Methods for efficient isolation and culture of neural stem cells

Abstract

The present invention relates to a method for efficiently isolating and culturing neural stem cells. This method can simplify the isolation and culturing process, shorten the isolation and culturing time, and increase the yield of neural stem cells. The method comprises the following steps: adding brain tissue to an enzyme solution to enzymatically treat the brain tissue; physically separating cell clusters from the enzymatically treated brain tissue; separating the cell clusters by size and removing impurities; and then seeding the cell clusters onto culture dishes for subculture.

Description

Technical Field

[0001] The present invention relates to a method for efficiently isolating and culturing neural stem cells. The method can shorten the isolation and culturing time and increase the yield of neural stem cells by simplifying the isolation and culturing methods of neural stem cells. Background Art

[0002] Biotechnology in the 21st century offers the potential for new solutions to food, environmental, and health issues, ultimately aimed at human well-being. Recently, stem cell-based technologies are opening a new chapter in the treatment of incurable diseases. Previously, organ transplantation or gene therapy were proposed as treatments for incurable human diseases, but these approaches were limited due to immune rejection, insufficient organ supply, inadequate vector development, and a lack of understanding of disease-related genes.

[0003] As interest in stem cell research grows, it has been recognized that pluripotent stem cells, capable of proliferating and differentiating into all organs, could fundamentally address organ damage and treat most diseases. Stem cells are cells that can self-renew and differentiate into two or more cell types, and are categorized as totipotent, pluripotent, and multipotent. Many scientists suggest that stem cells could be used in a variety of ways, including treating incurable conditions such as Parkinson's disease, various types of cancer, diabetes, and spinal cord injury, as well as regenerating nearly all human organs.

[0004] Neural stem cells are stem cells that can differentiate into neurons, astrocytes, and oligodendrocytes. They were first discovered in mice, a rodent. Since then, it has been known that neural stem cells exist in the human brain and participate in brain cell regeneration throughout life. Consequently, the development of therapeutic agents using neural stem cells has recently attracted attention for the treatment of degenerative neurological diseases.

[0005] At the beginning of the study of human-derived neural stem cells, neural stem cells were isolated from the fetal brain and cultured. Based on this research, clinical trials for the treatment of stroke or spinal cord injury have recently been conducted. In addition, research on the separation and cultivation of neural stem cells from embryonic stem cells has been conducted. It is known that embryonic stem cells can naturally differentiate into ectoderm, and methods for differentiating into neural stem cells have been identified. Recently, methods for differentiating into neural stem cells from induced pluripotent stem (iPS) cells have been studied, and expectations for the cultivation of neural stem cells that can be autologously transplanted are increasing.

[0006] However, there are ethical issues with using neural stem cells or embryonic stem cells derived from fetuses, including concerns about tumorigenicity. Furthermore, since neural stem cells derived from iPS cells also have tumorigenicity concerns, the clinical application of these stem cells requires verification of biological safety and fundamental solutions.

[0007] In previous studies, methods for isolating and culturing single cells from brain tissue have been widely used to isolate and culture neural stem cells.

[0008] However, methods for isolating and culturing neural stem cells, including single-cell separation using Percoll separation medium, take a long time and have problems with the amount of neural stem cells obtained. Summary of the Invention

[0009] [Technical Issues]

[0010] The present invention provides a method for efficiently isolating and culturing neural stem cells. The method can shorten the isolation and culturing time and increase the yield of neural stem cells by simplifying the isolation and culturing methods of neural stem cells.

[0011] However, the technical problems to be solved in the present invention are not limited to the above problems, and other problems not described herein will be fully understood by those of ordinary skill in the art from the following description.

[0012] [Technical Solution]

[0013] In one aspect, the present invention provides a method for efficiently isolating and culturing neural stem cells, comprising: placing brain tissue in an enzyme solution for enzyme treatment; physically dissociating cell clusters from the enzyme-treated brain tissue; separating the cell clusters by size and removing impurities; and then subculturing the cell clusters by inoculating them into culture dishes.

[0014] Additionally, the enzyme solution may include papain; which may be papain, deoxyribonuclease I (DNase I), and D,L-cysteine.

[0015] Additionally, the size of the cell cluster may be 40 to 70 μm, or greater than 70 μm and less than or equal to 100 μm.

[0016] In addition, the brain tissue may be adult brain tissue, and the method of isolating and culturing neural stem cells may not include single cell separation using a Percoll separation solution.

[0017] [Beneficial Effects]

[0018] The method for efficiently isolating and culturing neural stem cells according to the present invention can simplify the isolation and culturing process, shorten the isolation and culturing time, and increase the yield of neural stem cells. Furthermore, by shortening the isolation and culturing time, cell viability can be improved, and a large number of cells can be obtained in a short period of time, thereby increasing the success rate of culturing neural stem cells.

