Method for culturing stem cells using microcarriers

Abstract

The present invention relates to a method for culturing stem cells using a microcarrier.

Description

[Technical field]

[0001] The present invention relates to a method for culturing stem cells using a microcarrier. [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] An object of the present invention is to provide a method for culturing stem cells using a microcarrier. Effect of the Invention

[0006] The culture method of the present invention can maintain the size of stem cells small and can delay or inhibit aging. [Brief description of the drawings]

[0007] [Figure 1] This shows the results of measuring and comparing the size of stem cells cultured on plates and in microcarrier culture. [Diagram 2] The results show the results of measuring and comparing the levels of stem cell aging in plate-attached culture and microcarrier culture. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] 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.

[0009] Additionally, those of ordinary skill 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.

[0010] 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.

[0011] One aspect of the present invention provides a method for enhancing the efficacy of stem cells, comprising attaching and culturing stem cells on microcarriers.

[0012] In one embodiment, the stem cells are cultured attached to microcarriers suspended in a cell culture medium, the microcarriers being cross-linked with polygalacturonic acid (PGA), and the method provides for the stem cells to have one or more characteristics selected from (i) a reduction or maintenance of cell size, and (ii) delayed senescence, compared to plate culture.

[0013] 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 proliferation ability of small cell populations being relatively high and the expression of aging-related markers being low. Other studies have confirmed that stem cells with diameters below a certain size show relatively good 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.

[0014] 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.

[0015] The culture method of the present invention according to any one of the above-mentioned embodiments is characterized in that, compared to culturing stem cells on a plate, (i) the size of the stem cells can be maintained constant, or (ii) stem cell aging is suppressed or delayed.

[0016] The culture method of the present invention according to any one of the above-mentioned embodiments is characterized in that the size of stem cells is maintained constant or decreased, and stem cell aging is inhibited or delayed, compared to when stem cells are cultured on a plate.

[0017] In any one of the above-mentioned embodiments, the culture method of the present invention includes attaching stem cells to microcarriers and culturing the stem cells while the stem cells are attached to the microcarriers suspended in KSB-3 Complete Medium (registered trademark) Kit medium.

[0018] 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.

[0019] In one embodiment, the stem cells of the present invention are mesenchymal stem cells.

[0020] In any one of the above embodiments, the stem cells are cord blood derived mesenchymal stem cells.

[0021] 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:

[0022] 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.

[0023] 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.

[0024] In any one of the above-mentioned embodiments, the culture of the stem cells of the present invention is a subculture.

[0025] 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.

[0026] 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.

[0027] In one embodiment of the above-mentioned embodiments, the method of the present invention comprises culturing stem cells at a density of 2,000 cells / cm. 2 ~4,000 pieces / cm 2 In one embodiment, the culture method of the present invention comprises seeding and culturing stem cells at about 3,000 cells / cm. 2 The method includes the step of inoculating the

[0028] In any one of the above-mentioned specific examples, the culture method of the present invention uses microcarriers at a dose of 0.5 mg / ml to 2.0 mg / ml relative to the medium volume. In one specific example, the culture method of the present invention uses microcarriers at a dose of 0.5 mg / ml to 1.5 mg / ml relative to the medium volume. In one specific example, the culture method of the present invention uses microcarriers at a dose of about 1.0 mg / ml relative to the medium volume.

[0029] In the present invention, the term "about" is provided before a specific numerical value. The term "about" includes not only the exact number described after the term, but also an approximate number or a range close to the number. Whether the number is close to or approximately the specific number mentioned can be determined by considering the context in which the number is provided. As an example, the term "about" refers to a range of -10% to +10% of the numerical value. As another example, the term "about" refers to a range of -5% to +5% of the given numerical value. However, the present invention is not limited thereto.

[0030] In one embodiment of any one of the above-mentioned embodiments, the culture method of the present invention may be carried out in a bioreactor. The bioreactor may include a mechanism for stirring the culture medium.

[0031] In any one of the above-mentioned embodiments, the culture of the present invention may use KSB-3 Complete Medium® Kit medium.

[0032] 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.

[0033] Example 1: Comparative size analysis of mesenchymal stem cells derived from umbilical cord blood according to culture conditions For cell size comparison analysis of cord blood-derived mesenchymal stem cells in plate culture and microcarrier adherent culture conditions, cord blood-derived mesenchymal stem cells were cultured on a 150 mm plate at 3,000 cells / cm. 2 The cells were inoculated at 100°C and cultured in a 5% CO2 incubator from P3 to P6. The cord blood-derived mesenchymal stem cells were isolated as described in Korean Patent Registration No. 10-0950195 and used were stem cells that showed positive immunological properties against ZNF281.

