METHOD FOR MASS PROLIFERATION OF URINE-DERIVED PLURIPOTENT CELLS

Abstract

A method for the mass proliferation of urine-derived multipotent cells and a medium composition for the mass proliferation of urine-derived multipotent cells according to the present invention can be used to massively proliferate urine cells by efficiently isolating the same even from urine that has been left alone for a long period of time, and can be used to produce multipotent cells having characteristics of epithelial cells, mesenchymal cells, and stem cells.

Claims

1. A method of massively proliferating urine-derived multipotent cells, comprising: (a) isolating multipotent cells in urine by centrifuging the urine in a tube containing a precipitate solution for urine collection, prepared by adding serum; a growth factor; an antibiotic; insulin; an estrogen steroid hormone; a corticosteroid-based compound; a cytokine; a plasma-derived component; and the extracellular matrix to a basal medium for cell culture, and (b) culturing the isolated multipotent cells in a medium composition for massively proliferating urine-derived multipotent cells, prepared by adding a plasma-derived component; a growth factor; insulin, an estrogen steroid hormone; a corticosteroid-based compound; and a cytokine are added to a basal medium for cell culture.

2. The method of claim 1, wherein, in (a), the basal medium for cell culture is one selected from the group consisting of DMEM, RPMI, and Waymouth MB 752/1.

3. The method of claim 1, wherein, in (a), the serum is one or more selected from the group consisting of fetal bovine serum, calf serum, rabbit serum, goat serum, mouse serum, horse serum, sheep serum, pig serum, chicken serum, and human serum.

4. The method of claim 1, wherein, in (a), the antibiotic is penicillin or streptomycin.

5. The method of claim 1, wherein the estrogen steroid hormone is estradiol, and the corticosteroid-based compound is one or more selected from the group consisting of corticosterone, dexamethasone and hydrocortisone, wherein the cytokine is one or more selected from the group consisting of an interleukin, interferon-gamma and TGF-β, the plasma-derived component is one or more selected from the group consisting of albumin, hemoglobin and transferrin, and the extracellular matrix is one or more selected from the group consisting of collagen, laminin, fibronectin, gelatin, elastin and hyaluronic acid.

6. The method of claim 1, wherein the growth factor is one or more selected from the group consisting of an epidermal growth factor (EGF), a platelet-derived growth factor (PDGF), a vascular endothelial growth factor (VEGF), a fibroblast growth factor (FGF), an insulin-like growth factor (IGF) and a leukemia inhibitory factor (LIF).

7. The method of claim 1, wherein, in (a), the multipotent cells in urine are isolated from the urine after being left for 0 to 96 hours in the tube containing the precipitate solution for urine collection.

8. The method of claim 1, wherein the urine collected in tube containing the precipitate solution for urine collection has a pH of 7 to 8 after 0 to 96 hours.

9. The method of claim 1, wherein, in (a), the precipitate solution for urine collection and the urine are comprised in a content ratio of 1:100 to 1:0.01.

10. The method of claim 1, further comprising, after (b), (c) proliferating the cultured cells by subculture.

11. The method of claim 1, wherein the urine-derived multipotent cells have two or more characteristics selected from the group consisting of epithelial cell characteristics, mesenchymal cell characteristics and hematopoietic stem cell characteristics.

12. A medium composition for the mass proliferation of urine-derived multipotent cells, comprising: a basal medium; a plasma-derived component; a growth factor; insulin; an estrogen steroid hormone; a corticosteroid-based compound; and a cytokine.

13. The composition of claim 12, wherein the plasma-derived component is one or more selected from the group consisting of albumin, hemoglobin and transferrin, the growth factor is one or more growth factors selected from the group consisting of an epidermal growth factor (EGF), a platelet-derived growth factor (PDGF), a vascular endothelial growth factor (VEGF), a fibroblast growth factor (FGF), an insulin-like growth factor (IGF), and a leukemia inhibitory factor (LIF), the estrogen steroid hormone is estradiol, the corticosteroid-based compound is one or more selected from the group consisting of corticosterone, dexamethasone and hydrocortisone, and the cytokine is one or more selected from the group consisting of an interleukin, interferon-gamma and TGF-β.