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram showing a conventional method for isolating and culturing neural stem cells using a Percoll separation medium and a method for isolating and culturing neural stem cells using a pellet culture according to the present invention.

[0021] Figure 2A is a graph comparing cell yield per brain tissue weight (g) between a conventional separation and culture method (Percoll) and a separation and culture method according to an aspect of the present invention (pellet culture).

[0022] Figure 2B It is a graph showing the results of confirming cell growth according to subculture of neural stem cells obtained according to a conventional separation and culture method (Percoll) and a separation and culture method according to one aspect of the present invention (clump culture).

[0023] Figure 3A Shown are the morphologies of neural stem cells according to a conventional isolation and culture method (Percoll) and the morphologies of neural stem cells according to an isolation and culture method (clump culture) according to one aspect of the present invention.

[0024] Figure 3B Shown are the differentiation patterns of neural stem cells isolated and cultured according to a conventional isolation and culture method (Percoll) and an isolation and culture method according to an aspect of the present invention (clump culture) under differentiation conditions.

[0025] Figure 4A Only the method of separating the clumps to find the optimal size condition of the clumps according to the clump culture method is shown.

[0026] Figure 4B The morphologies of four types of aggregates (agglomerates I to IV) observed using a microscope are shown.

[0027] Figure 5A and Figure 5B Shown are the numbers of colonies observed on the bottom of culture dishes three and six days after incubation of type I to IV clumps derived from two patient groups (NS18-007TL, NS18-008TL).

[0028] Figure 5C Shown are the results of confirming the colony morphology observed in culture dishes after three and six days of incubation of type I to IV clumps.

[0029] Figure 6A is a graph comparing the growth curves of cells cultured using Type II pellets and the growth curves of single cells using regular Percoll.

[0030] Figure 6BThe morphology of cells subcultured with type II clumps is shown.

[0031] Figure 6C The results of immunofluorescence staining after differentiation are shown to confirm that neural stem cells cultured using pellet type II maintain neural differentiation ability.

[0032] Figure 7A Shown are experimental results on the angiogenic capacity of type II aggregate neural stem cells.

[0033] Figure 7B Shown are the results of immunofluorescence staining for α-SMA and CD31 to confirm the location of transplanted neural stem cells. DETAILED DESCRIPTION

[0034] The present invention aims to provide a method for efficiently isolating and culturing neural stem cells, which can shorten the isolation and culturing time and improve the acquisition rate of neural stem cells by simplifying the method for isolating and culturing neural stem cells.

[0035] In one embodiment of the present invention, as a result of culturing neural stem cells according to the method of the present invention (clump culture), it has been confirmed that more neural stem cells can be obtained per the same weight of brain tissue compared to using the conventional Percoll method (see Example 1).

[0036] In another embodiment of the present invention, it was confirmed that the neural stem cells according to the method of the present invention (clump culture) maintained neural differentiation ability (see Example 3).

[0037] In yet another embodiment of the present invention, by separating and culturing the aggregates by size, the results confirmed that type II aggregates (70 to 100 μm) showed the best efficiency (see Examples 4 and 5).

[0038] In another embodiment of the present invention, when type II aggregates are cultured according to the method of the present invention (aggregate culture), it has been confirmed that the time of the first passage is shortened, the cell morphology is normal, the neural differentiation ability is normal, and the characteristics of stem cells are also well maintained (see Example 6).

[0039] In yet another embodiment of the present invention, it was demonstrated that neural stem cells derived from type II aggregates have the ability to induce angiogenesis (see Example 7).

[0040] Hereinafter, the present invention will be described in detail.

[0041] The present invention provides a method for efficiently isolating and culturing neural stem cells, which comprises: placing brain tissue in an enzyme solution for enzyme treatment; physically dissociating cell clusters from the enzyme-treated brain tissue; separating the cell clusters by size and removing impurities; and then inoculating the cell clusters into culture dishes for subculture.

[0042] Methods for isolating and culturing neural stem cells, including the use of conventional Percoll to separate single cells, are time-consuming and pose challenges in the yield of neural stem cells. Therefore, the inventors have experimentally demonstrated that introducing a method for separating cells from aggregates, rather than using Percoll to separate single cells, shortens the isolation and culturing time and increases the yield of neural stem cells, leading to the completion of the present invention.

[0043] According to one aspect of the present invention, a method for efficiently isolating and culturing neural stem cells is provided, comprising: placing brain tissue in an enzyme solution for enzyme treatment; physically dissociating cell clusters from the enzyme-treated brain tissue; separating the cell clusters by size and removing impurities; and subculturing the cell clusters by inoculating them in a culture dish.