[0034] Specifically, the preparation and culture process of cord blood-derived mesenchymal stem cells is as follows: Frozen cells were thawed and mixed well by pipetting, then suspended in KSB-3 Complete Medium Kit and centrifuged at room temperature at 500 g for 5 minutes. The supernatant was then removed so that the cell pellet would not dissolve, and the pellet was resuspended in KSB-3 Complete Medium Kit again to measure the cell count and viability.

[0035] The culture process in the plate was as follows: After placing 15 mL of KSB-3 Complete Medium Kit in a 150 mm plate, the cord blood-derived mesenchymal stem cells were cultured at 3,000 cells / cm. 2 The mixture was added to a plate so that the amount of the cells was 100 μg / ml, and the mixture was cultured at 37°C in 5% CO2 for a total of 5 days.

[0036] The culture process in the bioreactor was as follows. 0.4 g of microcarriers (Dissolvable Microcarriers (Corning® Dissolvable Microcarriers)) were weighed out, and sterilized distilled water was added to proceed with hydration at 4°C. The hydrated microcarriers were placed in a vessel of a bioreactor (PBS Biotech Inc., 0.5 L bioreactor) in a volume of 400 ml using KSB-3 complete medium, and reacted at 20 rpm for 30 minutes. Umbilical cord blood-derived mesenchymal stem cells were cultured at 3,000 cells / cm. 2 The cells were added to the bioreactor vessel so that the total volume of the cells was 100 ml, and the cells were cultured in a 5% CO2 incubator at 37°C, 15 rpm for a total of 5 days from P3 to P6.

[0037] At each passage point, plate-cultured cells were triple-treated to obtain single cells, and bioreactor-cultured cells attached to microcarriers were triple-treated with pectinase to dissolve the microcarriers and obtain single cells. The obtained single cells were analyzed for cell size using NC200.

[0038] As a result, the diameter of cells in each culture condition in P4 was similar at 14.15μm and 14.50μm in plate culture and microcarrier culture conditions, respectively, but in P5 it was 16.20μm and 14.50μm, respectively, a difference of about 1.7μm, and in P6 it was 16.40μm and 14.15μm, respectively, a difference of about 2.25μm. Therefore, it was confirmed that the size of the cells derived from umbilical cord blood-derived mesenchymal stem cells in plate culture conditions increases as subculture proceeds under the same conditions, while the size of the cells derived from umbilical cord blood-derived mesenchymal stem cells in microcarrier adherent culture did not increase even when subculture proceeded under the same conditions (Figure 1).

[0039] Example 2: Comparative analysis of aging of mesenchymal stem cells derived from umbilical cord blood depending on culture conditions To compare and analyze the degree of aging of cord blood-derived mesenchymal stem cells under plate culture and microcarrier adherent culture conditions, the degree of aging of cord blood-derived mesenchymal stem cells under each culture condition was compared and analyzed through β-galactosidase staining.

[0040] The P4, P5, and P6 cells obtained in Example 1 under each culture condition were cultured in a 12-well plate at 1×10 5 The cells were seeded 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 0.5ml of β-galactosidase staining reagent per well. After staining at 37℃ for 48 hours, the cells were examined under a microscope.

[0041] As a result of β-galactosidase staining, the SA-βGal Positive (%) value at P4 was 15.07% and 19.28% in the plate culture condition and the microcarrier culture condition, respectively, at P4, 32.49% and 28.83% respectively, which was about 4% lower in the microcarrier culture condition, and at P6, 37.95% and 27.79% respectively, which was about 10% lower in the microcarrier culture condition. Therefore, it was confirmed that the aging of umbilical cord blood-derived mesenchymal stem cells was delayed more in the microcarrier repeated subculture condition than in the plate repeated subculture condition (Figure 2).

[0042] 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. A method for enhancing efficacy of stem cells, comprising attaching stem cells to microcarriers and culturing the stem cells, The stem cells are attached to microcarriers suspended in a cell culture medium and cultured; The microcarrier is a cross-linked polygalacturonic acid (PGA), The method is a method for enhancing stem cell efficacy, wherein the stem cells have one or more characteristics selected from (i) reduction or maintenance of cell size, and (ii) delayed senescence, compared to plate culture.

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

3. The method for enhancing efficacy of stem cells according to claim 1 , wherein the subculture is performed for two or more passages.

4. 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.

5. The method of claim 1, wherein the enhancement of stem cell efficacy comprises reducing or maintaining stem cell size and delaying stem cell aging.

6. 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.

7. The method involves culturing stem cells at 2,000 cells / cm ２ ~4,000 pieces / cm ２ 2. The method of claim 1, comprising inoculating and culturing with

8. 2. The method of claim 1, wherein the method uses microcarriers at a dosage of 0.5 mg / ml to 2.0 mg / ml relative to medium volume.

9. 10. The method of claim 1, wherein the method comprises culturing stem cells attached to microcarriers suspended in KSB-3 Complete Medium Kit medium.

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