Method for culturing stem cells using serum-free medium

Abstract

The present invention relates to a method for enhancing the efficacy of stem cells, comprising culturing the stem cells in a serum-free medium.

Description

[Technical field]

[0001] The present invention relates to a method for culturing stem cells using a serum-free medium. [Background technology]

[0002] Stem cells are undifferentiated cells obtained from various tissues of embryos, fetuses, and adults, and have characteristics such as self-renewing, differentiation, and immortality. Stem cells are classified into pluripotent, multipotent, and unipotent stem cells according to their differentiation ability. Adult stem cells, which are characterized by their pluripotency, are relatively free from the risk of cancer development, immune rejection, and ethical aspects compared to embryonic stem cells and IPS, which have pluripotent differentiation ability, and many researches and developments of therapeutic drugs are underway. In particular, umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs), which circulate in the cardiovascular system of fetuses during pregnancy and are obtained from the placenta and umbilical cord after delivery, have been attracting much attention in the research and medical fields after research results were reported that they have superior proliferation ability than stem cells derived from other tissues as they are mesenchymal stem cells derived from the blood of newborns. In particular, they are valuable in that they are isolated from tissues that would otherwise be discarded, so the burden on donors is small and medical waste can be recycled. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Korean published patent KR 10-2023-0017786 A [Patent Document 2] Korean Patent No. 10-0950195 [Patent Document 3] Korean Patent No. 10-1627907 Summary of the Invention [Problem to be solved by the invention]

[0004] This has led to a demand for methods for efficiently culturing stem cells. [Means for solving the problem]

[0005] One object of the present invention is to provide a method for enhancing the efficacy of stem cells, comprising culturing the stem cells in a serum-free medium.

[0006] Another object of the present invention is to provide a method for producing chondrocytes, comprising the steps of culturing stem cells in a serum-free medium; and differentiating the cultured stem cells into chondrocytes in a differentiation medium.

[0007] Another object of the present invention is to provide a method for producing a cell therapeutic agent, comprising the steps of culturing stem cells in a serum-free medium; and differentiating the cultured stem cells into chondrocytes in a differentiation medium and collecting the chondrocytes. Effect of the Invention

[0008] By culturing stem cells according to the method of the present invention, the size of stem cells can be maintained small, their differentiation potential can be increased, and aging can be delayed or inhibited. [Brief description of the drawings]

[0009] [Figure 1] The size of umbilical cord blood-derived mesenchymal stem cells was measured while they were subcultured in a conventional serum medium (KSB-3 Complete Medium (registered trademark) Kit) or a serum-free medium (StemVie (trademark) XF Medium Kit). [Diagram 2] The results show a comparison of stem cell aging during subculture of umbilical cord blood-derived mesenchymal stem cells in a conventional serum medium (KSB-3 Complete Medium (registered trademark) Kit) or a serum-free medium (StemVie (trademark) XF Medium Kit). [Diagram 3]This is a comparison of the chondrocyte differentiation ability of umbilical cord blood-derived mesenchymal stem cells cultured in a conventional serum medium (KSB-3 Complete Medium (registered trademark) Kit) or a serum-free medium (StemVie (trademark) XF Medium Kit). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention also applies to each other description and embodiment. That is, any combination of various elements disclosed in the present application belongs to the scope of the present invention. In addition, the following specific description is not considered to limit the category of the present application.

[0011] Additionally, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein, and such equivalents are intended to be encompassed by the present invention.

[0012] In addition, throughout this specification, a number of papers and patent documents are referenced and citations are displayed, the disclosure contents of which are incorporated by reference in their entirety into this specification in order to more clearly explain the state of the art to which this application pertains and the contents of the present invention.

[0013] One aspect of the invention is a method for enhancing stem cell potency comprising culturing stem cells in serum-free medium.

[0014] In the present invention, serum-free medium means a cell culture medium that does not contain or is substantially free of serum derived from humans or non-human animals.

[0015] In one embodiment, the serum-free medium is StemVie™ XF Medium Kit.

[0016] The StemVie™ XF Medium Kit is a commercially available medium and is available to those skilled in the art. The medium contained glycine, L-arginine, L-cysteine·2HCl, L-glutamic acid, L-histidine, L-isoleucine, L-leucine, L-lysine·2HCl, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine·2Na·2H2O, L-valine, and choline chloride. The medium may further contain inorganic salts, vitamins, buffers, isotonicity agents, and the like.

