METHOD FOR ISOLATING HUMAN BRAIN TISSUE-DERIVED NEURAL STEM CELL AT HIGH EFFICIENCY

Abstract

The present invention relates to a method for cultivation and isolation of neural stem cells whereby neural stem cells can be rapidly proliferated on mass scale and isolated at high efficiency and to a stroke patient-derived human adult neural stem cell, cultured and isolated thereby, for use in transplantation.

Claims

1. A method of culturing a neural stem cell, the method comprising: collecting cells from brain tissue; treating the collected cells with collagenase and DNase I, or papain, cysteine, and DNase Ito isolate single cells; dividing the single cells into two or more tubes, mixing the single cells in each tube with Percoll, and centrifuging each mixture to recover cells; primary culturing the recovered cells; and sub-culturing the primary cultured cells.

2. The method of claim 1, wherein the neural stem cell is a human adult neural stem cell for transplantation which is derived from a stroke patient.

3. The method of claim 1, wherein the primary culturing or the sub-culturing is performed using an adherent culture method.

4. The method of claim 1, wherein the sub-culturing is performed three times or less.

5. A human adult neural stem cell for transplantation which is derived from brain tissue cultured using the method according to any one of claims 1.

Description

DESCRIPTION OF DRAWINGS

[0014] FIG. 1 illustrates a process of culturing a neural stem cell according to an embodiment of the present invention.

[0015] FIG. 2 illustrates the number of cells according to Percoll treatment, according to an embodiment of the present invention.

[0016] FIG. 3 illustrates a method of differentiating a neural stem cell according to an embodiment of the present invention.

[0017] FIG. 4 is a set of images showing results of confirming the ability of a neural stem cell to differentiate into a neuron and an astrocyte according to differentiation conditions, according to an embodiment of the present invention.

MODES OF THE INVENTION

[0018] The present invention provides a method of culturing a neural stem cell, including: collecting cells from brain tissue; treating the cells with collagenase and DNase I, or papain, cysteine, and DNase I to isolate single cells; dividing the single cells into two or more tubes, mixing the single cells in each tube with Percoll, and centrifuging each mixture to recover cells; primary culturing the recovered cells; and sub-culturing the primary cultured cells.

[0019] The neural stem cell of the present invention is isolated from brain tissue extracted during surgery on a stroke patient, and may be autotransplanted into the stroke patient or allotransplanted.

[0020] Generally, single cells isolated from a biological tissue are subjected to a process of removing impurities using Percoll. In the method of the present invention, the single cells may be divided into two tubes and mixed with Percoll, followed by centrifugation to recover cells, whereby cell yield is increased about 2-fold compared to a case in which single cells are mixed with Percoll in a conventional single tube (see FIG. 2).

[0021] A medium used for primary culturing or sub-culturing of the present invention may be any medium generally used for culturing stem cells. For example, a medium containing serum (e.g., fetal bovine serum, horse serum, and human serum) may be used. The medium that may be used in the present invention may be, for example, the RPMI series such as RPMI 1640 (Moore et al., J. Amer. Med. Assoc. 199:519(1967)), Eagles's MEM (Eagle's minimum essential medium, Eagle, H. Science 130:432(1959)), -MEM (Stanner, C. P. et al., Nat. New Biol. 230:52(1971)), Iscove's MEM (Iscove, N. et al., J. Exp. Med. 147:923(1978)), 199 medium (Morgan et al., Proc. Soc. Exp. Bio. Med., 73:1(1950)), CMRL 1066, F12 (Ham, Proc. Natl. Acad. Sci. USA 53: 288 (1965)), F10 (Ham, R.G. Exp. Cell Res. 29: 515 (1963)), DMEM (Dulbecco's modification of Eagle's medium, Dulbecco, R. et al., Virology 8: 396 (1959)), a mixture of DMEM and F12 (Barnes, D. et al., Anal. Biochem. 102: 255 (1980)), Way mouth's MB752/1 (Waymouth, C. J. Natl. Cancer Inst. 22: 1003 (1959)), McCoy's 5A (McCoy, T. A., et al., Proc. Soc. Exp. Biol. Med. 100:115 (1959)) and the MCDB series (Ham, R.G. et al., In Vitro 14:11 (1978)), but the present invention is not limited thereto. The medium may include other components, for example, an antibiotic or antifungal agent (e.g., penicillin and streptomycin), glutamine, and the like.

[0022] Sub-culturing of stem cells is generally performed seven or nine times or more. In the method of the present invention, however, due to mixing with Percoll in two tubes and the use of an adherent culture method, the sub-culturing process may be performed three times or less, whereby a sufficient amount of neural stem cells for transplantation may be acquired.

[0023] Advantages and features of the present invention, and methods of achieving them will become apparent with reference to embodiments described below in detail. Hereinafter, the present invention will be described in further detail with reference to the following examples. However, these examples are provided to specifically explain the present invention and are not intended to limit the scope of the present invention.

EXAMPLE 1. ACQUISITION OF NEURAL STEM CELLS

Isolation and Culture of Human Neural Stem Cells

[0024] Brain tissue (brain tissue in an outer ceiling area of a lateral ventricle (brain tissue in the pathway for bleeding removal or ventricular puncture)) removed through a surgical operation (Department of Neurosurgery, Samsung Medical Center) from a stroke patient was obtained, and cultured according to processes as illustrated in FIG. 1.

[0025] First, the obtained brain tissue was immersed in a solution prepared by adding 3% antibiotic-antimycotic (Gibco) or 3% penicillin/streptomycin (Gibco) to Hank's balanced salt solution (HBSS, Welgene) and stored, and within a maximum of 12 hours after surgery, cells were isolated. In the case of difficulty in immediately performing cell isolation, the resulting brain tissue was kept refrigerated at 4 before being subjected to cell isolation.

