KR102476348B1 - A Method for Isolating Tissue-Resident uPAR+/Nestin+ Stem Cells and Their Uses - Google Patents

Abstract

The present invention relates to a method for separating and culturing tissue-intrinsic uPAR+/Nestin+ stem cells and a use thereof, and provides a method for isolating tissue-intrinsic stem cells present in solid tissues based on uPAR-plasmin activity. The uPAR-plasmin activity of stem cells is closely related to the growth, migration ability, physiological activity, and differentiation ability of stem cells, and can be used as a method for isolating high-titer stem cells. The uPAR+ stem cells isolated from the solid tissue of the present invention have great industrial potential as they can be applied to the production of biopharmaceuticals using secretions including cell therapy agents, tissue engineering therapeutic agents, and exosomes. In addition, in the present invention, it can be applied to cell biology, molecular biology basic research and new drug development research related to cell division, migration, growth, and differentiation of tissue endogenous stem cells.

Description

Method for isolating and culturing tissue-intrinsic uPAR+/Nestin+ stem cells and their uses {A Method for Isolating Tissue-Resident uPAR+/Nestin+ Stem Cells and Their Uses}

The present invention relates to a method for isolating and culturing uPAR+/Nestin+ tissue endogenous stem cells, which play a key role in tissue regeneration and tissue homeostasis in solid tissues, and uses thereof.

A conventional method for isolating and culturing stem cells present in solid tissue is a single cell suspension ( A process for manufacturing a single cell suspension) is required. In the tissue dissociation process, the yield of single cells obtained varies greatly depending on the type of tissue degradation enzyme used, the titer, the reaction time, the reaction temperature, and the type of tissue used. In addition, cell damage during the tissue dissociation process has been raised as an unavoidable problem. As a result, the tissue dissociation process showed a large difference in cell yield and degree of damage depending on the tissue type, and presented a disadvantage that it was difficult to standardize. In particular, the frequency of stem cells in solid tissue is less than 0.1%, and it is widely known that the yield of isolating an effective amount of stem cells through tissue degradation enzymes is extremely low.

A subsequent process of separating and purifying the tissue endogenous stem cells from the single cell suspension dissociated from the tissue is required. The process of separating and purifying stem cells uses markers such as c-Kit or Sca-1 to separate cells positive or negative for the marker. However, these markers are markers that are expressed not only in tissue-intrinsic stem cells but also in hematopoietic cells, so unwanted cells can be mixed and separated. Also, since stem cells negative for these markers definitely exist, tissue-intrinsic stem cells using specific markers Methods for isolating and purifying cells have limitations.

Frontiers in Cell and Developmental Biology. 2022; 10: 818616

An object of the present invention is to activate tissue endogenous uPAR+/nestin+ stem cells located in solid tissues such as adipose tissue, bone marrow tissue, myocardial tissue, peripheral nerve tissue, skeletal muscle tissue or synovial tissue, induce migratory growth, and isolate to provide a way to do it.

In addition, another object of the present invention is to provide tissue endogenous uPAR+ and nestin+ stem cells or a culture solution thereof separated and cultured according to the above method.

In addition, another object of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory diseases or autoimmune diseases, comprising the tissue-intrinsic uPAR+ and nestin+ stem cells or their culture medium as an active ingredient.

In addition, another object of the present invention is to provide a pharmaceutical composition for wound healing or vascular regeneration promotion, comprising the tissue-intrinsic uPAR+ and nestin+ stem cells or their culture medium as an active ingredient.

In order to achieve the above object, the present invention comprises the steps of (1) preparing a wound repair matrix mimic hydrogel; (2) incorporating the separated tissue sections into the wound repair matrix mimicking hydrogel; and (3) three-dimensionally culturing the wound repair matrix-mimicking hydrogel incorporating the tissue fragment in a culture medium to which a plasminogen activator inhibitor (PAI) is added. provides an activation method.

In addition, the present invention comprises the steps of (1) preparing a wound repair matrix mimic hydrogel; (2) incorporating the separated tissue sections into the wound repair matrix mimicking hydrogel; (3) three-dimensionally culturing the wound repair matrix mimicking hydrogel incorporating the tissue slice in a culture medium to which PAI is added; (4) removing and washing the three-dimensional culture medium to remove PAI; (5) degrading the wound repair matrix mimicking hydrogel by re-cultivating the PAI-free culture medium with a PAI-free culture medium; and (6) isolating stem cells released from the culture medium.

In addition, the present invention provides tissue endogenous uPAR+ and nestin+ stem cells or a culture solution thereof, separated and cultured according to the above method.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising the tissue-specific uPAR+ and nestin+ stem cells or their culture medium as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of autoimmune diseases comprising the tissue-specific uPAR+ and nestin+ stem cells or their culture medium as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for wound treatment comprising the tissue-endogenous uPAR+ and nestin+ stem cells or their culture medium as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for promoting blood vessel regeneration comprising the tissue-endogenous uPAR+ and nestin+ stem cells or their culture medium as an active ingredient.

The present invention relates to a method for separating and culturing tissue-intrinsic uPAR+/Nestin+ stem cells and a use thereof, and provides a method for isolating tissue-intrinsic stem cells present in solid tissues based on uPAR-plasmin activity. The uPAR-plasmin activity of stem cells is closely related to the growth, migration ability, physiological activity, and differentiation ability of stem cells, and can be used as a method for isolating high-titer stem cells. The uPAR+ stem cells isolated from the solid tissue of the present invention have great industrial potential as they can be applied to the production of biopharmaceuticals using secretions including cell therapy agents, tissue engineering therapeutic agents, and exosomes. In addition, in the present invention, it can be applied to cell biology, molecular biology basic research and new drug development research related to cell division, migration, growth, and differentiation of tissue endogenous stem cells.