[0017] In the method of the present invention, enhancing the efficacy of stem cells means any one or more selected from (i) reducing or maintaining cell size, (ii) increasing differentiation potential, and (iii) delaying senescence.

[0018] It has been reported that when stem cells are arbitrarily separated based on a certain size, the efficacy of stem cells is increased, such as the relatively high proliferation ability of small cell populations and low expression of aging-related markers. Other studies have confirmed that stem cells with diameters below a certain size show relatively excellent whitening effects, and other studies have reported that highly mobile stem cells have high differentiation ability and are characterized by their relatively small size. Therefore, maintaining the size of cells relatively small during stem cell culture leads to increased efficacy of stem cells.

[0019] It has been reported that lysosomal proteins, ROS (reactive oxygen species), and β-galactosidase activity increase in cells that have aged through stem cell culture. Therefore, to measure the level of aging, one or more of lysosomal proteins, ROS (reactive oxygen species), and β-galactosidase activity can be measured. For example, the level of aging of stem cells can be confirmed by measuring β-galactosidase activity.

[0020] By culturing stem cells in the serum-free medium of the present invention, (i) the size of the stem cells can be maintained constant, (ii) the ability to differentiate into target cells can be increased, and (iii) aging of the stem cells can be suppressed or delayed, compared to culturing the stem cells in a medium containing serum.

[0021] By culturing stem cells in the serum-free medium of the present invention, it is possible to (i) maintain a constant size of the stem cells, (ii) increase the ability to differentiate into target cells, and (iii) suppress or delay aging of the stem cells, compared to culturing the stem cells in a medium containing serum.

[0022] In any one of the above-mentioned specific examples, the method of the present invention can (i) maintain or reduce the size of stem cells, (ii) increase the differentiation ability into a target cell, or (iii) suppress or delay the aging of stem cells, compared to culturing stem cells in KSB-3 Complete Medium (registered trademark) Kit medium.

[0023] In the present invention, "differentiation" refers to the phenomenon in which a cell's structure or function is specialized. That is, it refers to the process in which the cells, tissues, etc. of an organism change to a form and function appropriate for performing the role assigned to each of them. For example, differentiation may include not only the differentiation of stem cells into bone cells, cartilage cells, or fat cells, but also the process in which embryonic stem cells change into ectodermal, mesodermal, and endodermal cells, and also includes improving the ability to differentiate into cells of a specific tissue when necessary.

[0024] In one embodiment, the differentiation of the present invention is the differentiation of stem cells into chondrocytes.

[0025] The "stem cells" of the present invention are cells capable of differentiating into various tissues, i.e., undifferentiated cells. The stem cells may be derived from humans or animals, and may be derived from umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, or placenta.

[0026] In one embodiment, the stem cells of the present invention are mesenchymal stem cells.

[0027] In one embodiment of any one of the above-mentioned embodiments, the stem cells of the present invention are cord blood derived mesenchymal stem cells.

[0028] In any one of the above-mentioned specific examples, the cord blood-derived mesenchymal stem cells are cord blood-derived pluripotent stem cells that exhibit positive immunological properties against the transcriptional regulator ZNF281 and have the following properties:

[0029] In any one of the foregoing embodiments, the umbilical cord blood derived mesenchymal stem cells exhibit any one or more of the following characteristics: (a) positive immunological signature for the transcription factor c-myc; (b) The cells attach to the extracellular matrix-coated bottom and grow, forming linear or spherical cell colonies 5 to 30 days after attachment; (c) shows a cumulative population doubling level (CPDL) of 30–45; (d) negative immunological characteristics for CD14, CD31, CD34, CD45, and HLA-DR; (e) capable of differentiating into mesodermal, endodermal, and ectodermal cells; and (f) secreting at least one cytokine or chemokine selected from the group consisting of TIMP-2, TGF-β, RANTES CINC-3, EOTAXIN, GM-CSF, IFN-γ, IL-1b, IL-3, IL-6, IL-8, IL-10, IL12p40, IL13, IL-16, IP-10, Leptin, MCP-2, MIG, MIP-3a, b-NGFm, sTNFRI, and PFGF-bb.

[0030] In any one of the above-mentioned embodiments, the cord blood-derived mesenchymal stem cells may be cells obtained by isolating as described in Korean Patent Registration No. 10-0950195. In relation to the cord blood-derived mesenchymal stem cells of the present invention, the contents disclosed in Korean Patent Registration Nos. 10-0950195 and 10-1627907 are incorporated by reference.

