KR101674253B1 - Method of isolating stem cells using transwell and chemokines - Google Patents

Abstract

The present invention relates to a method for separating stem cells having excellent tissue regeneration ability using a transwell and an inflammatory chemokine. According to the present invention, stem cells excellent in tissue regeneration ability can be easily and efficiently separated using a transwell and an inflammatory chemokine. The stem cells isolated according to the above method can be used as a pharmaceutical composition for tissue regeneration.

Description

TECHNICAL FIELD The present invention relates to a method for isolating stem cells using transwell and chemokine,

The present invention relates to a method for separating stem cells having excellent tissue regeneration ability using a transwell and an inflammatory chemokine.

Stem cell therapy market is growing very rapidly. Growth trend of stem cell sector in global industry trend is growing by 2.7% in 2012 and 5.1% in 2014. The global stem cell market is expected to reach about US $ 15 billion by 2013, and to exceed US $ 30 billion by 2018. Currently, development of stem cell treatment drugs is being actively carried out in Korea, and marketability is increasing.

Although there are many considerations in the development of stem cell therapies, it is important to selectively isolate cell lines that have excellent stem cell biologic characteristics including tissue regeneration ability.

Since early cells isolated from adult tissues are extracted and cultured as a heterogeneous group of cells containing a variety of cells including mesenchymal stem cells, a specific group of stem cells having excellent biological characteristics are extracted And it is very important to cultivate.

Conventional methods for isolating a specific population of stem cells involve several stages of subculture in order to isolate a population of cells with a purity above a certain level. However, this method has a problem that the biological characteristics of the stem cell can be degraded during the subculture. Another method is to isolate a specific antibody using a laser or an electromagnetic force. However, this method has a drawback that it does not reflect the biological characteristics including the regenerative ability of stem cells. In addition, these methods require several steps for cell separation and are not economically viable.

Therefore, there is a need for a technique for separating stem cells in a simple and efficient manner that can reflect the biological characteristics of the stem cells.

Korean Patent Application No. 2010-0124510

The present invention provides a method for separating stem cells having a simple and efficient tissue regeneration ability using a transwell and an inflammatory chemokine.

The present invention provides a method for producing a stem cell comprising the steps of: placing a cell mixture containing stem cells extracted from a living tissue in a transwell upper chamber;

Adding a culture medium and an inflammatory chemokine to a transwell lower chamber, and culturing the stem cell mixture; And

Separating the cells migrated to the transwell lower chamber and the cells remaining in the upper chamber of the transwell, and a method for separating stem cells having excellent tissue regeneration ability.

According to the present invention, stem cells excellent in tissue regeneration ability can be easily and efficiently separated using a transwell and an inflammatory chemokine. The stem cells isolated according to the above method can be used as a pharmaceutical composition for tissue regeneration.

1A is a schematic diagram showing a structure of an exemplary transwell plate and a method for separating stem cells.
FIG. 1B shows the morphological characteristics of the stem cell group (m-PDLSC) migrated to the lower chamber and the stem cell group (s-PDLSC) remaining in the upper chamber without migration.
Figures 1C and 1D show the results of observing the mobility of m-PDLSCs migrated according to chemokines.
FIGS. 1E and 1F show staining of Actin filaments in order to confirm cell migration ability in stem cells isolated according to chemokine.
FIG. 2A shows the results of analyzing the expression level of cell surface markers in the stem cells isolated according to the chemokine.
FIG. 2b shows the results of analysis of the cell proliferation rate of the stem cells isolated according to the chemokine.
FIGS. 3A and 3B show the results of analyzing the colony forming ability of the stem cells isolated according to the chemokine.
FIG. 3c shows the result of analyzing the diameter of the formed colonies in the stem cells isolated according to the chemokine.
FIGS. 4A and 4B show results of analyzing the pluripotency after inducing osteogenic differentiation in the stem cells isolated according to the chemokine.
FIGS. 4C and 4D show results of analyzing the pluripotency of the stem cells after inducing lipid differentiation according to the chemokine. FIG.
Fig. 5A shows the results of observing the regenerating ability of the isolated stem cells according to the chemokine.
FIG. 5B shows the results of analyzing the distribution of regenerated tissues in order to evaluate the tissue regenerating ability of the isolated stem cells according to the chemokine.
FIGS. 5C and 5D show the results of performing statistical analysis on the tissue regeneration ability of the isolated stem cells according to the chemokine.