Figure 1 shows the expression rates of pFAK, uPAR, nestin, and Ki-67 in constituent cells in organs after in vitro organ culture. 2D, monolayer organ culture; 3D, 3D organ culture; AT, adipose tissue; BM, bone marrow; Myocar, myocardium; PN, peripheral nerve; SM, skeletal muscle; Syn, Synovium. **, p < 0.01 versus 2D.
Figure 2 shows the characteristics of increasing pFAK+, uPAR+, and Nestin+ expressing cells in the myocardium according to 3D myocardial organ culture.
Figure 3 shows the characteristics of increasing ki-67 positive cells during cell division in the myocardium according to 3D organ culture.
4 shows that the number of pFAK, uPAR, nestin, and ki-67-positive cells in the myocardium increased proportionally according to the 3D myocardial organ culture period. **, p < 0.01 versus 0 d (before culture).
Fig. 5 shows uPAR, nestin and BrdU expression of cells grown in hydrogel after 3D myocardial organ culture.
Figure 6 shows the uPAR and nestin expression rates of cells grown by migration into hydrogels after 2 weeks of 3D organ culture. AT, adipose tissue; BM, bone marrow; Myocar, myocardium; PN, peripheral nerve; SM, skeletal muscle; Syn, Synovium.
7 shows uPAR mRNA (A) and plasmin activity (B) in organs before and after 3 days of organ culture. AT, adipose tissue; BM, bone marrow; Myocar, myocardium; PN, peripheral nerve; SM, skeletal muscle; Syn, Synovium. *, p < 0.05 versus pre-culture; **, p < 0.01 versus pre-culture.
Figure 8 shows that the hydrogel is degraded through the removal of PAI in the culture medium and washing of the culture medium after long-term culture, and the cells that have migrated and grown in the hydrogel are released into the culture medium.
Figure 9 shows that the cells that have migrated and grown in the hydrogel are separated from the hydrogel through the removal of PAI in the culture medium and washing of the culture medium, and the free cells are contracted and aggregated (phase contrast micrograph).
Figure 10 shows the isolation of free uPAR+ cells in the hydrogel after PAI removal and washing of the culture (paraffin section HE staining and uPAR immunohistochemical staining).
Figure 11 shows the cell yield and uPAR expression rate of the cells separated and harvested after PAI removal and culture washing (PAI withdrawn) and separation from the hydrogel after the addition of exogenous Urokinase. AT, adipose tissue; BM, bone marrow; Myocar, myocardium; PN, peripheral nerve; SM, skeletal muscle; Syn, Synovium. **, p < 0.01 versus urokinase.
FIG. 12 shows the cell yield obtained after repeatedly separating the tissue slices recovered from the hydrogel through repetitive long-term culture after removing PAI and washing the culture. AT, adipose tissue; BM, bone marrow; Myocar, myocardium; PN, peripheral nerve; SM, skeletal muscle; Syn, Synovium.
Figure 13 shows the cell attachment and growth in a monolayer culture environment after seeding the cells harvested after PAI removal and culture washing in a culture container. A, Cell aggregates released from the hydrogel after PAI removal and washing. B, Cell aggregates harvested from the hydrogel. CE, Attachment and growth of cells at 30 minutes (C), 1 hour (D), and 2 hours (D) after seeding the recovered cells in the culture container (phase contrast micrographs).
14 shows adipose tissue, bone marrow, myocardium, nerve, skeletal muscle, and synovial organ culture, PAI removal and culture washing, and seeding of cells advantageous to hydrogel and recovered cells, followed by attachment to a culture container and growth It shows the result of attaching and growing after seeding (bottom) (phase contrast micrograph).
Figure 15 shows adhesion and growth in a monolayer culture environment of cells released from the hydrogel after removal of PAI and washing of the culture after nerve and myocardial organ culture.
16 shows immunorepresentation characteristics of cells recovered from hydrogels. A, Immunoexpression characteristics of cells released after withdrawal from hydrogel after removal of PAI and washing of culture. B, Immunoexpression characteristics of cells released from the hydrogel after exogenous Urokinase treatment. AT, adipose tissue; BM, bone marrow; Myocar, myocardium; PN, peripheral nerve; SM, skeletal muscle; Syn, Synovium. **, p < 0.01 versus urokinase (B).
17 shows the expression rates of hematopoietic cell and vascular endothelial cell markers in cells recovered from the hydrogel. A, Expression rate of cells isolated and recovered after removal of PAI and washing of the culture. B, Expression rate of cells isolated and recovered after treatment with exogenous Urokinase. AT, adipose tissue; BM, bone marrow; Myocar, myocardium; PN, peripheral nerve; SM, skeletal muscle; Syn, Synovium. **, p < 0.01 versus urokinase (B).
18 shows the self-renewal ability of cells separated and recovered from the hydrogel. A; Representative photographs of Colony Forming Unit (CFU). B; CFU frequency of cells separated and harvested from hydrogels according to tissue origin. Cells isolated and recovered after treatment with urokinase and exogenous urokinase; Cells isolated and recovered after PAI withdrawal, PAI removal, and culture washing; AT, adipose tissue; BM, bone marrow; Myocar, myocardium; PN, peripheral nerve; SM, skeletal muscle; Syn, Synovium. **, p < 0.01 versus urokinase.
19 shows the in vitro growth ability of cells separated and recovered from the hydrogel. A; Population Doubling Time (PDT, cell doubling time). B; Population Doubling Level (PDL, cell doubling level). Cells isolated and recovered after treatment with urokinase and exogenous urokinase; Cells isolated and recovered after PAI withdrawal, PAI removal, and culture washing; AT, adipose tissue; BM, bone marrow; Myocar, myocardium; PN, peripheral nerve; SM, skeletal muscle; Syn, Synovium. *, p < 0.05 versus urokinase; **, p < 0.01 versus urokinase.
20 shows the Ki-67 expression rate of cells separated and recovered from the hydrogel. Cells isolated and recovered after treatment with urokinase and exogenous urokinase; Cells isolated and recovered after PAI withdrawal, PAI removal, and culture washing; AT, adipose tissue; BM, bone marrow, Myocar, myocardium; PN, peripheral nerve; SM, skeletal muscle; Syn, Synovium. **, p < 0.01 versus urokinase.
21 shows the differentiation ability of the cells separated and recovered from the hydrogel into osteoblasts (alizarin red) and adipocytes (Oil Red O). Cells isolated and recovered after treatment with urokinase and exogenous urokinase; Cells isolated and recovered after PAI withdrawal, removal of PAI, and washing of the culture.
22 shows a comparison of the differentiation abilities of cells separated and recovered from the hydrogel into osteoblasts (alizarin red) and adipocytes (Oil Red O). Cells isolated and recovered after treatment with urokinase and exogenous urokinase; Cells isolated and recovered after PAI withdrawal, PAI removal, and culture washing; AT, adipose tissue; BM, bone marrow; Myocar, myocardium; PN, peripheral nerve; SM, skeletal muscle; Syn, Synovium. **, p < 0.01 versus urokinase.
Figure 23 shows the tissue regeneration mRNA expression characteristics of the cells separated and recovered from the hydrogel. Cells isolated and recovered after treatment with urokinase and exogenous urokinase; Cells isolated and recovered after PAI withdrawal, PAI removal, and culture washing; CB-MSC, cord blood-derived mesenchymal stem cells; AT, adipose tissue; BM, bone marrow; Myocar, myocardium; PN, peripheral nerve; SM, skeletal muscle; Syn, Synovium. *, p < 0.05 versus urokinase; **, p < 0.01 versus urokinase.
24 shows the plasmin activity and nestin expression of cells separated and recovered from the hydrogel. uPAR-, uPAR-negative cells isolated and purified after treatment with exogenous urokinase; uPAR+, uPAR+ cells isolated and purified after treatment with exogenous urokinase; Cells isolated and harvested after PAI withdrawal, PAI removal and washing of the culture; AT, adipose tissue; BM, bone marrow; Myocar, myocardium; PN, peripheral nerve; SM, skeletal muscle; Syn, Synovium. *, p < 0.05 versus uPAR-; **, p, uPAR- versus 0.01. *, p < 0.05 versus uPAR-; **, p < 0.01 versus uPAR-.
25 shows self-renewal (CFU) and in vitro growth (Ki-67) abilities of cells separated and recovered from hydrogels. uPAR-, uPAR-negative cells isolated and purified after treatment with exogenous urokinase; uPAR+, uPAR+ cells isolated and purified after treatment with exogenous urokinase; Cells isolated and harvested after PAI withdrawal, PAI removal and washing of the culture; AT, adipose tissue; BM, bone marrow; Myocar, myocardium; PN, peripheral nerve; SM, skeletal muscle; Syn, Synovium. *, p < 0.05 versus uPAR-; **, p, uPAR- versus 0.01. *, p < 0.05 versus uPAR-; **, p < 0.01 versus uPAR-.
26 shows the anti-inflammatory ability of cells separated and recovered from the hydrogel. Inhibiting the secretion of TNFα and IL-1β in RAW 264.7 cells sensitized with LPS in the conditioned medium. uPAR-, uPAR-negative cells isolated and purified after treatment with exogenous urokinase; uPAR+, uPAR+ cells isolated and purified after treatment with exogenous urokinase; Cells isolated and harvested after PAI withdrawal, PAI removal and washing of the culture; AT, adipose tissue; BM, bone marrow; Myocar, myocardium; PN, peripheral nerve; SM, skeletal muscle; Syn, Synovium. *, p < 0.05 versus uPAR-; **, p, uPAR- versus 0.01. *, p < 0.05 versus uPAR-; **, p < 0.01 versus uPAR-.
27 shows the growth inducing effect of vascular endothelial cells (HUVEC) and fibroblasts (DF) of the cells separated and recovered from the hydrogel. uPAR-, uPAR-negative cells isolated and purified after treatment with exogenous urokinase; uPAR+, uPAR+ cells isolated and purified after treatment with exogenous urokinase; Cells isolated and harvested after PAI withdrawal, PAI removal and washing of the culture; AT, adipose tissue; BM, bone marrow; Myocar, myocardium; PN, peripheral nerve; SM, skeletal muscle; Syn, Synovium. *, p < 0.05 versus uPAR-; **, p, uPAR- versus 0.01. *, p < 0.05 versus uPAR-; **, p < 0.01 versus uPAR-.
28 shows the cytoprotective effect of vascular endothelial cells (HUVEC) and fibroblasts (DF) of cells separated and recovered from the hydrogel. uPAR-, uPAR-negative cells isolated and purified after treatment with exogenous urokinase; uPAR+, uPAR+ cells isolated and purified after treatment with exogenous urokinase; Cells isolated and harvested after PAI withdrawal, PAI removal and washing of the culture; AT, adipose tissue; BM, bone marrow; Myocar, myocardium; PN, peripheral nerve; SM, skeletal muscle; Syn, Synovium. *, p < 0.05 versus uPAR-; **, p, uPAR- versus 0.01. *, p < 0.05 versus uPAR-; **, p < 0.01 versus uPAR-.
29 shows a schematic diagram of the present invention compared to the existing technology.

