1 to 4 above, wherein the young pig is a young pig capable of undergoing cell transplantation into a human. 6. The stem cell according to any one of 1 to 5 above, isolated from bone marrow or pancreatic islet of a young pig. 7. The stem cell according to any one of 1 to 6 above, which is a stem cell for transplantation. 8. The stem cell according to 7 above, which is a stem cell for human transplantation. 9. The stem cell according to any one of 1 to 6 above, which is a stem cell for use as a feeder cell. 10. The stem cell according to 9 above, which is a stem cell to be used as a feeder cell for expanding stem cells for human transplantation. 11. A method for preparing stem cells, comprising isolating cells from a young pig. 12. The method for preparing stem cells according to 11 above, comprising a step of subculturing the stem cells 3 to 12 days after seeding. 13. The method for preparing stem cells according to 11 or 12 above, wherein the cells isolated from the young pig are cells of a mononuclear cell fraction. 14. The method for preparing stem cells according to any one of 11 to 13 above, wherein the isolation of cells from the young pig is isolation of cells from bone marrow or pancreatic islets of the young pig. 15. The method for preparing stem cells according to 13 or 14 above, which comprises a step of freezing the cells of the isolated mononuclear cell fraction. Effect of the Invention

[0010] The stem cells of the present invention have the advantages of having a significantly faster proliferation rate, excellent proliferation ability, and small cell size compared to conventional stem cells. Because the stem cells of the present invention have a significantly faster proliferation rate, it is possible to obtain a large amount of stem cells for use in transplantation, feeder cells, etc. in a short time and at low cost. In addition, although administration of stem cells can cause the stem cells to become stuck in the lungs and cause pulmonary embolism, the stem cells of the present invention have a small cell size and can prevent the formation of such pulmonary embolism. [Brief description of the drawings]

[0011] [Figure 1] Fig. 1A is a diagram showing the total cell mass in a specific culture period (days) when the stem cells of the present invention are cultured. Fig. 1B is a diagram showing the total cell proliferation rate in a specific culture period (days) when the stem cells of the present invention are cultured. In Fig. 1A and Fig. 1B, the dotted line and black circle represent juvenile porcine bone marrow-derived mesenchymal stem cells (npBM-MSCs), and the solid line and white circle represent human bone marrow-derived mesenchymal stem cells (hBM-MSCs). [Diagram 2] 2A and 2B are diagrams showing the differentiation of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and juvenile porcine bone marrow-derived mesenchymal stem cells (npBM-MSCs) into adipocytes, respectively. [Diagram 3] 3A and 3B are diagrams showing the differentiation of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and juvenile porcine bone marrow-derived mesenchymal stem cells (npBM-MSCs) into osteocytes, respectively. [Figure 4]4A and 4B are diagrams showing differentiation of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and juvenile porcine bone marrow-derived mesenchymal stem cells (npBM-MSCs) into osteocytes, respectively. [Diagram 5] 5A and 5B are diagrams showing differentiation of juvenile porcine bone marrow-derived mesenchymal stem cells (npBM-MSCs) into chondrocytes. [Figure 6] FIG. 6 shows the results of cell surface antigen analysis of immature pig bone marrow-derived mesenchymal stem cells (npBM-MSCs) using CD44, a marker for mesenchymal stem cells. [Figure 7] FIG. 7 shows the results of cell surface antigen analysis of immature porcine bone marrow-derived mesenchymal stem cells (npBM-MSCs) using CD90, a marker for mesenchymal stem cells. [Figure 8] Figures 8A to 8D show the results of cell surface antigen analysis of immature porcine islet-derived mesenchymal stem cells (npISLET-MSCs) using CD29, a marker for mesenchymal stem cells. Figure 8A shows the results of sample 11 (not frozen immediately after islet preparation), Figure 8B shows the results of sample 12 (frozen immediately after islet preparation), Figure 8C shows the results of sample 13 (not frozen on the third day of culture after islet preparation), and Figure 8D shows the results of sample 14 (frozen on the third day of culture after islet preparation). [Figure 9] Figures 9A to 9D show the results of cell surface antigen analysis of immature porcine islet-derived mesenchymal stem cells (npISLET-MSCs) using CD44, a mesenchymal stem cell marker. Figure 9A shows the results of sample 11 (not frozen immediately after islet preparation), Figure 9B shows the results of sample 12 (frozen immediately after islet preparation), Figure 9C shows the results of sample 13 (not frozen on the third day of culture after islet preparation), and Figure 9D shows the results of sample 14 (frozen on the third day of culture after islet preparation). [Figure 10]Figures 10A to 10D show the results of cell surface antigen analysis of immature porcine islet-derived mesenchymal stem cells (npISLET-MSCs) using CD90, a marker for mesenchymal stem cells. Figure 10A shows the results of sample 11 (not frozen immediately after islet preparation), Figure 10B shows the results of sample 12 (frozen immediately after islet preparation), Figure 10C shows the results of sample 13 (not frozen on the third day of culture after islet preparation), and Figure 10D shows the results of sample 14 (frozen on the third day of culture after islet preparation). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The stem cells of the present invention are stem cells isolated from young pigs. Note that, although the stem cells described in the Examples below were isolated from bone marrow or pancreatic islets of young pigs, stem cells derived from, for example, skin, fat, etc. of young pigs are also included in the present invention.

[0013] In the present invention, the term "young pig" refers to a pig that is less than one month old from the fetus, preferably less than 25 days old. The young pig is preferably for medical use, and more preferably a young pig that can be used for cell transplantation into humans. The type of pig is not particularly limited, and examples thereof include Landrace (e.g., Danish Landrace, American Landrace, British Landrace, Dutch Landrace, Swedish Landrace), Large Yorkshire, Berkshire, Duroc, Hampshire, Middle Yorkshire, and miniature pigs, with Landrace being preferred.