The present inventors have studied cell extraction techniques for improving the extraction purity of mesenchymal stem cell (MSCs) group having excellent in vivo regenerative potential. As a result, a method of selectively separating and extracting only a specific cell group having a high bioredeveloping ability from a MSCs cell group existing as a heterogeneous group cell group by application of the homing mechanism, which is the most important biological feature of stem cells, was completed.

The stem cells migrate to the injured area and undergo a series of proliferation and differentiation processes at the damaged area to regenerate the tissue. Such cell migration is referred to as homing. That is, according to the homing theory, the cell group migrated by homing is a cell group directly participating in regeneration, so it can be said to be a cell group having high regenerative ability (excellent in stem cell characteristics) different from other cells.

Accordingly, the present invention provides a method for producing a stem cell, comprising: placing a cell mixture containing stem cells extracted from a living tissue in a transwell upper chamber;

Adding a culture medium and an inflammatory chemokine to a transwell lower chamber, and culturing the stem cell mixture; And

Separating the cells migrated to the transwell lower chamber and the cells remaining in the upper chamber of the transwell, and a method for separating stem cells having excellent tissue regeneration ability.

The term 'cell mixture containing stem cells extracted from living tissue' used in the present invention refers to a stem cell group obtained from tissues or a primary stem cell group obtained from primary tissues in order to isolate stem cells, Cells that have been isolated. The first isolated stem cell population exists as a heterogeneous group including various stem cells, hematopoietic cells, mononuclear cells, neutrophils, and the like. In addition, the stem cell group obtained by subculturing the primary stem cell population is mixed with cells that differ in the tissue regeneration ability.

Methods for extracting stem cell mixtures from living tissue are well known in the art. For example, in the case of periodontal ligament stem cells, it can be isolated from the extracted healthy teeth. Specifically, it can be separated by the following process. After washing with α-MEM (GIBCO® Grand Island, NY, USA) containing 100 U / ml penicillin and 100 μg / ml streptomycin (GIBCO®), PDL tissue was stained with 100 U / ml penicillin and 100 μg / Gibco (R) (GIBCO®) was taken from the middle 1/3 to the end of the root, which was shaved thinly. Small pieces of tissue were digested with enzyme solution 4 to 5 times at 37 ° C at 20 minute intervals. The enzyme solution contained α-MEM, 2 mg / ml collagenase type I (Waco Pure Chemical Industries, Tokyo, Japan), and 1 mg / ml Dipassase (GIBCO®). The stem cell mixture, a single-cell suspension, was obtained using a 70-μm strainer (BD Falcon ™, BD Biosciences, Bedford, MA, USA).

The 'transwell' or 'transwell plate' used in the present invention is widely used in the art, and a commercially available product may be used. Typically, a product commercially available from Corning Incorporated may be used. Detailed information about the product can be obtained from the product catalog or website (www.corning.com) provided by Corning Incorporated. In fact, information and usage of the transwell plate can be obtained from a person skilled in the art It is within the skill level.

1A, the transwell plate has an upper chamber (also referred to as a product catalog or a transwell insert in the art) having a lower end made of a microporous membrane and a lower chamber ), The upper chamber is located inside the lower chamber, and the two chambers share the culture medium. Here, the microporous membrane is a support through which a cellular material such as a cell secretory material, a culture medium, and stem cells can permeate. The size of the microporous membrane can be suitably selected by those skilled in the art. For example, Or 8 탆.

The microporous membrane constituting the bottom surface of the transwell upper chamber is a permeable support capable of supporting cell culture and growth thereon, and may be a membrane made of materials conventionally used in the art. For example, polycarbonate , Polyester, collagen-coated polytetrafluoroethylene, or the like can be used as the microporous membrane.

In the present invention, the 'inflammatory chemokine' is a type of inflammatory cytokine, which functions as a chemotactic activity, that is, a humectant inducing factor, which collects certain kinds of cells. When a body is injured, an inflammatory chemokine, which is accompanied by inflammation of the wound area, carries an injury signal to collect cells for wound healing. That is, stem cells capable of regenerating tissue can be selectively isolated by a homing mechanism mediated by specific inflammatory chemokines.