The present invention comprises the steps of (1) preparing a hydrogel mimicking a wound repair matrix; (2) incorporating the separated tissue sections into the wound repair matrix mimicking hydrogel; and (3) three-dimensionally culturing the wound repair matrix-mimicking hydrogel incorporating the tissue fragment in a culture medium to which a plasminogen activator inhibitor (PAI) is added. provides an activation method.

Preferably, the wound repair matrix mimicking hydrogel is a fibrin hydrogel in which a 0.25 to 2.5% concentration of a fibrinogen solution and a 0.5 to 5 I.U./mL concentration of a thrombin solution are mixed, and a 0.1 to 0.5% concentration of collagen in the fibrin hydrogel. It may be a fibrin/collagen mixed hydrogel in which the solution is mixed or a fibrin/gelatin mixed hydrogel in which a 0.1 to 0.5% gelatin solution is mixed with the fibrin hydrogel, but is not limited thereto.

Preferably, the tissue may be adipose tissue, bone marrow tissue, myocardial tissue, peripheral nerve tissue, skeletal muscle tissue or synovial tissue, but is not limited thereto.

Preferably, the PAI may be tranexamic acid or aminomethyl benzoic acid, but is not limited thereto.

Preferably, the method activates integrin-FAK cell signal transduction of cells in the tissue to induce cell division and cell growth of tissue endogenous uPAR+ and nestin+ stem cells, but is not limited thereto.

Preferably, the method may induce cell migration and cell growth of the tissue-intrinsic uPAR+ and nestin+ stem cells into the wound repair matrix-mimicking hydrogel, but is not limited thereto.

In order to induce migration of uPAR+ and nestin+ tissue endogenous stem cells of the present invention to a wound repair matrix, a tissue-specific wound repair matrix is required. It can be prepared using biopolymers by mimicking wound repair matrix components formed by coagulation of blood plasma released from blood vessels after tissue damage. In the present invention, the wound repair matrix may be prepared by using fibrin, collagen, or gelatin alone or in combination. Engineered wound repair substrates have different uPA-plasmin activity depending on the organ, and wound repair substrates resistant to uPA-plasmin activity can be manufactured and used. The wound repair matrix can be prepared by adjusting the content or composition of biopolymers. That is, a wound repair matrix mixed with two or more components such as fibrin-collagen, fibrin-gelatin, or collagen-gelatin may be used. The wound repair matrix of the present invention may be constituted in an amount of 1.0 to 20.0 mg/ml of constituent polymers such as fibrinogen, collagen, and gelatin. The degree of crosslinking can be adjusted to enhance the structural properties of the wound repair matrix, and structural properties can be controlled by adjusting the degree of crosslinking of the wound repair matrix using a crosslinking agent such as Ca++ or Factor XIIIa.

Activation of cell signaling pathways is required to induce cell division and growth of stem cells in tissues through in vitro culture. It binds to the cell's integrin ligand and transmits and activates signals that can induce cell growth and migration through the wound repair matrix. It can induce cell division, growth and migration of stem cells in tissue as it supports tissue as a wound repair matrix that can directly bind to cell integrins and transmit signals into cells. Integrin-β1-FAK signaling pathway transmits signals through integrin to tissue-intrinsic stem cells and induces activation according to the support of the wound repair matrix, and tissue-intrinsic stem cells increase expression of uPAR and nestin according to the transmitted signals and can induce the division, growth and migration of regenerative stem cells in tissues. The present invention can achieve the above object by preparing a wound repair matrix mimicking hydrogel composed of a polymer having an RGD motif such as fibrin, collagen, gelatin, etc. as a wound repair matrix capable of activating the integrin-FAK cell signaling pathway.