[0014] Generally, the term "stem cell" refers to an immature cell that has the ability to self-renew and differentiate and proliferate. Stem cells include subpopulations such as pluripotent stem cells, multipotent stem cells, and unipotent stem cells, depending on their differentiation ability.

[0015] Pluripotent stem cells are cells that cannot become an individual by themselves, but have the ability to differentiate into all tissues and cells that make up a living body. Multipotent stem cells are cells that have the ability to differentiate into multiple types of tissues and cells, but not all types. Unipotent stem cells are cells that have the ability to differentiate into specific tissues and cells.

[0016] The stem cells of the present invention are preferably multipotent stem cells, including somatic stem cells such as mesenchymal stem cells, hematopoietic stem cells, neural stem cells, bone marrow stem cells, and germline stem cells, and are preferably mesenchymal stem cells.

[0017] The stem cells of the present invention include primary cultured cells and cells subcultured from the primary cultured cells, which are isolated from young pigs and can generate various cells expressing various differentiation markers. In addition, when the stem cells of the present invention are mesenchymal stem cells, the cell markers CD44 and CD90 are preferably both 60% or more positive, more preferably 70% or more positive, and even more preferably 80% or more positive. In addition, the cell marker CD29 is preferably 60% or more positive, more preferably 70% or more positive, and even more preferably 80% or more positive.

[0018] The doubling time of the stem cells of the present invention during the logarithmic growth phase is preferably 36 hours or less, more preferably 32 hours or less, even more preferably 28 hours or less, particularly preferably 24 hours or less, and most preferably 20 hours or less. Moreover, the doubling time during the logarithmic growth phase is preferably 14 hours or more, and more preferably 16 hours or more.

[0019] The stem cells of the present invention can be cultured in the logarithmic growth phase by, for example, seeding the stem cells of the present invention in a medium (e.g., MSC medium) containing vitamin C described below, and culturing them in a culture incubator in the presence of 5% CO2 at 37° C. The shorter the doubling time in the logarithmic growth phase, the more quickly and inexpensively it becomes possible to prepare a large amount of stem cells.

[0020] The stem cells of the present invention preferably have an average diameter of 17 μm or less, more preferably 16.5 μm or less, even more preferably 16 μm or less, particularly preferably 15.5 μm or less, and most preferably 15 μm or less. The average diameter is preferably 10 μm or more, more preferably 12 μm or more. The smaller the average diameter, the more the formation of pulmonary embolism caused by administration of stem cells can be prevented. The average diameter can be measured, for example, using Nucleo Counter NC-200 (trademark). Here, the average means the arithmetic mean.

[0021] The mesenchymal stem cells of the present invention can be induced to differentiate into adipocytes by culturing the mesenchymal stem cells of the present invention in the presence of, for example, insulin, MCGS (serum component, Mesenchymal Cell Growth Supplement), dexamethasone, indomethacin, isobutylmethylxanthine, etc.

[0022] For differentiation and maintenance into adipocytes, commercially available kits or media may be used, such as hMSC differentiation BulletKit (trademark)-adipogeni (PT-3004) manufactured by Lonza Walkersville, hMSC adipogenic induction medium (PT-3102B) manufactured by Lonza Walkersville, hMSC adipogenic maintenance medium (PT-3102B) manufactured by Lonza Walkersville, etc. Differentiation from mesenchymal stem cells into adipocytes can be confirmed using commercially available kits, such as Adipo Red (trademark) assay reagent manufactured by Lonza.

[0023] The differentiation of the mesenchymal stem cells of the present invention into bone cells can be induced by culturing the mesenchymal stem cells of the present invention in the presence of, for example, dexamethasone, ascorbate, MCGS, β-glycerophosphate, etc. Commercially available kits may also be used, such as hMSC differentiation BulletKit (trademark)-osteogenic, PT-3004, etc., manufactured by Lonza Walkersville. The differentiation of mesenchymal stem cells into bone cells can be confirmed using a commercially available alkaline phosphatase staining kit (e.g., manufactured by Cosmo Bio Co., Ltd., etc.), a commercially available calcification staining kit (e.g., manufactured by Cosmo Bio Co., Ltd., etc.), etc.

[0024] The differentiation of the mesenchymal stem cells of the present invention into chondrocytes can be induced by culturing the mesenchymal stem cells of the present invention in the presence of, for example, TGF-β3, dexamethasone, insulin-transferrin-selenium acid (ITS), sodium pyruvate, proline, and ascorbate. Alternatively, commercially available kits may be used, such as hMSC differentiation BulletKit (trademark)-chondrogenic and PT-3003 manufactured by Lonza Walkersville. The differentiation of mesenchymal stem cells into chondrocytes can be confirmed by Alcian blue staining or the like.

[0025] Stem cell transplantation can be easily performed by injecting a suspension of stem cells into the host body. Injection can be performed into the organ to be regeneratively treated or in the vicinity thereof, or intravenously. There is no particular limit to the number of stem cells to be injected, and it can be selected appropriately depending on the symptoms, the weight of the host, the method of administration, etc., but it is usually about 10 2 ~10 10 Approximately one piece.

[0026] As used herein, the term "feeder cells" refers to other types of cells that play a supporting role and are used to prepare culture conditions for the target cells to undergo proliferation and differentiation.

[0027] When used as a feeder cell, it is usually preferable to treat it with gamma irradiation or antibiotics in advance to prevent proliferation. As a feeder cell for stem cells, fibroblasts derived from mouse fetuses are mainly used, but various cell types such as fibroblasts such as 3T3 and SNL are used as feeder cells depending on the purpose of the experiment and the cells. The stem cells of the present invention can be used as feeder cells for stem cells for human transplantation by isolating them from young pigs that can be transplanted into humans.

[0028] The method for preparing stem cells of the present invention is characterized by comprising a step of isolating cells from a young pig. One embodiment of the method for preparing stem cells of the present invention includes, for example, a method comprising the following steps: (1) Collecting cells from young pigs (2) Culturing the cells collected in step (1) to prepare juvenile pig-derived stem cells. Each step will be described below.