Inflammatory chemokines as humming inducers are well known in the art and are known to induce the migration of endogenous cells present in the body by transplanting or direct injection of inflammatory chemokines into the damaged site, In the production of a stem cell therapeutic agent, it is intended to select a specific cell group having a high regenerative power at the cell extraction and culture stage, and it is different from the existing technology.

In one embodiment, the amount of cell mixture comprising stem cells extracted from a living tissue may be from 1 X 10 ⁴ cells to 1 X 10 ⁶ cells.

In one embodiment, the type of cell mixture containing the stem cells is not limited. For example, the cell mixture containing the stem cells may be extracted from one or more tissues selected from the group consisting of dental, alveolar bone, gingiva, dentate, periodontal ligament, bone marrow, fat, stomach, liver, pancreas and nerves.

In one embodiment, the culturing of the stem cell mixture may be for 3 to 5 days.

The growth medium to be added to the lower chamber can be used without limitation in the culture medium of stem cells known in the art. The types of such media are well known in the art. For example, the medium may be alpha-MEM.

In one embodiment, the inflammatory chemokine may be one or more selected from the group consisting of RANTES, IL-8, and SDF-1.

Depending on the type of inflammatory chemokine, the characteristics of the cell population moving to the lower chamber and the cell population remaining in the upper chamber may vary. The cell morphology, or cell mobility, of the cell group (m-PDLSC) migrating to the lower chamber and the cell group (s-PDLSC) remaining in the upper chamber without migration were analyzed, (Example 1). These results demonstrate that the isolation of stem cells by chemokines is efficacious.

The biological characteristics (Example 2), colony forming ability (Example 3) and multipotency (Example 4) of the separated stem cells according to each chemokine were analyzed. As a result, all of the separated cell groups showed similar biological characteristics. In particular, it was found that the m-PDLSC group migrated by RANTES had the best stem cell characteristics in terms of colony forming ability and multipotentiating ability. The m-PDLSC group migrated by RANTES maintained the least differentiated state.

In addition, tissue regeneration ability of the separated cell groups according to each chemokine was analyzed. As can be seen in Example 5 below, when the inflammatory chemokine is RANTES or IL-8, the stem cells migrated to the transwell lower chamber are superior to the stem cells remaining in the upper chamber. Therefore, the stem cells migrated to the lower chamber can be selected and separated to select stem cells having excellent tissue regeneration ability.

On the other hand, when the inflammatory chemokine is SDF-1, the stem cells remaining in the transwell upper chamber are a group of cells having excellent tissue regeneration ability. Therefore, the stem cell group remaining in the upper chamber can be selected and separated to select stem cells having excellent tissue regeneration ability (Example 5).

The stem cells isolated by the above method can be used variously for tissue regeneration since they are a group of cells having excellent tissue regeneration ability. That is, the stem cells isolated according to the above method are useful for patients who need tissue regeneration such as wound healing and soft tissue defects as a tissue regeneration pharmaceutical composition.

In the pharmaceutical composition for tissue regeneration, the content of the stem cells may be 1 x 10 ⁴ to 5 x 10 ¹⁰ cells per 1 ml of the composition, though not limited thereto. The total amount of the stem cells to be transplanted at the time of the procedure can be easily determined by those skilled in the art considering the condition of the patient.

The medicinal composition for tissue regeneration according to the present invention is very useful when cell transplantation is required for correction of soft tissue defects such as wound healing, wrinkles, filling for male sexual function improvement, breast enlargement for women, and incontinence treatment Can be used. Correction of soft tissue defects can be achieved by directly implanting the tissue regeneration pharmaceutical composition of the present invention in areas such as face, face including non-contiguous wrinkles, lines around mouth, machete line, dermis, And the implantation site is not limited thereto.

The pharmaceutical composition for tissue regeneration of the present invention can be administered intradermally, subcutaneously, or intramuscularly.