The present invention provides a method for inducing cell division, growth and migration of tissue endogenous stem cells by activating the Integrin-β1-FAK-uPAR signaling pathway. Signal transduction efficiency to stem cells in tissues is proportional to the density of receptors that can bind integrin ligand. Receptor density that can bind integrin ligand can be achieved by increasing the contact surface with the tissue. By providing a 3-dimensional environment rather than a 2-dimensional environment, as a result, the area that can be bonded to cells can be increased, thereby increasing integrin ligand and receptor binding. As a result, the efficiency of signal transduction to stem cells in tissues can be increased. The present invention provides a method for activating the Integrin-β1-FAK-uPAR signaling pathway by providing an artificial wound repair matrix as a three-dimensional support to tissue. To this end, we provide a method of strengthening the signal transduction pathway by providing a three-dimensional bond to tissue slices using a hydrogel capable of sol-gel phase transition as a wound repair substrate.

uPA-plasmin activity can vary depending on the tissue, degree of damage and cause. Compared to fat, placenta, and umbilical cord, nervous system tissue has higher uPA-plasmin activity, and as a result, the degree of degradation of wound repair matrix is higher in nervous system tissue. Decomposition of the wound repair matrix can be controlled by adjusting the content of components constituting the wound repair matrix, the molecular weight of the polymer, and the degree of crosslinking. The present invention provides a method for controlling the resistance to uPA-plasmin activity to be increased by increasing the content, molecular weight, and degree of crosslinking of the polymers constituting the wound repair matrix. In addition, it provides a method of continuously inducing the activity, growth and migration of stem cells in tissue in the wound repair matrix by regulating uPA-plasmin activity through the addition of PAI. PAI applicable to the present invention may be selected from the group consisting of aminocaproid acid, tranexamic acid, aprotinin, and aminomethylbenzoic acid. PAI applied to the present invention can be used by adding 10 μg to 10 mg per ml of hydrogel volume. As a result, the present invention provides a method for inducing and supporting the growth and migration of tissue endogenous stem cells during the long-term culture period by adding PAI and maintaining the structural stability of the wound repair matrix.

The activity of uPAR-uPA-plasmin in excessive stem cells results in the rapid decomposition of the wound repair matrix-mimicking hydrogel during the in vitro culture process, resulting in the degradation and loss of tissue fragments and the wound repair matrix around the cells. As a result, stem cells can attach and migrate Loss of the repair substrate may result, resulting in loss or reduction or loss of cell migration from the tissue into the hydrogel. The present invention controls the excessive plasmin activity of stem cells and tissue slices by adding PAI to control the degradation and loss of excessive wound repair matrix mimicking hydrogel, so that the wound repair matrix mimicking hydrogel physically destroys the tissue slice during the long-term culture period. Provided is a method for maintaining a role as a support substrate and at the same time maintaining a structural and functional role as a substrate on which activated tissue endogenous stem cells can migrate and grow.

In addition, the present invention comprises the steps of (1) preparing a wound repair matrix mimic hydrogel; (2) incorporating the separated tissue sections into the wound repair matrix mimicking hydrogel; (3) three-dimensionally culturing the wound repair matrix mimicking hydrogel incorporating the tissue slice in a culture medium to which PAI is added; (4) removing and washing the three-dimensional culture medium to remove PAI; (5) degrading the wound repair matrix mimicking hydrogel by re-cultivating the PAI-free culture medium with a PAI-free culture medium; and (6) isolating stem cells released from the culture medium.

Preferably, the wound repair matrix mimicking hydrogel is a fibrin hydrogel in which a 0.25 to 2.5% concentration of a fibrinogen solution and a 0.5 to 5 I.U./mL concentration of a thrombin solution are mixed, and a 0.1 to 0.5% concentration of collagen in the fibrin hydrogel. It may be a fibrin/collagen mixed hydrogel in which the solution is mixed or a fibrin/gelatin mixed hydrogel in which a 0.1 to 0.5% gelatin solution is mixed with the fibrin hydrogel, but is not limited thereto.

Preferably, the tissue may be adipose tissue, bone marrow tissue, myocardial tissue, peripheral nerve tissue, skeletal muscle tissue or synovial tissue, but is not limited thereto.

Preferably, the PAI may be tranexamic acid or aminomethyl benzoic acid, but is not limited thereto.

Preferably, the step (5) induces an increase in uPAR expression in the tissue and decomposes the wound repair matrix mimicking hydrogel through an increase in plasmin activity, but is not limited thereto.

Preferably, the tissue-intrinsic uPAR+ and nestin+ stem cells may have enhanced self-renewal ability, in vitro growth ability, differentiation potential, or tissue regeneration inducing ability, but are not limited thereto.

Preferably, the tissue slices recovered from the culture medium in step (5) may further include repeating steps (2) to (5) 1 to 10 times, but is not limited thereto.

The present invention provides a method for selectively isolating uPAR+ stem cells that have migrated and grown into the wound repair matrix. Although the degradation of the wound repair matrix due to excessive uPAR-plasmin activity can be controlled through the addition of PAI, a method for recovering cells from the wound repair matrix after the targeted migration and growth of stem cells is achieved is provided. As PAI is removed after washing, the wound repair matrix is degraded using the uPAR-plasmin activity characteristic inherent in stem cells, and the stem cells that have migrated and grown in the matrix are liberated and recovered. The present invention provides a method for isolating uPAR+ stem cells from solid tissue without the use of any exogenous protease and subsequent purification, wherein the wound repair matrix is degraded according to uPAR expression rate and plasmin activity in stem cells by uPAR-plasmin. .

The present invention provides a method for recovering tissue slices used for organ culture in a state in which the structure and function are preserved by not using any exogenous tissue degradation enzymes. Tissue fragments recovered after long-term culture are preserved in their structure, and thus, a method of inducing migration of uPAR+ tissue endogenous stem cells into the wound repair matrix through repeated long-term culture is provided.

The present invention provides a composition of a wound repair matrix that differs in uPAR-uPA-plasmin activity of tissue endogenous stem cells depending on the tissue and is not lost due to degradation by uPAR-uPA-plasmin activity according to the tissue, and controls excessive degradation. Provide the type and concentration of PAI according to the tissue capable of

Provided is a method for isolating high-purity stem cells without a subsequent purification process by PAI washing and removal (PAI withdrawal) of uPAR+/nestin+ tissue endogenous stem cells that have migrated and grown in the final wound repair matrix-mimicking hydrogel. uPAR+/nestin+ tissue endogenous stem cells can be used to manufacture stem cell therapeutic agents and stem cell-derived biopharmaceuticals with high self-replication, in vitro growth, differentiation ability, tissue regeneration-inducing gene expression, blood vessel regeneration and wound repair effects.