[0044] The enzyme solution is not particularly limited and may include papain, DNase I, and D,L-cysteine.

[0045] Additionally, after dissociation from brain tissue, a mesh can be used to further separate the cell clumps by size. The size of the cell clumps can be 40 to 70 μm, or greater than 70 μm and less than or equal to 100 μm.

[0046] Furthermore, the brain tissue is preferably adult brain tissue.

[0047] As described above, the method for efficiently isolating and culturing neural stem cells according to the present invention does not include the use of Percoll to separate single cells. The method for isolating and culturing neural stem cells according to the present invention has the following advantages: by excluding the use of Percoll to separate single cells, the method for isolating and culturing neural stem cells is simplified, thereby shortening the isolation and culturing time and improving the yield of neural stem cells. Furthermore, due to the shortened isolation and culturing time, cell viability is increased, and a large number of cells can be obtained in a short period of time, thereby improving the success rate of culturing neural stem cells.

[0048] Hereinafter, in order to help understand the present invention, exemplary embodiments will be presented. However, the following embodiments are provided only to make it easier to understand the present invention, rather than to limit the present invention.

[0049] [Example]

[0050] Example 1. Primary culture of neural stem cells

[0051] After measuring the weight of human brain tissue, PBS was used to wash 2-3 times. After the tissue was homogenized to an appropriate size, the tissue was added to an enzyme solution (10 units / ml papain, 0.1mg / ml DNase I, 4mg / ml D, L-cysteine) and physically cut into smaller fragments using blades and surgical scissors. After being treated at 37°C for 30 minutes, the resulting fragments were broken up using a disposable pipette, filtered using a 70μM filter, and then washed with phosphate-buffered saline (PBS).

[0052] When Percoll was used, 10-fold concentrated PBS and Percoll were mixed at a ratio of 1:9 to prepare a Percoll solution, 20 mL of the cell suspension and 10 mL of Percoll were mixed and centrifuged at 20,000 rpm and 18°C ​​to separate the cells from the cell layer, and then washed twice again with PBS and cultured on a culture dish coated with poly-L-ornithine (PLO).

[0053] When the cells reached 70% to 80% confluence, they were dissociated using Accutase cell digestion solution and then subcultured. Cell number and viability were calculated using Tryphan Blue solution, and the population doubling time (PDL) was measured to plot a cell growth curve. The morphology of the isolated and cultured neural stem cells was observed under a microscope.

[0054] Results, such as Figure 2A As shown in FIG, it was confirmed that a larger number of neural stem cells can be obtained per the same weight of brain tissue according to the separation and culture method (pellet culture) compared with the method using Percoll, and as Figure 2B As shown, it was confirmed that the cell growth effect according to the separation and culture method (clump culture) was generally better than the cell growth effect according to the conventional method using Percoll.

[0055] In addition, if Figure 3A As shown, as a result of observing the cultured cells, it was also confirmed that according to the separation culture method (clump culture) of the present invention, the cell morphology was similar to that of the conventional method using Percoll.

[0056] Example 2. Differentiation induction under neural differentiation conditions

[0057] Neural stem cells were cultured to approximately 70% to 80% confluence and washed twice with PBS. After treatment with neural differentiation conditions such as DMEM / F12, 0.5% FBS / B27, 0.5 mM IBMX, and P / S for 48 hours, the cells were fixed and then immunostained.

[0058] Example 3. Immunostaining

[0059] Neural stem cells were cultured on PLO-coated 8-well chamber slides (Thermo) and fixed in 4% paraformaldehyde for 5 minutes. The cells were rinsed with 0.1% Triton-X 100 diluted in PBS (0.1% PBST), treated with blocking solution (5% normal goat serum, 5% normal donkey serum) for approximately 1 hour, and reacted with primary antibodies overnight. The next day, the cells were washed twice with PBS and treated with Alexa488- or Alexa594-conjugated secondary antibodies for 1 hour. After counterstaining with DAPI and mounting, the cells were observed under a fluorescence microscope.

[0060] Results, such as Figure 3B As shown, after neural stem cell differentiation, it was confirmed that nestin expression decreased and the expression of neurons (Tuj1, MAP2) and astrocytes (GFAP) increased.

[0061] Example 4. Size-dependent separation and morphological observation of aggregates

[0062] To find the optimal conditions for the pellet culture method, human brain tissue was treated with an enzyme solution, physically divided, and then filtered through meshes of various sizes to obtain pellets of varying sizes (see Figure 4A ). The agglomerates were categorized by size into types I to IV as described below and then observed under a microscope.