[0031] In one embodiment, the culture of stem cells may be subculture.

[0032] The "subculture" of the present invention is a method for growing cells, in which the cells are periodically transferred from the previous culture medium to a new (fresh) medium, for example, every 1 to 7 days. Subculture is a culture method for preserving cell lines and passing on the generations of cells, and this method is called subculturing or passaging. Through such subculture, accumulated toxic metabolic substances can be removed, depleted nutrients can be supplied, cell death can be suppressed, and growth and proliferation can be promoted.

[0033] In any one of the above embodiments, the subculture is for 2 or more passages, hi one embodiment, the subculture is for 3, 4, or 5 passages, or more.

[0034] Another aspect of the present invention is a method for producing chondrocytes, comprising the steps of culturing stem cells in a serum-free medium; and differentiating the cultured stem cells into chondrocytes in a differentiation medium.

[0035] Another aspect of the present invention provides a method for producing a cell therapeutic agent, comprising the steps of culturing stem cells in a serum-free medium; and differentiating the cultured stem cells into chondrocytes in a differentiation medium and collecting the chondrocytes.

[0036] In one embodiment, the serum-free medium is StemVie™ XF Medium Kit.

[0037] In the present invention, the term "cell therapeutic agent" refers to a pharmaceutical product (as defined by the U.S. FDA) that is used for therapeutic, diagnostic and preventive purposes on cells and tissues that have been isolated from an individual, cultured and specially processed, and that is used for therapeutic, diagnostic and preventive purposes through a series of actions such as expanding and selecting living autologous, allogeneic or xenogeneic cells in vitro, or changing the biological properties of cells in other ways, in order to restore the functions of cells or tissues.

[0038] The cell therapy agent of the present invention contains 1.0 × 10 to 1.0 × 10 cells per ml. 9The cell therapy agent of the present invention may contain, but is not limited to, cells. The cell therapy agent of the present invention may be used without freezing, or may be frozen for later use. If freezing is required, standard cryopreservatives (e.g., DMSO, glycerol, Epilife Cell Freezing Medium (Cascade Biologics)) may be added to the cell population before freezing. The cell therapy agent may also be administered by formulating it into a unit-dose formulation suitable for administration into the body of a patient according to conventional methods in the pharmaceutical field, and the formulation contains an effective dosage amount for one or several administrations. As dosage forms suitable for such purposes, injections such as injection ampoules, infusions such as infusion bags, and sprays such as aerosol formulations may be used as parenteral administration preparations. The injection ampoules may be mixed and prepared with an injection solution just before use, and the injection solution may be physiological saline, glucose, mannitol, Ringer's solution, etc. In addition, the infusion bag may be made of polyvinyl chloride or polyethylene material, and examples of such bags include infusion bags from Baxter, Becton Dickinson, Medcep, National Hospital Products, and Terumo.

[0039] The cell therapy agent may further include one or more pharma- ceutically acceptable conventional inactive carriers, such as preservatives, pain-relieving agents, solubilizers, or stabilizers in the case of injections, and bases, excipients, lubricants, or preservatives in the case of formulations for local administration.

[0040] The cell therapy agent of the present invention thus prepared may be administered together with other stem cells used for transplantation and other applications, or in the form of a mixture with such stem cells, using administration methods commonly used in the art, and may be directly engrafted or transplanted into the diseased site of a patient in need of treatment, or directly transplanted or injected into the abdominal cavity, but is not limited thereto. In addition, the administration may be non-surgical administration using a catheter, or surgical administration such as injection or transplantation after incision of the diseased site. In addition, in addition to parenteral administration according to conventional methods, for example, administration directly to a lesion, transplantation by intravascular injection, which is a common method for hematopoietic stem cell transplantation, is also possible.

[0041] The cell therapy agent can be administered once or in several doses. However, it should be understood that the actual dose of the active ingredient must be determined in light of various relevant factors such as the disease to be treated, the severity of the disease, the administration route, the patient's weight, age and sex, etc., and therefore the dose does not limit the scope of the present invention in any way.

[0042] The present invention will be described in more detail below with reference to examples and experimental examples. However, these examples and experimental examples are merely for illustrative purposes and the scope of the present invention is not limited to these examples and experimental examples.