In addition, the present invention provides tissue endogenous uPAR+ and nestin+ stem cells or a culture solution thereof, separated and cultured according to the above method.

In the present invention, "culture medium" includes a medium capable of supporting the growth and survival of stem cells in vitro, secretions of cultured stem cells contained in the medium, and the like. The medium used for the culture includes all conventional mediums used in the art suitable for culturing stem cells. Media and culture conditions can be selected according to the type of cell. The medium used for culture is preferably a cell culture minimum medium (CCMM), and generally contains a carbon source, a nitrogen source, and trace elements. Such cell culture minimal media include, for example, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal essential Medium), BME (Basal Medium Eagle), RPMI1640, F-10, F-12, αMEM (α Minimal essential Medium), GMEM. (Glasgow's Minimal essential Medium), Iscove's Modified Dulbecco's Medium, etc., but are not limited thereto.

On the other hand, the present invention is a form containing all of the stem cells, their secretions, and medium components, a form containing only secretions and medium components, a form in which only secretions are separated and used alone or together with stem cells, or by administering only stem cells It is also possible to use it in a form that produces secretions in the body.

The stem cells may be obtained using any method commonly known in the art.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising the tissue-specific uPAR+ and nestin+ stem cells or their culture medium as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of autoimmune diseases comprising the tissue-specific uPAR+ and nestin+ stem cells or their culture medium as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for wound treatment comprising the tissue-endogenous uPAR+ and nestin+ stem cells or their culture medium as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for promoting blood vessel regeneration comprising the tissue-endogenous uPAR+ and nestin+ stem cells or their culture medium as an active ingredient.

Tissue endogenous uPAR+ and nestin+ stem cells of the present invention can be used as a cell therapy agent for the treatment of specific diseases, and the treatment can be direct treatment or pre-treatment of the molecules.

The "cell therapy product" means a product that changes the biological properties of cells by proliferating or selecting living autologous, allogenic, or xenogenic cells in vitro in order to restore the function of cells and tissues or by other methods. Drugs used for treatment, diagnosis, and prevention through a series of actions such as

The cell therapy may be administered to the human body through any general route as long as it can reach the target tissue.

The pharmaceutical composition of the present invention can be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvant includes an excipient, a disintegrant, a sweetener, a binder, a coating agent, an expanding agent, a lubricant, and a glidant. Alternatively, a solubilizer such as a flavoring agent may be used. The pharmaceutical composition of the present invention may be preferably formulated as a pharmaceutical composition by including one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration. In compositions formulated as liquid solutions, acceptable pharmaceutical carriers are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added if necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare formulations for injections such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets.

The pharmaceutical formulation form of the pharmaceutical composition of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions, and sustained-release preparations of active compounds. can The pharmaceutical composition of the present invention can be administered in a conventional manner through intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalational, topical, rectal, oral, intraocular or intradermal routes. can be administered with An effective amount of the active ingredient of the pharmaceutical composition of the present invention means an amount required for preventing or treating a disease. Therefore, the type of disease, the severity of the disease, the type and amount of the active ingredient and other ingredients contained in the composition, the type of formulation and the patient's age, weight, general health condition, sex and diet, administration time, administration route and composition It can be controlled by various factors including secretion rate, duration of treatment, and drugs used concurrently.

The present invention provides a method for isolating stem cells without the use of any exogenous tissue-degrading enzyme and without tissue dissociation during the process of isolating endogenous stem cells from solid tissues. Since no exogenous tissue-degrading enzyme is used, the function and structure of the tissue after isolating the stem cells can be preserved, thus providing a method that can be used as a tissue source capable of repeatedly isolating stem cells from the tissue.

Tissue endogenous stem cells are known to account for a very small percentage of cells constituting tissues and are usually less than 0.01%. After tissue damage, the number of somatic cells decreases, and stem cells for replacing or regenerating these cells are activated, and the frequency of stem cells increases compared to before damage. After damage, stem cells in the tissue reproduce themselves through cell division and produce daughter progenitor cells, and daughter stem cells migrate to the damaged area. This patent provides a method for inducing self-renewal and cell division of stem cells in tissue slices by activating the tissue homeostasis mechanism in vivo through in vitro culture, and mobilizing the self-replicating and dividing stem cells in vitro to damaged sites.

After tissue damage, a provisional matrix is formed around the damaged tissue, and the matrix-cellular integrin pathway is activated through this matrix. The regenerative mechanism is initiated. The present invention maximizes the matrix-cellular integrin interaction through three-dimensional support of the wound repair matrix mimicking hydrogel around the tissue slice to activate stem cells in the tissue, cell division, growth and migration to the wound repair matrix mimic hydrogel and provides a method for isolating stem cells that have migrated into the hydrogel.

In response to tissue damage, tissue endogenous stem cells are activated, and the activated stem cells have been reported to have increased expression of uPAR or nestin. Expression of uPAR and nestin has been found to play an important role in the growth and migration of stem cells, and uPAR+ and nestin+ cells are known to play a pivotal role in tissue regeneration. Since the expression of uPAR and nestin is increased in reparative stem cells, uPAR and nestin can be applied as major target markers for stem cell isolation. The present invention provides a method for selectively separating and harvesting uPAR+ stem cells by inducing cell division, growth, and migration of tissue-intrinsic uPAR+ and nestin+ stem cells as a result of matrix-cellular integrin pathway activation through in vitro culture.

In addition, according to the present invention, as a result of the physiological response to the activation and stimulation of stem cells, according to the activity of uPAR-plasmin-MMP, migration and growth are induced into the hydrogel mimicking the wound repair substrate provided during 3-dimensional culture, and then the stem that moves and grows within the hydrogel Provided is a method for separating and harvesting cells from hydrogels without exogenous tissue degradation enzymes according to the characteristics of uPAR expression and plasmin activity of the cells.

The present invention provides a common method for isolating tissue-intrinsic uPAR+ and nestin+ stem cells from representative solid tissues such as fat, bone marrow, myocardium, nerve, skeletal muscle, and synovial membrane. Tissue-intrinsic uPAR+ and nestin+ stem cells can be used as regenerative medicine treatments because they have self-renewal, high in vitro growth, multipotential characteristics, and high tissue regeneration ability.

Hereinafter, the present invention will be described in more detail through the following examples. However, the present invention is not limited by these examples.

< Example 1> wound healing temperament copy hydrogel Produce

The wound repair-imitating hydrogel is prepared by mixing fibrinogen, collagen, gelatin or these polymers. Plasma-derived fibrinogen is dissolved in phosphate-buffered saline (PBS) containing 10 to 50 mM CaCl ₂ at a concentration of 2.5 to 10 mg/ml to prepare a fibrinogen solution. Thrombin is dissolved in PBS at a concentration of 1 to 10 units/ml to prepare a thrombin solution.