Description

DESCRIPTION OF DRAWINGS

[0064] FIG. 1 is a schematic diagram of a method of massively proliferating urine-derived multipotent cells using a precipitate solution for urine collection.

[0065] FIG. 2A shows a process of collecting urine in a tube, in which the left panel shows the result of leaving urine that has been collected in a tube containing a precipitate solution for urine collection for 72 hours, and the right panel shows the result of leaving urine that has been collected in a tube not containing a precipitate solution for urine collection for 72 hours, and FIG. 2B shows the precipitation result for the precipitate solution for urine collection and the urine cells by centrifugation of the collected urine.

[0066] FIG. 3 shows the process of primarily culturing the collected urine-derived cells in a culture plate, in which FIG. 3A shows the result of culturing the urine cells isolated from the urine that has been left for 72 hours in a tube not containing a precipitate solution for urine collection, and FIG. 3B shows the result of culturing the urine cells isolated from the urine that has been left for 72 hours in a tube containing a precipitate solution for urine collection.

[0067] FIG. 4 shows that the number of cells primarily cultured in a culture plate increases over time.

[0068] FIG. 5 shows the change in the number of viable cells that are proliferating after subculture.

[0069] FIG. 6 shows the result of observing the morphology of urine-derived cells cultured in a medium not containing a plasma-derived component, a growth factor and a cytokine.

[0070] FIG. 7 shows the result of observing the morphological characteristic of cells after the urine-derived cells of the present invention have been cultured for 3 or more passages, a breast cancer epithelial cell line MCF7, and adipose-derived cells, which are mesenchymal cells, are cultured for the same period and under the same culture conditions.

[0071] FIG. 8 shows the result of confirming whether an epithelial cell marker (CK18) and mesenchymal cell markers (fibronectin and vimentin) are expressed after the urine-derived cells of the present invention have been cultured for 3 or more passages, a breast cancer epithelial cell line MCF7, and adipose-derived cells, which are mesenchymal cells, are cultured for the same period and under the same culture conditions.

[0072] FIG. 9 is the result showing that the urine-derived cells of the present invention express an epithelial cell marker (CK18) and mesenchymal cell markers (fibronectin and vimentin) at the same time.

[0073] FIG. 10 is the result showing that, as the urine-derived cells of the present invention are subcultured, a hematopoietic stem cell marker (CD133) and an epithelial stem cell marker (CD24) are expressed in certain amounts.

[0074] FIG. 11 shows that the urine-derived cells of the present invention are isolated and cultured in a group expressing an epithelial stem cell marker (positive) and a group not expressing an epithelial stem cell marker (negative) using a flow cytometer (FACS). a: the expression of epithelial stem cell marker (CD24) in urine-derived cells; b: the expression of epithelial stem cell marker (CD24) after isolating epithelial stem cell marker (CD24)-negative (CD24-) and -positive (CD24+) cells; c: epithelial stem cell marker-negative (CD24-) and -positive (CD24+) cells cultured in culture plates after isolation.

[0075] FIG. 12 is the result showing that, after staining urine-derived cells with the gene staining reagent Hoechst 33342, which is used for hematopoietic stem cell analysis and isolation, there is a hematopoietic stem cell population (side population) of urine-derived cells through flow cytometry. a: urine-derived cells with various sizes; b: showing that the proportion of a hematopoietic stem cell population (side population) depends on a cell size.

MODES OF THE INVENTION

[0076] Hereinafter, the present invention will be described in detail with reference to examples of the present invention. The following examples only illustrate the present invention, but the scope of the present invention is not limited to the following examples.

EXAMPLES

Example 1: Isolation and Mass Proliferation of Urine-Derived Multipotent Cells

1) Collection of Urine Cells

[0077] For urine collection, a precipitate solution for urine collection was prepared in DMEM, which is a basal medium, so that components for a composition shown in Table 1 below are contained in DMEM to have the corresponding final concentrations. The prepared precipitate solution for urine collection was put into a sterilized 250-ml tube, and then 200 ml of urine was collected (FIG. 2A). Here, 150 ml of the urine in the tube was isolated and left at room temperature for 72 hours. To compare a cell yield according to whether or not to leave the urine, urine was collected in a sterilized 250-ml tube not containing a precipitate solution for urine collection and then left at room temperature for 72 hours. A precipitate solution in which urine cells were precipitated was obtained by transferring the urine collected in the tube to a clean bench and adding the urine into a 50-ml tube, and centrifuging the urine in a centrifuge at a room temperature and 2,000 rpm for 5 minutes (FIG. 2B).