The tissue regeneration medicinal composition of the present invention can be administered to a subject by any means known to those skilled in the art and can be administered intradermally, subcutaneously, or intramuscularly, for example, by being loaded in a delivery device such as a syringe, Or other techniques that allow delivery to a particular target site of the subcutaneous space within the breast. Preferably a needle or a catheter, or directly by a surgical implant, and a composition for cell implantation comprising stem cells cultured in an embodiment of a surgical implant may be used together with additives such as a preformed matrix.

The medicinal composition for tissue regeneration of the present invention can be used as a medicinal composition for regenerating tissues, such as a cell carrier, a fragment of another cell, a tissue, a tissue, a growth factor or a cytotoxic agent necessary for cell adhesion, a living or inert compound, a reabsorbable scaffold, And other additives for enhancing potency, tolerance, or function.

Hereinafter, the present invention will be described in detail with reference to examples. The following examples illustrate the invention and are not intended to limit the scope of the invention.

[ Example ]

[ Example  One] Transwell  And inflammatory Chemokine  Isolation of stem cells used

FIG. 1A shows a schematic diagram of a method for separating stem cells according to the present invention. The human periodontal ligament stem cell (hPDLSC) group (1 × 10 ⁴ cells to 1 × 10 ⁶ cells) obtained by extracting from the root surface of teeth and subculturing was inoculated into an upper chamber And a growth medium containing inflammatory chemokines (50 ng / ml IL-8, 150 ng / ml RANTES, 150 ng / ml SDF-1 and 10 ng / ml FGF-2, respectively) And cultured for 3 days. As the growth medium, α-MEM growth medium (containing 15% fetal bovine serum, 100 uM L-ascorbic acid-2-phosphate, 2 mM L-glutamine, 100 U / ml penicillin, 100 ug / ml streptomycin) was used.

As a result, some cell groups were separated into m-PDLSC (migrated periodontal ligament stem cell) and s-PDLSC (static-PDLSC), which were not migrated, depending on the characteristics of the chemokine. The morphological differences of the stem cells were observed and the actin filaments showing the number of cells moved to the lower chamber and the cell mobility were stained and examined to confirm whether the separated stem cells exhibited different characteristics according to mobility. The morphological observation results using 150 ng / ml of RANTES are shown in Fig. 1B. As a result, it was found that the m-PDLSC cells migrated morphologically different phenotypes compared to the non-migrated s-PDLSC cells in the upper chamber (Fig. 1B).

The results of observation of cell mobility using 50 ng / ml IL-8, 150 ng / ml RANTES, 150 ng / ml SDF-1 and 10 ng / ml FGF-2 are shown in Fig. 1c and Fig. 1d. The m-hPDLSC stained with m-hPDLSC passed through the chamber was observed to show that the shape of the m-hPDLSC transferred after chemokine treatment was larger than that of s-hPDLSC, which did not migrate, (Fig. 1 (c)).

In the m-hPDLSC, the number of cells in arbitrarily selected 10 areas was measured and the average value was calculated. As a result, a statistically significant shift of hPDLSC was observed in the chemokine treated group compared to the control group without chemokine treatment (Fig. 1d).

These results indicate that the m-PDLSC cells have different characteristics to be transferred according to the characteristics of the chemokine given to the lower chamber. In particular, it was confirmed that the most cells migrated by the chemokine gradient of RANTES (Fig. 1C and Fig. 1D).

HPDLSC and s-hPDLSC isolated according to 50 ng / ml IL-8, 150 ng / ml RANTES, 150 ng / ml SDF-1 and 10 ng / ml FGF- , Cultured in a 2-well slide chamber for 5 days, stained with F-actin for observation (FIG. 1 e), and the cell fluorescence intensities of 10 randomly selected regions were measured to calculate an average value ).

As a result, m-hPDLSC under RANTES / CCL5 treatment showed the strongest staining intensity of F-actin compared to other chemokine treatment groups. s-hPDLSC showed low immunofluorescence staining intensity in a small and thin cell shape, and m-hPDLSC showed high staining intensity in a large, branched cell type.

These results indicate that the actin filament composition of m-PDLSC and s-PDLSC varies depending on the type of chemokine, among which actin filaments of m-PDLSC induced by RANTES are most developed (Fig. 1E and Fig. 1E) 1f).