[0029] (1) Collecting cells from young pigs In step (1), cells are collected from bone marrow, fat, skin, pancreas, etc. of young pigs. Specifically, for example, when cells are collected from the bone marrow of a young pig, bone marrow cells can be collected from the femur, iliac crest, sternum, etc. of the young pig. For example, the femur is collected from the young pig, both ends are cut, a needle is inserted, and the bone is flushed with a physiological buffer solution (e.g., phosphate buffer, hereinafter also referred to as PBS) to which heparin has been added, and the effluent is collected from the opposite side as bone marrow fluid. When the amount of effluent decreases, the bone is turned over, a needle is inserted into the opposite side, and the bone is flushed again with PBS to prepare bone marrow fluid, which is a cell-containing solution.

[0030] Furthermore, the cell-containing solution prepared above may be centrifuged to isolate a mononuclear cell fraction derived from young pigs. The cell-containing solution prepared above is diluted with PBS or the like, and the diluted cell-containing solution is placed on top of a medium layer in a tube containing a medium for human lymphocyte separation (e.g., Ficoll-Paque PLUS manufactured by GE Healthcare Life Sciences, etc.).

[0031] The tube is centrifuged to separate layers, and the layer containing the young pig-derived mononuclear cells is collected. The collected solution is further centrifuged, the supernatant is removed, and the solution is diluted with PBS or the like and centrifuged again to isolate the mononuclear cell fraction. The mononuclear cell fraction cells thus isolated may be cryopreserved before culturing. By freezing the isolated young pig-derived mononuclear cell fraction cells, cells that are less susceptible to the effects of freezing and thawing can be selectively prepared. When cryopreserving before culturing, the temperature is preferably -80°C or lower, more preferably -150°C or lower.

[0032] Furthermore, for example, when cells are collected from the pancreas of a young pig, pancreatic islets are collected from the young pig, and in some cases, the islets are cultured in suspension to prepare cell masses to be used for adhesion culture for the purpose of preparing stem cells.

[0033] For example, when collecting cells from the fat of young pigs, fat is collected from the young pigs and finely chopped with scissors, followed by enzyme treatment. The mixture is filtered through a cell strainer and centrifuged at low speed. The cells that settle to the bottom of the tube are used for culture. For example, when collecting cells from the skin (including hair) of young pigs, skin is collected from the young pigs and subjected to enzyme treatment. After enzyme treatment, the hair is removed from the skin and the bulge portion is collected and used for culture. 3T3 feeder cells are used for culture.

[0034] (2) Culturing the cells collected in step (1) to prepare juvenile pig-derived stem cells. The cells, cell fractions, or cell aggregates collected in step (1) above contain many unintended cells other than stem cells. Usually, a culture method is used to remove these unintended cells by using a basal medium (e.g., MSC basal medium described below) that does not contain vitamin C, which is essential for the survival of these unintended cells.

[0035] In step (2) of the present invention, the cells, cell fractions, or cell aggregates collected in step (1) above are cultured in a culture incubator preferably at 35 to 39°C, more preferably 36 to 38°C, and most preferably 37°C, in the presence of preferably 4 to 6%, more preferably 4.5 to 5.5%, and most preferably 5% CO2, to remove undesired cells other than stem cells and to proliferate the stem cells of the present invention.

[0036] Since the stem cells of the present invention have a significantly high proliferation rate, the stem cells of the present invention can be prepared by culturing the cells to remove the above-mentioned unintended cells using only a medium containing vitamin C (e.g., MSC medium described below) without using a basal medium containing no vitamin C. Note that the stem cells of the present invention can also be prepared by culturing the cells to remove the above-mentioned unintended cells using a basal medium containing no vitamin C, and then exchanging the medium for one containing vitamin C to proliferate the stem cells of the present invention.

[0037] Specifically, the stem cells of the present invention are cultured, for example, by the following method: Using a culture vessel coated with gelatin (e.g., a plate coated with 0.1% gelatin) or a culture vessel not coated with gelatin (e.g., a plate), a basal medium not containing vitamin C (e.g., MSC basal medium described below) or a medium containing vitamin C (e.g., MSC medium described below) is used, preferably at a concentration of 5.0 × 10 5 pieces~5.0×10 7 Cells / 9.6cm 2are seeded and incubated, for example, at 37°C under conditions of 5% CO2 and 90% humidity to obtain primary cultured cells. The culture period for obtaining primary cultured cells is preferably 3 to 12 days, more preferably 3 to 11 days, and most preferably 3 to 10 days after seeding. Primary cultured cells may be passaged. Stem cells obtained by passage are also called subcultured cells. Primary cultured cells or subcultured cells are passaged after the stem cells have reached 30% to 100% confluence, preferably 50% to 95% confluence, more preferably 60% to 90% confluence, and most preferably 70% to 85% confluence, preferably 2 to 6 days, more preferably 2 to 5 days, even more preferably 2 to 4 days, and most preferably 3 days after seeding. The stem cells are seeded at a density of preferably 5.0 × 10 in a culture vessel coated with gelatin (e.g., a plate coated with 0.1% gelatin) or a culture vessel (e.g., a plate) without gelatin coating, using a medium containing vitamin C (e.g., MSC medium described below). 5 pieces~5.0×10 7 Cells / 9.6cm 2 The stem cells are cultured, for example, under conditions of 37° C., 5% CO 2 and 90% humidity. During the culture of the stem cells, the medium is replaced as necessary to proliferate the stem cells of the present invention.