Dermal-derived collagen (Matrix BioScience, Germany) or gelatin (befMatrix Collagen, Nitta Gelatin, Japan) is dissolved in 0.1% (wt./vol.) acetic acid, and a collagen solution having a concentration of 1.0 to 20.0 mg/ml is used. 10X buffer solution (Reconstitution Buffer) for preparing a neutral gelatin or collagen solution is prepared with 50 mM NaHCO ₃ , 40 mM HEPES, 0.01 N NaOH and mixed with collagen or gelatin solution in a 9:1 volume to obtain a neutral collagen or gelatin solution. manufacture

The wound repair matrix mimicking hydrogel is prepared by preparing a fibrin hydrogel with a 0.25 to 2.5% fibrinogen solution and a 0.5 to 5 I.U./mL thrombin solution, and a 0.1 to 0.5% concentration range of collagen or gelatin solution. are mixed during production of fibrin hydrogel to prepare fibrin/collagen or fibrin/gelatin mixed hydrogel.

< Example 2> Monolayer (2-dimension, 2D) and 3-dimensional (3-dimension, 3D) organ culture

The organs used for culture are adipose tissue (AT), bone marrow (BM), myocardium (Myocar), peripheral nerve (PN), muscle (skeletal muscle (SM)), synovium (Syn) was used. Approval was obtained from the Institutional Ethics Review Committee for research using brain-dead tissues, and tissues donated from brain-dead individuals were used. Hematoma and fibrous tissue attached to the tissue are removed with scissors, and then the donated tissue is cut into 0.2 to 2 mm ³ pieces using a surgical scalpel. Thereafter, the tissue section is suspended in PBS, centrifuged at 1,000 rpm, and the washing process of removing the supernatant is repeated three times.

After washing, the tissue sections are suspended in a culture medium and cultured by two organ culture methods. The first method was defined as a monolayer (2D) organ culture method in which tissue fragments suspended in a culture solution were seeded in a culture container and cultured on the surface of the culture container.

The second method is defined as a 3D organ culture method in which tissue sections are embedded in fibrin, fibrin/collagen or fibrin/gelatin wound repair matrix-mimicking hydrogel and then cultured in a 3-dimension (3D) environment. did In the three-dimensional organ culture method, tissue sections were mixed with 0.25-2.5% fibrinogen solution, mixed with the same amount of 0.5-5 unit/mL thrombin solution in a 1:1 volume, and then polymerized at 37°C for 1 hour. After roughening, the tissue sections were embedded into the fibrin hydrogel. Fat, bone marrow, heart, and muscle were embedded in tissue sections in a fibrin hydrogel mimicking wound repair matrix with a final 0.25–1.25% fibrinogen solution and 0.25–2.5 unit/mL thrombin solution. Nerve and synovial tissue slices were embedded in a wound repair matrix-mimicking fibrin/collagen or gelatin hydrogel consisting of 0.25–2.5% fibrinogen, 0.1–0.5% collagen or gelatin neutral solution, and 0.25–2.5 unit/mL thrombin solution.

The tissue sections mixed with the wound repair matrix mimicking hydrogel solution were transferred to a 100-mm to 150-mm culture container and then placed in a 37° C. incubator for 1 hour to convert to a gel after polymerization. Long-term culture medium is 45% v/v DMEM, 45% v/v Ham's F12, 10% fetal bovine serum (FBS, Invitrogen), 20 ng/ml EGF, 2 ng/ml bFGF, 10 ng/ml IGF , 10 μg/ml gentamicin (Invitrogen). The culture solution was added in an amount corresponding to twice the gel capacity, and after the addition of the culture solution, the culture vessel was placed on an orbital shaker and cultured for 14 days while stirring at a speed of 30 rpm. The culture medium was replaced twice a week.

In order to inhibit hydrogel degradation, tranexamic acid or aminomethyl benzoic acid, a PAI, was added to the culture solution every day during the cultivation period at a concentration of 10 to 500 μg/mL. For adipose, bone marrow, heart and skeletal muscle organ cultures, tranexamic acid or aminomethyl benzoic acid at a concentration of 100 to 250 μg/mL was added to the culture medium to suppress wound repair matrix mimic hydrogel degradation. In the case of peripheral nerve and synovial membrane, tranexamic acid or aminomethyl benzoic acid at a concentration of 250 ~ 500 μg/mL was added to the culture medium and cultured.

< Example 3> In organs according to organ culture pFAK , uPAR , nestin , Ki -67 expression

After 7 days of 2D and 3D organ culture, cultured adipose tissue, bone marrow, cardiac muscle, nerve, skeletal muscle, and synovial tissue sections were recovered from the culture vessel or hydrogel. After fixing for 2 hours using a 4% neutral formalin solution, a paraffin block was prepared. Immunochemical staining was performed after thinning 4 μm-thick sections from paraffin blocks.

anti-phospho FAK (3283, Cell Signaling Technology, USA; pFAK), anti-uPAR (MAB807, R&D Systems, USA) and Ki-67 (M7240) to evaluate the activity level of the integrin-FAK cell signaling pathway in cultured tissue , DAKO, USA) and the like were used to evaluate expression. After reacting with the primary antibody, it was washed with water three times, reacted with the HRP-conjugated secondary antibody (ImmPRESS One-Step Polymer Systems, Vector Laboratories, USA), and then color developed using DAB as a substrate, followed by Hematoxylin (H-3401-500, Vector Laboratories) was used to evaluate the expression rate and the location of positive cells after counterstaining.

In accordance with in vitro organ culture, the constituent cells in the tissue slice are activated, and the activated cells proliferate after cell division to replace or regenerate damaged or lost cells. The integrin cell signal transduction pathway is one of the main mechanisms that induce tissue regeneration, and the integrin cell signal transduction is activated according to the binding of cell ligands, extracellular matrix and receptors. In this example, after direct seeding of tissue slices in a culture vessel, monolayer culture (2-dimension, 2D) cultured on the surface of the culture vessel and inside of tissue slices cultured in a 3D (3-dimension, 3D) environment after incorporation into a hydrogel Expressions of pFAK, uPAR, and nestin proteins, which are target factors that are activated as a result of integrin cell signals, were analyzed in constitutive cells.

It was confirmed that the expression rate of pFAK in tissue slices after 3D organ culture was significantly higher than that of 2D organ culture. Differences in pFAK expression rates after organ culture according to tissue types were confirmed, and pFAK expression was significantly higher in myocardium, nerve and skeletal muscle after 3D organ culture (FIG. 1A).

Expression of uPAR, a sub-target factor of the integrin signaling pathway, was also significantly high in the 3D organ culture environment, similar to pFAK, and high expression was confirmed in cardiac muscle, nerve and skeletal muscle (FIG. 1B).