That is, it is a result of using transwells to isolate a specific cell group according to the chemokine and to show that the characteristics of the isolated stem cells are different.

[ Example  2] To chemokine  Identification of biological characteristics of isolated stem cells

In order to analyze the stem cell biology characteristics of m-PDLSC and s-PDLSC separated by chemokine, we analyzed the expression levels of CD44, CD73, CD90 and CD105 as surface markers that characterize stem cells.

Human periodontal ligament stem cells were cultured in the medium containing each chemokine under the same conditions as in Example 1. After 3 days of culture, m-hPDLSC, which was moved through the 8-um pore of the chamber, was separated from the un-migrated s-hPDLSC of the upper chamber and analyzed by using flow cytometry to determine the cell surface markers CD44, CD73, Immunophenotypic analysis of CD90 and CD105 was performed.

As a result, both of the surface markers were expressed in good numbers in both the m-PDLSC and s-PDLSC cell groups, and there was no significant difference according to the chemokine (Fig. 2A). This shows that the cell sorting by chemokine does not affect the stem cell characteristics represented by surface markers.

In addition, cell proliferation was analyzed by another biological characteristic of stem cells. After 3 days of culture, m-hPDLSC and untransferred s-hPDLSC in the upper chamber were separated and the proliferation rates for 1, 4 and 7 days were confirmed by MTT assay.

As a result, on day 7 of culture, the cell proliferation rate was increased in all the cell groups (Fig. 2B). This means that the biological properties of the stem cells are maintained in all groups regardless of the type of chemokine treated in the lower chamber. In addition, there is no difference in the basic stem cell characteristics according to the type of chemokine, but it shows that there is a difference in cell activity.

[ Example  3] To chemokine  Stem cell characterization of isolated stem cells - Colony Formability  Confirm

The colony forming ability (CFE) was analyzed to determine whether the stem cell characteristics of the isolated stem cells varied depending on the type of chemokine.

Human periodontal ligament stem cells were cultured under the same conditions as in Example 1. After 3 days, the m-hPDLSC moved through the 8-um pores of the chamber and the un-migrated s-hPDLSC of the upper chamber were separated and stained with crystal violet in a 55 cm ² dish for 14 days, Respectively. The number and diameter of each colonies were measured and the mean value was calculated. The larger number of colonies and the larger the diameter, the higher the colony forming ability.

Microscopic observations indicated that the CFE was increased in the chemokine treated groups compared to the control without chemokine, and the size of the colonies was larger (FIG. 3a). Also, counting the number of colonies (number of colonies) showed statistically significant increase in chemokine treated groups compared to the untreated control group (FIG. 3b). In addition, it was confirmed that the size of colonies and colonies was larger in m-hPDLSC than in s-hPDLSC (Fig. 3C). These results indicate that cells transferred by chemokines exhibit stem cell character and exhibit higher activity than m-PDLSC.

[ Example  4] To chemokine  Identification of Stem Cell Features of Isolated Stem Cells - Multivability ( 다피턴ency ) Confirm

Human periodontal ligament stem cells were cultured under the same conditions as in Example 1, and the pluripotency of the stem cells isolated according to the chemokine was analyzed as another stem cell characteristic.

m-hPDLSC and s-hPDLSC were separated and cultured in 6-well plate for 3 weeks. After 3 weeks, calcified nodules formed by alizarin red staining were identified, and 10 regions randomly selected were measured and the average value was calculated.

As a result, the mineralized nodule formed after induction of osteogenic differentiation was measured lower in m-PDLSC, which was generally migrated, and the lowest in m-PDLSC moved by RANTES / CCL5 and SDF-1 / CXCL12 (Figs. 4A and 4B).

In addition, m-hPDLSC and s-hPDLSC were separated and cultured for induction of lipid differentiation in 6-well plate for 3 weeks. After 3 weeks, lipid droplets formed by oil red O staining were observed, and 10 regions randomly selected were measured and the average value was calculated.

In addition, when lipid differentiation was analyzed, the formation of fat droplets of migrated m-PDLSC was lower compared to s-PDLSC (Fig. 4c and Fig. 4d) (but not statistically significant).