[0038] As the MSC basal medium and MSC medium, those known in the art can be used, or commercially available ones can be used. An example of the MSC basal medium is a medium obtained by adding 55 mL of Gibco's Fetal bovine serum (FBS) and 5.5 mL of Sigma-Aldorich's Penicillin-Streptomycin to 500 mL of Gibco's MEMα (Nucleosides, no Ascorbic acid). An example of the MSC medium is a medium obtained by adding 55 mL of Gibco's Fetal bovine serum (FBS), 5.5 mL of Sigma-Aldorich's Penicillin-Streptomycin, and 22.2 μL of Sigma-Aldorich's FGF-Basic, recombinant, expressed in E. coli, suitable for cell culture (final concentration: 1 ng / mL) to 500 mL of Gibco's MEMα (Nucleosides).

[0039] It is preferable to perform passage at least once. The number of passages is not particularly limited as long as the stem cells of the present invention can be obtained, but is preferably 1 to 3 times, and more preferably 1 to 20 times.

[0040] The stem cells of the present invention can be cryopreserved. The timing of cryopreservation is not particularly limited, but is preferably after 1 to 20 passages, and more preferably after 2 to 10 passages. Conventionally known methods can be used for cryopreservation and thawing.

[0041] Specifically, the stem cells can be cryopreserved by dispersing them in a cryopreservation solution and cryopreserving them at -80°C or lower in a freezer or in liquid nitrogen until needed. Examples of cryopreservation solutions include a solution in which OPF-301 [lactated Ringer's solution containing 3% trehalose and 5% dextran (International Publication No. 2014 / 208053)] and dimethyl sulfoxide (DMSO) are mixed in a ratio of 9:1, serum-containing or serum-free preservation solutions that can be used for cryopreservation of animal cells, or commercially available cell cryopreservation reagents [preferably Cellbanker, such as CELLBANKER (registered trademark) manufactured by Takara Bio Inc.]. EXAMPLES

[0042] Test Example 1 [Collection of bone marrow cells from young pigs] Bone marrow was collected from the femur of a young pig. Femurs were collected from young pigs (medical Landrace pigs aged 23 days), cut at both ends, inserted a 12G needle, flushed with 50 mL of heparinized PBS (3 mL of heparin (1000 U / mL), 47 mL of PBS), and 50 mL of bone marrow effluent (hereinafter also abbreviated as bone marrow fluid) was collected from the opposite site. When the amount of effluent decreased, the bone was turned over and the needle was inserted into the opposite side, and the bone marrow fluid was collected by flushing again with PBS. A 50 μL sample was taken in 1950 μL of PBS (40-fold dilution) in a 15 mL conical tube for counting, and the cell count was measured with a cell counter.

[0043] [Isolation of the juvenile porcine mononuclear cell (npMNC) fraction] The bone marrow fluid obtained by the above procedure was gently resuspended. The entire bone marrow fluid was divided into four 50 mL tubes, 10 mL each, and each was diluted to 30 mL with PBS. After confirming that cells were not attached to the tube, the tubes were mixed well. 10 mL of Ficoll-Paque PLUS (GE Healthcare Life Sciences) was added to four new 50 mL tubes, and 30 mL of bone marrow fluid mixed with PBS was placed on top of the Ficoll-Paque PLUS layer.

[0044] The tubes were centrifuged at 400×g for 30 min at 20°C and then slowly accelerated (1 / 3 full speed) without braking to form three distinct layers. The mononuclear cell fraction was located in a floating white ring, so the entire white ring was collected in 50 mL tubes (×4) containing 25 mL of PBS. Centrifuged at 400×g for 7 min at room temperature and the supernatant was removed. PBS was added up to 40 mL and centrifuged again at 400×g for 7 min at room temperature. The cell count was measured as above, with 25-30% of the total bone marrow cells being the mononuclear cell fraction, each at (20-30)×10 6 were isolated.

[0045] [Freezing and preserving cells from the juvenile porcine mononuclear cell (npMNC) fraction] The isolated mononuclear cell fraction cells were cultured at 10 7 cells / mL DMSO in a cryovial containing mixed FBS (90% FBS and 10% DMSO) to a total volume of 1 mL of cell suspension [number of cells / 10 × 10 6 = volume of FBS mixed with DMSO (mL). The cryovials were stored at -20°C for 1 h, then at -80°C for 24 h, and finally transferred to a liquid nitrogen tank for long-term storage.

[0046] [Culture of young porcine mononuclear cell (npMNC) fraction cells and preparation of young porcine bone marrow-derived mesenchymal stem cells (npBM-MSC)] A cell suspension containing cells of the mononuclear cell (npMNCs) fraction derived from young pigs, which had been frozen and stored in a cryovial, was quickly thawed in a water bath at 37°C, and the thawed cell suspension was gently added to 30mL of MSC basal medium (500mL of Gibco's MEMα (Nucleosides, no Ascorbic acid) supplemented with 55mL of Gibco's Fetal bovine serum (FBS) and 5.5mL of Sigma-Aldorich's Penicillin-Streptomycin, hereinafter the same) that had been adjusted to temperature equilibration (37°C) using a micropipette. The mixture was centrifuged at 500×g for 5 minutes at room temperature, and the pellet was resuspended in 4mL of MSC basal medium equilibrated at temperature and gently pipetted up and down. The total cell number and viable cell number were measured, and the total cell number was 4.18×10 6 , number of viable cells 6.6×10 5 The survival rate was 15.8%.

[0047] Coat 6-well plates with 0.1% gelatin and place in an incubator (37 °C, 5% CO2) for 10-15 min, then remove the gelatin before use. Add the cell suspension to each prepared 0.1% gelatin-coated 6-well plate and gently rock to disperse the cell suspension on the growth surface (gelatin coat), and add 2.09 × 10 cells in 2 mL of MSC basal medium. 6Cells were seeded at 100 cells / well. They were cultured in a CO2 incubator at 37°C under 5% CO2 and 90% humidity conditions. After 3 days, the medium was replaced with MSC medium [500 mL of Gibco MEMα (nucleosides) supplemented with 55 mL of Gibco Fetal bovine serum (FBS), 5.5 mL of Sigma-Aldorich Penicillin-Streptomycin, and 22.2 μL of Sigma-Aldorich FGF-Basic, recombinant, expressed in E. coli, suitable for cell culture (final concentration: 1 ng / mL), hereinafter the same] to grow the cells. Thereafter, the MSC medium was replaced once every 3 days. After 10 days, the immature pig bone marrow-derived mesenchymal stem cells (npBM-MSCs) became confluent. When a plate without gelatin coating was used, the immature pig bone marrow-derived mesenchymal stem cells (npBM-MSCs) similarly became confluent after 10 days.