[0043] Example 1: Comparative size analysis of mesenchymal stem cells derived from umbilical cord blood according to culture medium conditions To compare the cell size of cord blood-derived mesenchymal stem cells when cultured in serum-free medium (StemVie™ XF Medium Kit) and conventional serum medium (KSB-3 Complete Medium Kit), cord blood-derived mesenchymal stem cells were cultured on a 150 mm plate at 3,000 cells / cm. 2The cells were seeded at 1000 x 1000 and cultured in a 5% CO2 incubator at 37℃ from P3 to P6. Mesenchymal stem cells derived from umbilical cord blood were isolated and used from stem cells that showed positive immunological characteristics for the transcription factor ZNF281 as described in Korean Patent 10-0950195. At each passage point, cells under each culture condition were triple treated to obtain single cells.

[0044] The obtained single cells were analyzed for cell size using NC200. As a result, the diameter of the cells in each culture condition in P4 was similar at 13.7 μm and 13.8 μm in the culture conditions of serum-free medium (StemVie™ XF Medium Kit) and existing serum medium (KSB-3 Complete Medium® Kit), respectively, but in P5 it was 14 μm and 13.5 μm, respectively, with a difference of about 0.5 μm, and in P6 it was 15.4 μm and 13.7 μm, respectively, with a difference of about 1.7 μm.

[0045] Therefore, it was confirmed that cord blood-derived mesenchymal stem cells cultured in existing serum medium (KSB-3 Complete Medium (registered trademark) Kit) increased in size as they were passaged under the same conditions, whereas cord blood-derived mesenchymal stem cells cultured in serum-free medium (StemVie (trademark) XF Medium Kit) did not increase in size even when passaged under the same conditions (Figure 1).

[0046] Example 2: Comparative analysis of senescence of mesenchymal stem cells derived from umbilical cord blood according to culture medium conditions In order to comparatively analyze the degree of aging of cord blood-derived mesenchymal stem cells when cultured in serum-free medium (StemVie™ XF Medium Kit) or conventional serum medium (KSB-3 Complete Medium Kit), the degree of aging of cord blood-derived mesenchymal stem cells under each culture condition was comparatively analyzed through β-galactosidase staining.

[0047] P4 and P6 cells obtained in Example 1 under each culture condition were cultured in a 12-well plate at 1x10 5The cells were inoculated at 100 / mL and cultured at 37℃ in a 5% CO2 incubator for one day. After that, the cells were washed once with PBS and fixed with 4% paraformaldehyde for 10 minutes. After fixation, the cells were washed twice with PBS and treated with 0.5 ml of β-galactosidase staining reagent per well. After staining at 37℃ for 48 hours, the cells were examined under a microscope.

[0048] As a result of β-galactosidase staining, the SA-βGal Positive (%) value at P4 was 2.91% and 1.81% under culture conditions using serum-free medium (StemVie™ XF Medium Kit) and existing serum medium (KSB-3 Complete Medium Kit), respectively, which was approximately 1% lower under culture conditions using serum-free medium (StemVie™ XF Medium Kit), and at P6 it was 12.38% and 2.97%, respectively, which was approximately 10% lower under culture conditions using serum-free medium (StemVie™ XF Medium Kit).

[0049] Therefore, it was confirmed that repeated subculture in serum-free medium (StemVie™ XF Medium Kit) delayed aging of umbilical cord blood-derived mesenchymal stem cells compared to culture conditions in existing serum-free medium (KSB-3 Complete Medium Kit) (Figure 2).

[0050] Example 3: Comparative analysis of the differentiation potential of cord blood-derived mesenchymal stem cells depending on culture medium conditions To compare the differentiation potential of cord blood-derived mesenchymal stem cells when cultured in serum-free medium (StemVie™ XF Medium Kit) or conventional serum-free medium (KSB-3 Complete Medium Kit), chondrogenic differentiation was analyzed in cells cultured in each medium condition.

[0051] Specifically, the P6 cells cultured under the culture conditions of each culture medium obtained in Example 1 were cultured at 3 x 10 per 15 mL tube. 5After dispensing at 1 mL / cell and centrifuging, the pellets were cultured at 37℃ in a 5% CO2 incubator for one day. The pellets were then transferred to a 96-well round plate and induced to differentiate for three weeks by replacing the medium with chondrogenesis differentiation medium (StemPro™ Chondrogenesis Differentiation Kit) three times a week. After differentiation, the pellets were washed with PBS and fixed with 4% PFA, and then stained with Safranin-O by an analysis company. As a result, it was confirmed that the chondrogenesis differentiation ability was significantly increased when cultured in serum-free medium (StemVie™ XF Medium Kit) compared to the existing serum medium (KSB-3 Complete Medium™ Kit) (Figure 3).