[0063] Type I clumps >100 μm

[0064] 70μm<Type II agglomerates<100μm

[0065] 40μm<Type III agglomerates<70μm

[0066] Type IV masses <40 μm

[0067] Results, such as Figure 4B As shown, it can be confirmed that each type is divided into agglomerates of uniform size.

[0068] Example 5. Comparison of the effects of isolation and culture of type I to IV clumps

[0069] In order to compare the effects of separation and culture based on the size of the clumps, type I to IV clumps from two patients were cultured according to the method of Example 1 to confirm the number of colonies and observe the colony morphology using a microscope.

[0070] Results, such as Figure 5A and 5B As shown, the largest number of colonies was observed in type II aggregates on days 3 and 6 of isolation and culture, confirming that type II aggregates had the best culture efficiency.

[0071] Example 6. Subculture of Type II Aggregates and Confirmation of Neural Differentiation Ability

[0072] In order to compare the effects of culturing type II aggregates by the method according to the present invention and culturing single cells using Percoll, cells were cultured according to the method of Example 1, and then a growth curve was plotted, and then the cell morphology according to the subculture of type II aggregates was observed using a microscope.

[0073] Furthermore, in order to confirm whether the neural stem cells cultured using the type II aggregate method maintain neural differentiation ability, immunofluorescence staining was performed according to the method of Example 3 after neural differentiation.

[0074] Results, such as Figure 6A As shown, it has been confirmed that in the case of type II aggregate culture, the time of the first subculture is shortened to 10 9 The time for each cell is also shortened, as well as Figure 6B As shown, the cell morphology was well maintained during subculture of type II aggregates.

[0075] In addition, as a result of immunofluorescence staining, e.g. Figure 6C As shown, the undifferentiated marker nestin was strongly expressed before neural differentiation but was barely expressed after differentiation. Furthermore, the neuronal markers Tuj1 and Map2 were not expressed before differentiation but were observed after differentiation. The astrocyte marker GFAP was barely expressed before differentiation but was observed after differentiation.

[0076] This result means that neural stem cells cultured using type II aggregates maintain good stem cell properties.

[0077] Example 7. Demonstration of angiogenesis of neural stem cells derived from type II aggregates

[0078] Umbilical vein endothelial cells were purchased from Promocell and used the manufacturer's proprietary culture medium. 1 × 10 6 Endothelial cells and 1×10 6 Neural stem cells were mixed in 200 μL of phenol red-free Matrigel matrix glue (BD) and then subcutaneously transplanted into 4 to 6 week old Balbc-nu mice. After 3 to 4 days, Matrigel was removed and then fixed with 4% PFA for 24 hours to produce clumps. For histological analysis, hematoxylin and eosin (H&E) staining was performed, and in order to stain the new blood vessels generated in the Matrigel matrix glue, vascular endothelial cells and neural stem cells were immunofluorescently stained with CD31 and α-smooth muscle actin (α-SMA), respectively.

[0079] Results, such as Figure 7AAs shown in , neural stem cells alone hardly form blood vessels, but when vascular endothelial cells and neural stem cells are mixed and transplanted, blood vessel formation can be confirmed. Figure 7B As shown, neural stem cells have been shown to be located around blood vessels and contribute to angiogenesis.

[0080] It should be understood by those skilled in the art that the above description of the present invention is exemplary, and that the exemplary embodiments disclosed herein can be easily modified into other specific forms without departing from the technical spirit or essential features of the present invention. Therefore, the above exemplary embodiments should be interpreted as illustrative and not limiting in any respect.

[0081] [Industrial Applicability]

[0082] The method for efficiently isolating and culturing neural stem cells according to the present invention can shorten the separation and culture time by simplifying the method for isolating and culturing neural stem cells, and increase the yield of neural stem cells. In addition, the advantage of the present invention is that the cell viability is increased due to the shortened separation and culture time, and the success rate of neural stem cell culture is improved by obtaining a large number of cells in a short time, so it is expected that the use of neural stem cells will contribute to the treatment of central nervous system diseases to achieve direct or indirect neural regeneration. In addition, it is expected that the present invention can also be used in fields related to direct or indirect neural regeneration by proteins or enzymes released from neural stem cells (purification of conditioned medium, development of exosomes, etc.).

Claims

1. A method for efficiently isolating and culturing neural stem cells, comprising: Adult brain tissue was placed in an enzyme solution for enzymatic treatment; Cell clusters were physically dissociated from enzymatically treated brain tissue; separating the cell aggregates by size and removing impurities; and By seeding cell clusters with a size of 70 to 100 μm into culture dishes for subculture, Wherein, the method for isolating and culturing neural stem cells does not include isolating single cells.

2. The method according to claim 1, wherein The enzyme solution contains papain, deoxyribonuclease I and D,L-cysteine.

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