Through integrin signaling, it is possible to induce cell division and growth of long-term constituent cells. Cell regeneration ability and cell growth ability were evaluated through the expression of nestin and Ki-67, which are characteristically expressed in regenerating or dividing cells. Expression of nestin in tissue slices after 3D organ culture was significantly higher than expression of nestin in tissues after 2D organ culture (Fig. 1C). The expression rate of Ki-67, a cell division marker, was also significantly higher in tissue sections after 3D organ culture (FIG. 1D).

Cell division and growth of cells constituting organs can be induced through in vitro organ culture, integrin-binding ligand can be maximized through 3D culture environment, and downstream target factors , pFAK and uPAR expression can be enhanced, and as a result, it was confirmed that 3D organ culture induces cell division and cell growth of constituent cells in tissue slices at a higher efficiency compared to 2D organ culture. .

3D physical stimulation of the wound repair matrix-mimicking hydrogel can provide a way to significantly increase cell division and growth by activating the signaling pathways of the constituent cells in the organ.

< Example 4> In tissue after 3D organ culture pFAK +, uPAR + and nestin + Location and characteristics of cells

Myocardial tissue sections cultured before (0 d) or after 3, 5, 7, and 14 days of 3D organ culture were recovered from the hydrogel. After fixing for 2 hours using a 4% neutral formalin solution, a paraffin block was prepared. Immunochemical staining was performed after thinning 4 μm-thick sections from paraffin blocks. After culture, anti-phospho FAK (3283, Cell Signaling Technology, USA; pFAK), anti-uPAR (MAB807, R&D Systems, USA), nestin (MAB5326, Expression was evaluated using primary antibodies such as Millipore, USA) and Ki-67 (M7240, DAKO, USA). After reacting with the primary antibody, it was washed with water three times, reacted with the HRP-conjugated secondary antibody (ImmPRESS One-Step Polymer Systems, Vector Laboratories, USA), and then color developed using DAB as a substrate, followed by Hematoxylin (H-3401-500, Vector Laboratories) was used to evaluate the expression rate and the location of positive cells after counterstaining.

After embedding the myocardium into the wound repair matrix-mimicking hydrogel and performing 3D organ culture, the characteristics of pFAK+, uPAR+, and nestin+ cells in the myocardium were identified. The expression rates of pFAK, uPAR and nestin before culture were less than 6%. However, the number of pFAK, uPAR, and nestin-positive cells increased significantly in proportion to the culture period after 3D organ culture. After 2 weeks of culture, the expression rates of pFAK, uPAR, and nestin in tissue slices were 30% or more (FIGS. 2 and 3). pFAK, uPAR, and nestin were expressed in cells in the interstitial space between mature somatic cells, and pFAK, uPAR, and nestin were expressed in pericytes around capillaries rather than capillary vascular endothelial cells.

Ki-67, a cell division marker, was not expressed in mature somatic cells and was expressed in less than 1% before culture. However, after 3D organ culture in vitro, the expression rate of Ki-67 increased in proportion to the culture period, and after 2 weeks of organ culture, the expression rate of Ki-67 in the myocardium was 45.7%. Through this, it was confirmed that division and growth of cells in the myocardium could be induced, and Ki-67 was also mainly expressed in pericytes (FIG. 4).

In this embodiment, cell division and growth can be induced in the myocardium through the 3D wound repair matrix mimicking hydrogel, and division and growth of pFAK+, uPAR+ and nestin+ pericytes can be induced through long-term culture. confirmed that

< Example 5> In tissue through 3D organ culture uPAR + and nestin + cellular wound healing temperament copy hydrogel Moving into and inducing growth

After embedding fat, bone marrow, myocardium, nerve, skeletal muscle, and synovial tissue sections in fibrin or fibrin/collagen hydrogel, 3D organ culture was performed. The long-term culture solution was added twice as much as the hydrogel capacity, and the culture vessel was placed on an orbital shaker and cultured for 14 days while stirring at a speed of 30 rpm. PAI was added to the culture medium to inhibit hydrogel degradation. During in vitro culture, 5 μM bromodeoxyuridine (B5002, Sigma-Aldrich, USA) was added to the culture medium every day for 7 days in order to label the cells that had undergone cell division. The culture medium was cultured for 2 weeks by replacing the culture medium twice a week.

After 3, 7 and 14 days of 3D organ culture, the cultured tissue and hydrogel were simultaneously recovered. After fixing for 2 hours using a 4% neutral formalin solution, a paraffin block was prepared. Immunochemical staining was performed after thinning 4 μm-thick sections from paraffin blocks.

Anti-uPAR (MAB807, R&D Systems, USA), -nestin (MAB5326, Millipore, USA) and -BrdU (347580, BD Biosciences, USA) ) primary antibody was used. After reacting with the primary antibody, it was washed with water three times, reacted with the HRP-conjugated secondary antibody (ImmPRESS One-Step Polymer Systems, Vector Laboratories, USA), and then colored using DAB as a substrate, and hematoxylin (H-3401-500 , Vector Laboratories) was used to evaluate the expression rate after counterstaining.

The wound repair matrix mimicking hydrogel serves as an extracellular matrix that can support cell migration and growth, and this can be achieved through the provision of cell adhesion factors such as fibronectin, collagen, and fibrin, which are essential for cell migration. . 3D wound repair matrix hydrogel activates the integrin-pFAK cell signal signal transmission pathway through receptor-ligand binding to induce cell division and growth of uPAR+ and nestin+ cells in the organ, and mimic uPAR+ and nestin+ cells as the wound repair matrix Mobilization, migration and growth can be induced with hydrogels.

It was confirmed that the number of cells moving and growing into the hydrogel increased in proportion to the period of 3D myocardial organ culture (FIG. 5). It can be seen that the cells that have moved and grown in the hydrogel combine with the hydrogel in a spindle shape and expand the cytoplasm.

It can be seen that the cells migrated from the myocardium and grown in the hydrogel had uPAR and nestin expression rates of 88.5% and 95.4%, respectively, and showed the same uPAR+ and nestin+ characteristics as cells that were divided and grown after organ culture in the tissue. More than 89.4% of the cells that migrated into the hydrogel were BrdU-positive, which means that the cells synthesized DNA by uptake of BrdU added during organ culture, so most of the cells in the hydrogel were Prove that the cells in the tissue are cells that have migrated after division and growth. BrdU+ cells were not detected in the myocardium before organ culture, and uPAR+ and nestin+ cells were less than 2%.

After 2 weeks of 3D organ culture of fat, bone marrow, myocardium, nerve, skeletal muscle, and synovial tissue sections, the cells in the wound repair matrix-mimicking hydrogel are around 90% uPAR-positive and more than 90% BrdU-positive. Through culture, it was confirmed that tissue-intrinsic cells were cells that had undergone cell division, growth, and migration to the hydrogel, and no significant difference was observed depending on the tissue (FIG. 6).