[0052] From the above description, a person skilled in the art to which the present invention pertains will understand that the present invention can be embodied in other specific forms without changing the technical idea or essential features of the present invention. In this regard, it should be understood that the above-described embodiments are merely illustrative and not limiting. The scope of the present invention should be interpreted as including all modifications and variations derived from the meaning and scope of the claims below, and their equivalent concepts, rather than the above detailed description.

Claims

1. 1. A method for enhancing efficacy of stem cells, comprising culturing stem cells in a serum-free medium, The serum-free medium is StemVie™ XF Medium Kit; The enhancement of the efficacy of stem cells is A method for enhancing stem cell efficacy, which is selected from one or more of: (i) reducing or maintaining cell size; (ii) increasing differentiation potential; and (iii) delaying aging.

2. The method for enhancing efficacy of stem cells according to claim 1, wherein the serum-free medium is StemVie(TM) XF Medium Kit, and the method reduces or maintains the size of stem cells, increases their differentiation potential, and delays aging, as compared to culturing stem cells in a medium containing serum.

3. 2. The method of claim 1, wherein the serum-free medium is StemVie™ XF Medium Kit, and the method reduces or maintains the size of stem cells, increases their differentiation potential, and delays aging, as compared to culturing stem cells in KSB-3 Complete Medium Kit medium.

4. The method for enhancing efficacy of stem cells according to claim 1 , wherein the differentiation potential is the potential to differentiate into chondrocytes.

5. The serum-free medium contains glycine, L-arginine, L-cysteine.2HCl, L-glutamic acid, and Acid), L-Histidine, L-Isoleucine, L-Leucine, L-Lysine.2HCl, L-Methionine, L-Phenylalanine, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine.2Na.2H2O, L-Valine, Choline Chloride, D-Pantothenic Acid Acid (hemiCa)), Folic Acid, Myo-inositol, Niacinamide, Pyridoxine.HCl, Riboflavin, Thiamine.HCl, Magnesium Sulfate (anhydrous), Potassium Chloride, Sodium Chloride 2. The method for enhancing efficacy of stem cells according to claim 1, comprising administering to said stem cells a medicament comprising: D-glucose (dextrose); and D-choline chloride; and D-glucose (dextrose).

6. The method for enhancing efficacy of stem cells according to claim 1 , wherein the stem cells are mesenchymal stem cells derived from umbilical cord blood.

7. The method for enhancing efficacy of stem cells according to claim 1 , wherein the culture is a subculture.

8. The method for enhancing efficacy of stem cells according to claim 7 , wherein the subculture is performed for two or more passages.

9. The method for enhancing efficacy of stem cells according to claim 1, wherein the stem cells are umbilical cord blood-derived pluripotent stem cells that exhibit positive immunological characteristics against the transcriptional regulator ZNF281 and have the following characteristics: (a) exhibiting a positive immunological signature for the transcription factor c-myc; (b) The cells attach to the extracellular matrix-coated bottom and grow while forming linear or spherical cell colonies 5 to 30 days after attachment; (c) exhibiting a cumulative population doubling level (CPDL) of 30 to 45; (d) exhibiting negative immunological characteristics for CD14, CD31, CD34, CD45 and HLA-DR; (e) capable of differentiating into mesodermal, endodermal and ectodermal cells; and (f) secreting at least one cytokine or chemokine selected from the group consisting of TIMP-2, TGF-β, RANTES CINC-3, EOTAXIN, GM-CSF, IFN-γ, IL-1b, IL-3, IL-6, IL-8, IL-10, IL12p40, IL13, IL-16, IP-10, Leptin, MCP-2, MIG, MIP-3a, b-NGFm, sTNFRI, and PFGF-bb.

10. 1. A method for producing chondrocytes, comprising: culturing stem cells in a serum-free medium; and differentiating the cultured stem cells into chondrocytes in a differentiation medium, The method for producing chondrocytes, wherein the serum-free medium is StemVie™ XF Medium Kit.

11. 1. A method for producing a cell therapeutic agent, comprising: culturing stem cells in a serum-free medium; and differentiating the cultured stem cells into chondrocytes in a differentiation medium and collecting the chondrocytes, The method for producing a cell therapeutic agent, wherein the serum-free medium is StemVie(TM) XF Medium Kit.

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