[0048] [Subculture] After the juvenile porcine bone marrow-derived mesenchymal stem cells (npBM-MSCs) reached nearly 100% confluence, we harvested the cells from two wells and reseeded them into T75 flasks with or without a 0.1% gelatin coat.

[0049] The cells were washed with 2 mL of PBS (calcium and magnesium free), 320 μL of 0.25% trypsin was added per well, and the wells were left in an incubator for several minutes. Once the cells had detached, they were neutralized with 1680 μL of MSC medium. The cell suspension was collected into a 50 mL tube using a 1 mL pipette, and 16 mL (8 mL x 2 wells) of MSC medium was added, followed by centrifugation at 500 × g for 5 minutes at room temperature. The resulting pellet was gently resuspended in temperature-equilibrated MSC medium (2 mL) using a pipette. The total cell number and viable cell number were counted, resulting in a total cell number of 2.05 × 10 6 pcs, number of viable cells 2.02×10 6 The survival rate was 98.5%.

[0050] MSC medium was added to T75 flasks with and without 0.1% gelatin coating, and 4.5 × 10 5 The cells were reseeded to obtain live cells / flask in a T75 flask and cultured in a CO2 incubator at 37°C under conditions of 5% CO2 and 90% humidity. These cells were passage 1. Three days after the first passage, the cells reached 100% confluence, regardless of whether or not they were coated with 0.1% gelatin.

[0051] [Preparation of juvenile porcine bone marrow-derived mesenchymal stem cells (npBM-MSC)] After the juvenile porcine bone marrow-derived mesenchymal stem cells (npBM-MSCs) reached nearly 100% confluence, the cells were harvested from two flasks of T75 flasks with or without 0.1% gelatin coating. The cells were washed with 8 mL of PBS(-), 2.4 mL of 0.25% trypsin was added per well, and the wells were left in an incubator for a few minutes. Once the cells had detached, they were neutralized with 12.6 mL of MSC medium. The cell suspension was collected in a 50 mL tube and centrifuged at 500 x g for 5 minutes at room temperature.

[0052] Temperature-equilibrated MSC medium (10 mL) was added to the resulting pellet, which was then gently resuspended by pipetting up and down. The total cell count and viable cell count were measured, and the results are shown below. Cells from 0.1% gelatin-coated flasks (x2): total cell count 1.62 x 10 7 pcs, number of viable cells 1.60×10 7 pieces, survival rate: 98.8% Cells from non-gelatin-coated flasks (x2): Total cell count 1.48 x 10 7 pcs, number of viable cells 1.46×10 7 pieces, survival rate: 98.6%

[0053] [Freezing and preserving young porcine bone marrow-derived mesenchymal stem cells (npBM-MSC)] In addition to the above culture, early passage juvenile porcine bone marrow-derived mesenchymal stem cells (npBM-MSCs) were frozen to prepare cell stocks. Trypsinized npBM-MSC pellets were resuspended in a 9:1 mixture of CELLBANKER®1 or OPF-301 (lactated Ringer's solution containing 3% trehalose and 5% dextran (WO 2014 / 208053) at the desired concentration and DMSO, and 1.5 × 10 6 The cells were stored at 1 mL per vial. The vials were placed in a bicell and stored at -80°C for 24 hours, after which the cells were transferred from -80°C to liquid nitrogen for long-term storage.

[0054] [CFU assay] Juvenile porcine bone marrow-derived mesenchymal stem cells (npBM-MSCs) (P2) were cultured at 21 cm 2 Culture dishes (without gelatin coating or with 0.1% gelatin coating) were cultured at 630 cells at 30 cells / cm. 2 The cells were seeded at a density of 100 μg / ml and cultured in MSC medium. The MSC medium was changed every 3 days. After 6 days of culture, the adherent cells were washed twice with 4 mL of PBS and fixed with 4 mL of ice-cold methanol for 15 min at 4 °C. To visualize the colonies, the cells were stained with 4 mL of Giemsa diluted 1:19 in phosphate buffer for 30 min, then washed at room temperature (RT) and twice with HO.

[0055] Next, the number of colonies with more than 50 cells was counted, and the colony formation efficiency of the cells was calculated by dividing the number of colonies per dish by the number of cells seeded per dish (630 cells). The results are shown in Table 1. The values ​​in Table 1 show the mean ± SD (n = 3).

[0056] [Table 1]

[0057] As shown in Table 1, the results of the CFU assay demonstrated that the obtained immature porcine bone marrow-derived mesenchymal stem cells (npBM-MSCs) were capable of forming colonies regardless of the presence or absence of a gelatin coat.

[0058] [Average cell diameter] The results of measuring the average cell diameter for human bone marrow-derived mesenchymal stem cells (hBM-MSCs, passage number P4) and the resulting immature pig bone marrow-derived mesenchymal stem cells (npBM-MSCs) are shown in Table 2. The average cell diameter was measured using a Nucleo Counter NC-200 (trademark), and the average value (n=3) was calculated.

[0059] [Table 2]

[0060] As shown in Table 2, the obtained juvenile porcine bone marrow-derived mesenchymal stem cells (npBM-MSCs) were found to have a smaller average diameter compared to human bone marrow-derived mesenchymal stem cells.