TABLE-US-00001 Component Final concentration Growth factor bFGF 10 ng/ml hEGF 10 ng/ml LIF 10 ng/ml Insulin Insulin 1 .Math.g/ml Cytokine IL-2 10 ng/ml Corticosteroid-based compound Hydrocortisone 10 ng/ml Estrogen steroid hormone 17-B-estradiol 10 nM Plasma-derived component Albumin 10 ng/ml Hemoglobin 10 .Math.g/ml Antibiotic Penicillin or streptomycin 10 .Math.l/ml Serum Fetal bovine serum 10% Extracellular matrix Collagen 10 .Math.g/ml Gelatin 100 .Math.g/ml Hyaluronic acid 10 .Math.g/ml

2) Primary Culture of Acquired Urine Cells (7 Days)

[0078] The cells acquired from the tube containing the precipitate obtained in 1) and the tube that did not contain the precipitate were put into a medium for primary culture of the composition of Table 2 below, transferred to a 100-pi cell culture plate by pipetting, and cultured in a 5% CO.sub.2, 37° C. incubator for 7 days (FIGS. 3A and 3B). According to the culture, it was confirmed that urine cells isolated from the urine left for 72 hours at room temperature in the tube not containing the precipitate solution for urine collection were not cultured and proliferated in an incubator (FIG. 3A), and urine cells isolated from the urine cells left for 72 hours in the tube containing the precipitate solution for urine collection were cultured and proliferated (FIG. 3B).

TABLE-US-00002 Composition of medium for mass proliferation Component Final concentration Growth factor bFGF 10 ng/ml hEGF 10 ng/ml LIF 10 ng/ml Insulin Insulin 1 .Math.g/ml Cytokine IL-2 10 ng/ml Corticosteroid-based compound Hydrocortisone 10 ng/ml Estrogen steroid hormone 17-B-estradiol 10 nM Plasma-derived component Albumin 10 ng/ml Hemoglobin 10 .Math.g/ml Antibiotic Penicillin or streptomycin 10 .Math.l/ml Serum Fetal bovine serum 10%

3) Primary Culture of Acquired Urine Cells (14 Days)

[0079] Urine cells acquired from urine were primarily cultured in the same manner as above except that the cells in 2) were cultured for 14 days (FIG. 4).

4) Mass Proliferation of Cells

[0080] In order to massively proliferate the primarily cultured cells in 2) and 3), the primarily cultured cells were transferred to a clean bench, and cell states were observed through a microscope. Afterward, the cells were washed three times with PBS as a washing solution, and a trypsin-EDTA solution was added to a culture plate and left in an incubator for 10 minutes. After taking out the plate from the incubator and inactivating the trypsin-EDTA action using a cell culture medium, cells were collected, transferred to a tube and then centrifuged. During this process, to confirm cell proliferation, a part of the cell culture was taken, and then the number of viable cells was measured at each subculture. The subculture was performed every 7 days, and the number of viable cells measured at each subculture was compared with the initial number of viable cells just before the first suspension. As a result, it was confirmed that the number of viable cells increased at a constant rate at each subculture (FIG. 5). In addition, the morphology of cells was observed when cells were cultured in a medium that did not contain a “plasma-derived component, a growth factor and a cytokine,” which were contained in a medium for the mass proliferation of urine-derived multipotent cells according to the present invention (FIG. 6). As a result, the urine-derived cells cultured in the medium that did not include the “plasma-derived component, the growth factor and the cytokine” show the characteristics of typically aged cells that increase in size and grow slower as the culture period elapses, and therefore, when urine-derived cells were cultured in the medium for mass proliferation of the urine-derived cells according to the present invention, a mass proliferation effect, that is, an increased number of cells, can be confirmed.