Taken together, these results indicate that m-PDLSCs, which are transferred by chemokines, exhibit distinctive biological properties. Among them, the m-PDLSCs migrated by RANTES possessed more distinctive characteristics, and they were found to be less differentiated than the other groups.

[ Example  5] To chemokine  The structure of the separated stem cells Playability  Confirm

The regenerative capacity of isolated stem cells according to chemokine was compared and analyzed. Cells of S-hPDLSC or m-hPDLSC (6X10 ⁶ ) were transplanted into the subcutaneous tissues of nude mice along with 80 mg of hydroxyapatite / tricalcium phosphate cell donors and after 8 weeks of healing time After grafting, grafts were histologically analyzed. After histological treatment such as extraction fixation, demineralization, and paraffin embedding, tissue slides were prepared for each of the extractions, and histone analysis was performed by optical microscopy with Masson-Trichrome staining. Histologic regeneration of hPDLSC is the most important factor in cementum formation, so that neoplastic cementum regeneration can be assessed as tissue regeneration ability. Histologic measurements were made and analyzed by each group. As a result, it was confirmed that different tissue regeneration ability was shown depending on the separated cell group (FIG. 5A).

When the distribution of the regenerated tissues was measured, differential regeneration characteristics were shown according to the separated cell groups (Fig. 5B). Regarding the distribution of regenerated tissues, 10 regions were selected from each tissue slide, and a 5 × 5 grid template (60 × 80 μm / grid) was superimposed on the new cementum grid to calculate the grid, Respectively. The width measured on each slide was defined as the distribution of the slide. This procedure was performed using computer-aided image-analysis software. As a result, the m-PDLSC group after treatment with IL-8 and RANTES showed higher tissue regeneration ability than the s-PDLSC group. In particular, m-PDLSC cells migrated by RANTES exhibited significantly higher tissue regeneration ability, whereas the non-migrated s-PDLSC tissue regeneration capacity was relatively small. This means that the m-PDLSCs migrated by RANTES have very high tissue regeneration capability.

On the other hand, the cell group separated by SDF-1 showed the opposite phenomenon. The m-PDLSCs migrated by SDF-1 had little tissue regeneration ability, whereas the s-PDLSC tissue regeneration ability was significantly higher. This suggests that SDF-1 inhibits the migration of cells with high tissue regeneration ability.

To confirm the tissue regeneration ability as described above, metrological statistical analysis was performed. ANOVA and post hoc t test (ANOVA and post hoc t test) were performed for each group and unpaired t-test was performed for comparison between the two groups. As a result, the same results as those of the tissue regeneration ability confirmation test were obtained (Figs. 5C and 5D).

Compared to the s-PDLSC group which did not migrate to the lower chamber after treatment with the inflammatory chemokines IL-8 and RANTES, the m-PDLSC group migrated to the lower chamber showed superior tissue regeneration ability, . In contrast, when SDF-1 was treated, it was confirmed that s-PDLSC, which did not migrate to the lower chamber, had superior tissue regeneration ability as compared with the m-PDLSC group which migrated to the lower chamber.

These results indicate that stem cells with superior tissue regeneration ability can be isolated according to the inflammatory chemokines, and that such separated cells can be used for tissue regeneration purposes.

Claims (7)

Placing a cell mixture containing stem cells extracted from a living tissue in a transwell upper chamber;
Adding a culture medium and an inflammatory chemokine to a transwell lower chamber, and culturing the stem cell mixture;
Separating the cells migrated to the transwell lower chamber and the cells remaining in the transwell upper chamber; And
Selecting stem cells that have migrated to the transwell lower chamber when the inflammatory chemokine is RANTES and selecting stem cells remaining in the transwell upper chamber when the inflammatory chemokine is SDF-1, A method for separating excellent stem cells.
The stem cell-containing cell mixture according to claim 1, wherein the cell mixture containing stem cells is extracted from at least one living tissue selected from the group consisting of dental, alveolar bone, gingiva, dentate, periodontal ligament, bone marrow, fat, stomach, liver, pancreas, A method of separating stem cells having excellent tissue regeneration ability.
The method according to claim 1, wherein the culture medium is? -MM.
delete The method of separating stem cells according to claim 1, wherein the cell mixture containing the stem cells is cultured for 3 to 5 days.
delete delete

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