[0061] [Evaluation of growth rate] For human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and juvenile porcine bone marrow-derived mesenchymal stem cells (npBM-MSCs), cells were cultured at 5000 cells / cm in a T25 flask. 2 (1.25×10 5 The cells were seeded at a density of 1000 x 1000 cells / flask and cultured in MSC medium. The MSC medium was replaced every 3 days. The total numbers of viable and dead cells were counted 1, 2, 4, and 8 days after the start of culture. The results are shown in Tables 3 and 4 and in Figures 1A and 1B. The values ​​in Tables 3 and 4 are the mean ± SD (n = 4).

[0062] [Table 3]

[0063] [Table 4]

[0064] As shown in Tables 3 and 4 and Figures 1A and 1B, the resulting immature porcine bone marrow-derived mesenchymal stem cells (npBM-MSCs) were found to have a significantly faster cell proliferation rate than human bone marrow-derived mesenchymal stem cells.

[0065] [Differentiation into adipocytes] Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and juvenile porcine bone marrow-derived mesenchymal stem cells (npBM-MSCs) were induced to differentiate into adipocytes using the hMSC differentiation BulletKit™-adipogeni, PT-3004 (manufactured by Lonza Walkersville) according to the protocol. The results of staining with Sigma-Aldorich Oil Red on the 17th day after the start of induction are shown in Figures 2A and 2B, respectively.

[0066] As shown in Figures 2A and 2B, the obtained immature porcine bone marrow-derived mesenchymal stem cells (npBM-MSCs) were found to be capable of differentiating into adipocytes, similar to human bone marrow-derived mesenchymal stem cells.

[0067] [Differentiation into bone cells] Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and juvenile porcine bone marrow-derived mesenchymal stem cells (npBM-MSCs) were induced to differentiate into osteocytes using the hMSC differentiation BulletKit™-osteogenic, PT-3002 (manufactured by Lonza Walkersville) according to the protocol. The results of staining using the Cosmo Bio alkaline phosphatase staining kit on the 14th day after the start of induction are shown in Figures 3A and 3B, respectively, and the results of staining using the Cosmo Bio mineralization staining kit are shown in Figures 4A and 4B, respectively.

[0068] As shown in Figures 3A and 3B and Figures 4A and 4B, the obtained juvenile porcine bone marrow-derived mesenchymal stem cells (npBM-MSCs) were found to be capable of differentiating into bone cells, similar to human bone marrow-derived mesenchymal stem cells.

[0069] [Differentiation into chondrocytes] Juvenile porcine bone marrow-derived mesenchymal stem cells (npBM-MSCs) were induced to differentiate into osteocytes using hMSC differentiation BulletKit™-chondrogenic, PT-3003 (manufactured by Lonza Walkersville) according to the protocol. The results of HE staining and Alcian blue staining on the 19th day after the start of induction are shown in Figures 5A and 5B, respectively.

[0070] As shown in Figures 5A and 5B, the obtained juvenile porcine bone marrow-derived mesenchymal stem cells (npBM-MSCs) were found to be capable of differentiating into chondrocytes.

[0071] Test Example 2 [Culture of young pig-derived mononuclear cell (npMNC) fraction and preparation of young pig bone marrow-derived mesenchymal stem cells (npBM-MSC)] The MSC basal medium or MSC medium was left in an incubator (37°C, 5% CO2) for 10 to 15 minutes before use. As in Test Example 1, a cell suspension containing cells of the juvenile pig-derived mononuclear cell (npMNC) fraction that had been frozen and stored in a cryovial was quickly thawed in a 37°C water bath. Using a micropipette, the thawed cell suspension was gently added to 30 mL of temperature-equilibrated (37°C) MSC basal medium, and 15 mL was dispensed into two 50 mL tubes.

[0072] After centrifugation at 500×g for 5 minutes at room temperature, the pellet was resuspended in 2 mL of temperature-equilibrated MSC basal medium or MSC medium and gently pipetted up and down. The total cell number and viable cell number were counted and the results are shown below. 2mL of MSC basal medium: total cell number 2.60 x 10 6 , number of viable cells 4.8×10 5 pieces, survival rate 18.5% 2 mL of MSC medium: 2.55 x 10 total cells, 4.5 x 10 viable cells 5 pieces, survival rate 17.6%

[0073] An amount of cell suspension calculated to give the seeding cell numbers as shown below was added to a 6-well plate (without gelatin coating) containing the medium described below per well, and the plate was gently rocked to distribute the cell suspension over the growth surface. 2mL of MSC basal medium: 2.60 x 10 6 Cells seeded at 1 cell / well 2mL MSC medium: 2.55 x 10 6 Cells seeded at 1 cell / well

[0074] The cells were incubated in a CO2 incubator at 37°C under conditions of 5% CO2 and 90% humidity. Three and six days after seeding, the medium was replaced with MSC medium to proliferate the cells, and the cells were passaged on the eighth day after seeding.

[0075] [Subculture] After the juvenile porcine bone marrow-derived mesenchymal stem cells (npBM-MSCs) reached approximately 50-60% confluence, we harvested the cells from one well and re-seeded them into a T75 flask without gelatin coating.

[0076] The cells were washed with 2 mL of PBS(-), 320 μL of 0.25% trypsin was added per well, and the wells were left in an incubator for several minutes. Once the cells had detached, they were neutralized with 1680 μL of MSC medium. The cell suspension was collected in a 50 mL tube, 8 mL of MSC medium was added, and the wells were centrifuged at 500 × g for 5 minutes at room temperature.