Example 2: Cell Characterization

1) Morphological Characterization

[0081] In order to confirm the characteristics of the cells cultured and proliferated in Example 1, breast cancer cell line MCF7, which is a representative cell line having an epithelial cell characteristics, adipose-derived cells, which are mesenchymal cells, and the urine-derived cells of Example 1, which were subcultured three or more times, were cultured using the same medium for the same period of time (72 hours) on 18-mm coverslips coated with 10 .Math.g/ml of type IV collagen in 24-well plates. Four hours after culture, all cells were attached, and 72 hours after culture, their morphological characteristics were observed. As a result, it was seen that the urine-derived cells, compared to the adipose cells, which are mesenchymal cells, have a smaller size and overall oval shapes. In addition, the epithelial cancer cells MCF7 showed small and layered cells, whereas the urine-derived cells of the present invention showed the characteristics of larger and oval-shaped squamous cells, compared to the MCF7 cells (FIG. 7).

2) Confirmation by Immunostaining

[0082] In order to confirm the characteristics of the cells cultured and proliferated in Example 1, the cells were fixed with paraformaldehyde, and subjected to immunocytochemistry. Cytokeratin 18 (CK18) was used as an epithelial cell marker, and fibronectin and vimentin were used as mesenchymal cell markers. Immunocytochemistry was performed as follows: the cultured cells on the 18-mm cover slip were washed with PBS, and fixed with a 4% formaldehyde solution at room temperature for 15 minutes. After washing with PBS three times, 0.5% Triton X-100 was permeated into the cells at room temperature for 15 minutes, and then the cells were washed with PBS three times. After blocking with 10% normal goat serum at room temperature for 1 hour, the cells were washed several times with PBS. The cells were left with a primary antibody for 1 hour. The cells were washed several times with PBS, left with a secondary antibody for 1 hour, and then washed three times with PBS. Afterward, the immunostained cells were stored in a VECTASHIELD mounting medium, and whether or not the markers were expressed was confirmed using a confocal microscope. As an observation result, the adipose-derived cells, which are mesenchymal cells, expressed the mesenchymal cell markers such as fibronectin and vimentin, the epithelial cells MCF7 did not express fibronectin and vimentin but expressed only the epithelial cell marker CK18 (FIG. 8). On the other hand, it was confirmed that the urine-derived cells according to the present invention express all of the mesenchymal cell markers such as fibronectin and vimentin, and the epithelial cell marker CK18 (FIG. 9). From the above result, it was able to be confirmed that the urine-derived cells according to the present invention have the characteristics of multipotent cells.

3) Confirmation of Multipotency

[0083] In order to confirm the multipotency of the urine-derived cells cultured and proliferated in Example 1, the expression of the hematopoietic stem cell marker CD133 and the epithelial stem cell marker CD24 was confirmed by a flow cytometer. The cells collected for flow cytometry were washed three times with PBS without fixing. The cells were blocked with 10% normal goat serum at room temperature for 1 hour, and washed several times with PBS. The cells were left with a secondary antibody-binding primary antibody for 1 hour at 4° C. without light. After washing three times with PBS, phenol red-free 10% FBS was added to confirm expression through flow cytometry (FIG. 10).

[0084] As a result of the observation, it was able to be confirmed that, even when the cells are subcultured, the expression pattern of a hematopoietic stem cell marker and an epithelial cell marker is increased or maintained (FIG. 10).

[0085] Based on the above result, it was able to be confirmed that the urine-derived cells according to the present invention have the characteristics of mesenchymal cells and hematopoietic stem cells as well as epithelial stem cells. In addition, it was able to be confirmed that positive cells show the morphology of epithelial cells, and negative cells show the morphology of mesenchymal cells by dividing these cells into the positive and negative cells based on the expression of an epithelial stem cell marker through flow cytometry (FIG. 11).

[0086] From the above results, it can be confirmed that epithelial stem cells and mesenchymal cells can be isolated from the urine-derived cells and proliferated.

[0087] In addition, as a result of observing a side population using Hoechst 33342, which is a gene staining reagent for confirming hematopoietic stem cells, it was observed that there are side populations in all cell groups regardless of cell size (FIG. 12). This shows that hematopoietic stem cells can be isolated and cultured.