[0026] The obtained brain tissue was weighed and then rinsed two or three times with a sterilized PBS solution, and then mechanically pulverized with scissors or a razor blade, and stored in an enzyme solution prepared by mixing Collagenase (0.4 mg/ml, Gibco) and DNase I (0.01 mg/ml to 1 mg/ml, Roche) or mixing papain (10 unit/ml, Sigma), DL-Cysteine (400 ng/ml, Sigma), and DNase I (0.01 mg/ml to 1 mg/ml, Roche) in a CO.sub.2 incubator at 37 for 1 hour. Thereafter, the enzyme solution was treated with DMEM:F12(Gibco) and a 1% FBS solution in an amount equal to or greater than the enzyme solution to inactivate the enzymes, followed by pipetting with a pipette, pulverization, and passing through a 70 M nylon mesh to obtain single cells.

[0027] Percoll (Sigma) was warmed in a 37 water bath for about 5 minutes, and then 9 mL of Percoll and 1 mL of 10 PBS were added to a 50 mL sterile ultracentrifuge tube to adjust a concentration thereof to 1. The obtained single cell suspension was diluted with 1 PBS to a total volume of 40 mL, and then divided into two 50 mL conical tubes at a volume of 20 mL each, and Percoll was added to each tube to adjust a total volume of each tube to 30 mL. Then, each tube was centrifuged at 20,000 rpm and 18 for 20 minutes to remove erythrocytes and other tissues and cells. A white layer formed after centrifugation was separated using a pipette, and then washed twice with a DMEM:F12(Gibco) solution.

[0028] The final cells were suspended in a DMEM:F12 (Gibco) solution-based culture solution containing 0.5% to 1% FBS, a 1 B27 supplement (Gibco), bFGF (R&D), and EGF (R&D), the number of the cells was confirmed, and then 100 pi dishes were pre-treated with poly-L-ornithine (Sigma) before cell culture, followed by culturing to a density of 410.sup.5 cells/dish. At this time, only half of the culture solution was replaced at intervals of 3 days to 4 days, and 10 days to 14 days were generally taken until primary sub-culturing.

[0029] As a comparative example, neural stem cells were cultured in the same manner as in the above example, except that single cells were mixed with Percoll in a single tube, and the result was compared with that of the above example in which Percoll was mixed in two tubes. As can be seen in FIG. 2, while the number of cells was 510.sup.5 in the case in which Percoll was mixed in a single tube, 110.sup.6 cells were shown in the case in which Percoll was mixed in two tubes according to the present example.

Subculture

[0030] The cells were sub-cultured when the cells occupied about 70% to 80% of a total area of each dish in the above culture process.

[0031] First, the existing cell culture solution was removed, followed by washing once with DPBS. Then, the cells were treated with 0.05% Trypsin/EDTA (T/E, Gibco) or Accutase such that the cells were immersed therein, stored in a 5% CO.sub.2 incubator at 37 for 2 minutes to 3 minutes, and then treated with a solution to which DMEM:F12 (Gibco) and 1% FBS were added, to inactivate the enzyme. The cells were pelleted using a centrifuge, and then the supernatant was removed and suspended in the cell culture solution.

[0032] The cells were counted and then 410.sup.5 cells were placed in each of 100 pi dishes and cultured. When sub-cultured once, the number of cells was increased by average of 10 times and 10.sup.3-fold cells were obtained when sub-culturing was performed three times.

[0033] The average time for sub-culturing once is 3 days to 4 days and sub-culturing three times may be done within 2 weeks, and thus 110.sup.8 cells were obtained within one month.

[0034] When sub-culturing is performed 7 times or more, the growth of cells slows down and there are many cases in which cells take a form similar to aging such as cells increasing in length and the like, and accordingly, the characteristics of stem cells are gradually lost. In addition, long-term culture causes an increase in genetic mutations.

[0035] When culture is performed using the method of the present invention, a sufficient number of cells for multi-dose administration and autotransplantation may be obtained performing sub-culturing three times or less.

EXAMPLE 2. DIFFERENTIATION OF NEURAL STEM CELLS

[0036] To confirm a differentiation ability of the neural stem cells obtained in Example 1, as illustrated in FIG. 3, the neural stem cells were differentiated.

[0037] First, the neural stem cells were cultured on poly-L-ornithine (PLO)-coated culture dishes for 3 days, and when the cells occupied 70% to 80% of a total area of each dish, the culture medium was replaced with a differentiation medium (DMEM/F12, 1% P/S, 1B27, 0.5% FBS, 100 ng/mL bFGF, 100 ng/mL EGF, and 0.5 mM IBMX). On day 2 and day 4 after differentiation, the cells were immobilized and immunofluorescence-stained with Nestin, which is an undifferentiation marker, MAP2, which is a neuron-specific marker, and GFAP, which is an astrocytic marker, and then observed with a fluorescence microscope.

[0038] As can be seen in FIG. 4, it was confirmed that the neural stem cells differentiated into neurons and astrocytes. The ability of the neural stem cells to differentiate into neurons (MAP2+) and astrocytes (GFAP+) was confirmed under differentiation conditions, i.e., before differentiation (0 DIV) and after differentiation (2, 5, 9 DIV), and it was also confirmed that the expression of Nestin, which is an undifferentiation marker, was reduced as differentiation proceeded.

[0039] The present invention has been described with reference to exemplary embodiments thereof. It will be understood by those skilled in the art that various changes may be made in forms and details without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in an illustrative sense rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereto should be construed as being within the scope of the present invention.