[0077] Temperature-equilibrated MSC medium (2 mL) was added to the resulting pellet, which was then gently resuspended by pipetting up and down. The total cell count and viable cell count were measured, and the results are shown below. Group with MSC basal medium at P0 seeding: total cell number 5.0 × 10 5 pcs, number of viable cells 5.0×10 5 pieces, survival rate: 100% Group with MSC medium at P0 seeding: total cell number 3.3 × 10 5 pcs, number of viable cells 3.3×10 5 pieces, survival rate: 100%

[0078] 15 mL of MSC medium was added to a T75 flask (without gelatin coating), and immature pig bone marrow-derived mesenchymal stem cells (npBM-MSCs) were reseeded to the following cell counts and cultured in an incubator. These cells were the first passage. MSC basal medium group at P0 seeding: 5.0 x 10 viable cells 5 pcs / flask MSC medium group at P0 seeding: 3.3 x 10 viable cells 5 pcs / flask

[0079] [Preparation of juvenile porcine bone marrow-derived mesenchymal stem cells (npBM-MSC)] After the cells replated by the above procedure reached approximately 80-90% confluence, cells were collected from one T75 flask (without gelatin coating). The cells were washed with 8 mL of PBS(-), 0.25 mL / well of 2.4 mL of trypsin was added, and the cells were left in an incubator for several minutes. Once the cells had detached, they were neutralized with 12.6 mL of MSC medium. The cell suspension was collected in a 50 mL tube and centrifuged at 500 × g for 5 minutes at room temperature.

[0080] Temperature-equilibrated MSC medium (5 mL) was added to the resulting pellet, and the cells were gently resuspended by pipetting up and down. The total cell count and viable cell count were measured, and the results are shown below. Cells from one flask (MSC basal medium 3 days after seeding at P0): Total cell count 5.12 x 10 6 , number of viable cells 5.09×10 6 pieces, survival rate: 99.5% Cells from one flask (in MSC medium from seeding at P0): Total cell count 4.76 x 10 6 pcs, number of viable cells 4.73×10 6 pieces, survival rate: 99.4%

[0081] [Freezing and preserving young porcine bone marrow-derived mesenchymal stem cells (npBM-MSC)] Separately from the above-mentioned culture, cells from early passages were frozen in the same manner as in Test Example 1 to prepare cell stocks.

[0082] Test Example 3 The cell surface antigens of the young pig-derived mononuclear cells (npMNC) prepared in Test Example 1 and Test Example 2 were analyzed. The preparation method of each sample used in the analysis is shown in Table 5. In Table 5, "Switch" indicates that MSC basal medium (vitamin C-free) was used for initial culture, and the medium was changed to MSC medium (containing vitamin C), which is a proliferation medium, for proliferation culture.

[0083] [Table 5]

[0084] [Analysis of cell surface antigens] Each cell sample was removed from the liquid nitrogen tank, the lid was loosened to release the pressure, the lid was closed again, and the cells were thawed in a thermostatic bath preheated to 37°C with gentle stirring for 1 to 2 minutes. The thawed cells were transferred to a 15 mL centrifuge tube containing 5 mL of Stain Buffer (BD), centrifuged at 500 x g for 5 minutes at 4°C, and the supernatant was removed. 5 mL of Stain Buffer was added, centrifuged at 500 x g for 5 minutes at 4°C, and washed twice.

[0085] The cells were resuspended in 2 mL of Stain Buffer (BD) and the number of viable cells was counted. The cells were centrifuged again (500 × g, 5 min, 4 °C) to obtain a cell count of 1 × 10 7 The cells were resuspended in Stain Buffer (BD) to a concentration of 2 × 10 cells / mL and then added to 20 μL (cell count: 2 × 10 5 Each piece was dispensed into a 1.5 mL tube, and a total of four tubes were prepared: unstained control, CD44, CD90, and isotype control.

[0086] 4 μL of Anti-CD44, Mouse (MEM-263), PE (GeneTex), 1 μL of PE Mouse Anti-Human CD90 (BD) (cross-reactive with pigs), and 4 μL of PE Mouse IgG1, κ Isotype Control (BD) were added to each tube and incubated on ice in the dark for 45 minutes. Unstained controls were also stored on ice.

[0087] 1 mL of Stain Buffer (BD) was added to each tube, and the tubes were centrifuged at 500×g for 5 minutes at 4°C and washed twice. The cell pellets were loosened by tapping, resuspended in 500 μL of Stain Buffer (BD), and transferred to a flow cytometer test tube through a filter just before analysis. The cells were stored on light-shielded ice until analysis, and analyzed using flow cytometry. The results for CD44 are shown in Figure 6, and the results for CD90 are shown in Figure 7.

[0088] As shown in Figures 6 and 7, the mesenchymal stem cell markers CD44 and CD90 were positive in all samples. It is also believed that the desired mesenchymal stem cells could be established even without coating with gelatin during initial culture. In addition, no nonspecific reactions were observed in the measurement of the isotype control in any case.

[0089] Test Example 4 [Preparation of mesenchymal stem cells derived from young pig islets] Pancreatic islets were collected from young pigs and cultured in suspension to prepare cell masses, which were then frozen and stored in the same manner as in Test Example 1. The young pig pancreatic islets that had been frozen and stored in a cryovial were quickly thawed in a 37°C water bath.

[0090] Using a micropipette, the thawed islet suspension was gently added to 30 mL of MSC basal medium that had been adjusted to temperature equilibration (37°C). The suspension was centrifuged at 210 x g for 1 minute at 4°C. If the islets were not frozen, the islets were allowed to settle by gravity at room temperature, and the supernatant was removed. The pellet was resuspended in 4 mL of MSC basal medium that had been equilibrated to temperature, and gently pipetted up and down.

[0091] Add the islet suspension to a 6-well plate and gently rock to distribute the cell suspension over the growth surface (without gelatin coating) and seed islets ranging from 1650 IEQ to 2125 IEQ / well in 2 mL of MSC basal medium.

[0092] The cells were cultured in a CO2 incubator at 37°C under conditions of 5% CO2 and 90% humidity, and after 3 days, the medium was replaced with MSC medium to allow the cells to grow. Thereafter, the MSC medium was replaced once every 3 days. Table 6 shows the sample preparation conditions. Regardless of whether or not the cells were initially frozen, they reached 100% confluence 6 days after seeding.

[0093] [Table 6]

[0094] [Subculture] After the juvenile porcine islet-derived mesenchymal stem cells (npISLET-MSCs) reached approximately 80% to nearly 95% confluence, we harvested the cells from 2 wells and re-seeded them into a T75 flask without a gelatin coat.

[0095] The cells were washed with 2 mL of PBS (calcium and magnesium free), 320 μL of 0.25% trypsin was added per well, and the wells were left in an incubator for several minutes. Once the cells had detached, they were neutralized with 1680 μL of MSC medium. The cell suspension was collected into a 50 mL tube using a 1 mL pipette, and 16 mL (8 mL x 2 wells) of MSC medium was added, followed by centrifugation at 500 × g for 5 minutes at room temperature. The resulting pellet was gently resuspended in temperature-equilibrated MSC medium (2 mL) using a pipette.

[0096] [Average cell diameter] 20 mL of the MSC medium was added to a T75 flask without gelatin coating, and the cells were reseeded and cultured in a CO2 incubator at 37°C under conditions of 5% CO2 and 90% humidity. These cells were the first passage. Three days after the first passage was seeded, the cells reached 100% confluence, regardless of whether or not they were initially frozen. This shows that the proliferation rate of mesenchymal stem cells prepared from the islets of young pigs is comparable to that of mesenchymal stem cells prepared from the bone marrow of young pigs. The average diameter of the obtained mesenchymal stem cells derived from the islets of young pigs is shown in Table 7.

[0097] [Table 7]

[0098] As shown in Table 7, it was found that mesenchymal stem cells derived from young porcine islets could be prepared regardless of the freezing conditions used in preparing the islets, and that the average diameter was similar regardless of whether the cells were frozen or not.

[0099] [Analysis of cell surface antigens] Each cell sample was removed from the liquid nitrogen tank, the lid was loosened to release the pressure, the lid was closed again, and the cells were thawed in a thermostatic bath preheated to 37°C with gentle stirring for 1 to 2 minutes. The thawed cells were transferred to a 15 mL centrifuge tube containing 5 mL of Stain Buffer (BD), centrifuged at 500 x g for 5 minutes at 4°C, and the supernatant was removed. 5 mL of Stain Buffer was added, centrifuged at 500 x g for 5 minutes at 4°C, and washed twice.

[0100] The cells were resuspended in 2 mL of Stain Buffer (BD) and the number of viable cells was counted. The cells were centrifuged again (500 × g, 5 min, 4 °C) to obtain a cell count of 1 × 10 7 The cells were resuspended in Stain Buffer (BD) to a concentration of 2 × 10 cells / mL and then added to 20 μL (cell count: 2 × 10 5 Each piece was dispensed into a 1.5 mL tube, and a total of four tubes were prepared: unstained control, CD29, CD44, and CD90.

[0101] 1 μL of Mouse Alexa Fluor 647 Mouse Anti-Pig CD29 (BD), 4 μL of Anti-CD44, Mouse (MEM-263), PE (GeneTex), and 1 μL of PE Mouse Anti-Human CD90 (BD) (cross-reactive with pigs) were added to each tube and incubated on ice in the dark for 45 minutes. Unstained controls were also stored on ice.

[0102] 1 mL of Stain Buffer (BD) was added to each tube, and the tubes were centrifuged at 500×g for 5 minutes at 4°C and washed twice. The cell pellets were loosened by tapping, resuspended in 500 μL of Stain Buffer (BD), and transferred to a test tube for flow cytometry through a filter just before analysis. The cells were stored on ice in the dark until analysis, and analyzed using flow cytometry.

[0103] The results for CD29 are shown in Figures 8A to 8D, those for CD44 in Figures 9A to 9D, and those for CD90 in Figures 10A to 10D. Figures 8A, 9A, and 10A show the results for sample 11 (not frozen immediately after islet preparation); Figures 8B, 9B, and 10B show the results for sample 12 (frozen immediately after islet preparation); Figures 8C, 9C, and 10C show the results for sample 13 (not frozen on the third day of culture after islet preparation); and Figures 8D, 9D, and 10D show the results for sample 14 (frozen on the third day of culture after islet preparation).

[0104] As shown in Figures 8A to 10D, high positivity rates were observed for mesenchymal stem cell markers CD29, CD44, and CD90 in all samples. Furthermore, it appears that the desired mesenchymal stem cells were established regardless of whether or not the cells were frozen during initial culture.

[0105] Although the present invention has been described in detail with reference to specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the present invention. This application is based on a U.S. provisional application (US62 / 555,913) filed on September 8, 2017, the entirety of which is incorporated by reference. In addition, all references cited herein are incorporated in their entirety.

Claims

1. A mesenchymal stem cell isolated from a young pig aged 0 to 25 days, The average diameter is 17 μm or less, The doubling time during logarithmic growth is 36 hours or less; The mesenchymal stem cells are produced by freezing cells isolated from the bone marrow or pancreatic islets of a young pig that is between 0 and 25 days old, thawing and seeding the frozen cells, and then culturing the cells.

2. The mesenchymal stem cell according to claim 1, wherein the immature pig is an immature pig capable of undergoing cell transplantation into a human.

3. The mesenchymal stem cell according to claim 1 or 2, which is a stem cell for transplantation.

4. The mesenchymal stem cell according to claim 3, which is a stem cell for human transplantation.

5. The mesenchymal stem cell according to claim 1 or 2, which is a stem cell for use as a feeder cell.

6. The mesenchymal stem cell according to claim 5, which is a stem cell to be used as a feeder cell for growing stem cells for human transplantation.

7. A method for preparing mesenchymal stem cells, comprising the steps of isolating cells from bone marrow or pancreatic islets of young pigs that are between 0 and 25 days old, freezing the isolated cells, thawing and seeding the frozen cells, and subculturing the mesenchymal stem cells 3 to 12 days after seeding.

8. The method for preparing mesenchymal stem cells according to claim 7, wherein the cells isolated from the young pig are cells of a mononuclear cell fraction.

9. The method for preparing mesenchymal stem cells according to claim 7 or 8, comprising a step of freezing the cells of the isolated mononuclear cell fraction.

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