< Example 6> After 3D organ culture hydrogel Isolation of intra-migration-grown cells

After embedding fat, bone marrow, myocardium, nerve, skeletal muscle, and synovial tissue sections in fibrin or fibrin/collagen hydrogel, 3D organ culture was performed. The long-term culture solution was added twice as much as the hydrogel capacity, and the culture vessel was placed on an orbital shaker and cultured for 14 days while stirring at a speed of 30 rpm. In order to inhibit hydrogel degradation, PAI was added every day during the culture period to suppress wound repair matrix mimic hydrogel degradation according to the PA activity of cells that migrated and grew in the hydrogel.

The activity of uPAR mRNA and plasmin in tissue sections collected after 3D organ culture was evaluated. After preparing tissue lysate from 10 mg tissue sections, plasmin activity was analyzed using the Plasmin Activity Assay Kit (ab204728, abcam). Plasmin activity was measured by measuring the fluorescence intensity after adding a fluorescent substrate to the tissue lyate and reacting at 37 ° C for 20 minutes. Plasmin activity was calculated by the following formula (ΔRFU360/450nm = (RFU ₂ -RFU _2BG )-(RFU ₁ -RFU _1BG )). The uPAR mRNA expression rate in tissue sections was analyzed by real time qPCR after total RNA was extracted from 10 mg tissue sections and cDNA was generated after reverse transcriptase reaction.

After 14 days of 3D organ culture, the culture medium was removed. After adding DMEM, washing was performed while stirring at 30 rpm for 30 minutes, and the washing solution was removed. This washing process was repeated 3 times. After washing, the cells that migrated and grew in the hydrogel were separated and recovered in the following two ways. In the first method, after adding 1,000 unit/mL urokinase to the culture medium, the hydrogel was degraded for 2 hours after being added to the culture medium, and the cells and tissue fragments freed from the disintegrated hydrogel were transferred to a tube and then 10 at 3,000 rpm. After centrifugation for 10 minutes, the supernatant was removed, and the collected cell and tissue section pellets were suspended in the culture medium.

Cells can be separated and collected from the hydrogel by the PAI withdrawal method. After 14 days of long-term culture, the culture medium was removed and washed three times using DMEM to remove the remaining PAI in the culture medium and hydrogel. After adding fresh culture medium, the culture was incubated for 1 hour while stirring at a speed of 30 rpm, and PAI in the culture medium was not added. Cells separated from the hydrogel were collected using a transfer pipette, transferred to tubes, centrifuged at 3,000 rpm for 10 minutes, and the supernatant was removed. The collected cells and tissue fragment pellet were suspended in a culture medium.

The cells separated and collected from the hydrogel were suspended in a culture medium, and the total number of cells was calculated using a hemocytometer, and the uPAR expression rate of the collected cells was analyzed using flow cytometry.

After 3D long-term culture, uPAR+/nestin+ cells grown after cell division in the tissue induce migration and growth into the hydrogel, and the cells that migrate and grow within the hydrogel become hydrogels according to the plasmin activity of uPAR+/nestin+ cells themselves without exogenous protease. It is intended to provide a method for selectively separating and collecting uPAR+/nestin+ cells by dissolving the gel through this embodiment.

uPAR mRNA expression and plasmin activity were evaluated in tissue sections before and after organ culture. Even before in vitro culture, uPAR mRNA expression and plasmin activity were different according to organs, and uPAR mRNA expression and plasmin activity were higher in myocardium, nerve, and skeletal muscle than in other tissues (FIG. 7). In all tissue sections evaluated after organ culture, uPAR mRNA expression and plasmin activity significantly increased compared to before culture. In particular, mRNA expression and plasmin activity were the highest in myocardium, nerve and skeletal muscle. Since Plasmin activity is proportional to uPAR expression, it is possible to induce an increase in uPAR expression in tissues through organ culture, and provides a method to increase plasmin activity in tissues through increased uPAR after organ culture. do.

Plasminogen Activator Inhibitor (PAI) is added in the culture medium during 3D organ culture, and the added PAI can inhibit hydrogel degradation by controlling excessive plasmin and protease activity in organs and cells. Through long-term culture, cell division and growth in tissue are induced to move and grow into the hydrogel, and then the PAI is removed from the culture medium and hydrogel through washing and washing. and protease activity alone can induce hydrogel disassembly, and as a result, cells that have migrated and grown in the hydrogel are released, and the freed cells can be separated and collected.

PAI withdrawal is performed by removing the PAI-containing culture medium, washing it with DMEM three times to remove the PAI remaining in the hydrogel and organs through the washing process, and adding twice the amount of the hydrogel to the culture medium without PAI. did After 30 minutes of incubation, the hydrogel began to degrade as a result of PAI withdrawal. As the hydrogel disintegrated, the cells that migrated within the hydrogel were released from the hydrogel and aggregated between cells, and after 2 hours of culture, tissue sections and cells that migrated grew. The surrounding hydrogel is completely degraded, resulting in dissociation of the cells within the hydrogel (FIG. 8). Through this example, only with PAI withdrawal, the wound repair matrix mimicking hydrogel is lost due to cell-specific plasmin and protease activity, and the cells in the hydrogel are released into the culture medium. As a result, the cells released in the culture medium have their cytoplasm contracted and aggregated It can be separated and recovered in the form.

Hydrogel degradation can be inhibited through the addition of PAI during long-term culture, and the cells in the hydrogel show a spindle-shaped shape as a result of cytoplasmic expansion. However, degradation begins from the hydrogel around the cells through PAI withdrawal in the culture medium, and as a result, the cytoplasm of the cells shrinks, and as a result of cell-cell junctions, the cells form aggregates, and the aggregated cells are released into the culture medium. (FIG. 9).

In the histological method, cells in the hydrogel before PAI withdrawal can confirm the expanded shape of the cytoplasm, but when the hydrogel begins to degrade after PAI withdrawal, the hydrogel to which cells can attach is lost, resulting in the disruption of cell-cell junctions. It can be verified that the resultant cells are aggregated and separated from the hydrogel (FIG. 10). All cells released from the hydrogel and aggregated showed uPAR-expressing characteristics, and the higher the uPAR expression, the higher the plasmin activity, and it was confirmed that uPAR+ cells can be separated from the hydrogel only with PAI withdrawal (FIG. 10). .

Cells that have migrated and grown in the hydrogel can be isolated and harvested after PAI withdrawal or urokinase treatment. The present invention compared and evaluated the usefulness of a method for isolating migratory and grown cells in a hydrogel with PAI withdrawal without urokinase treatment.

Through the example, it was confirmed that even with the PAI withdrawal method, cells equivalent to the number of cells could be separated and collected through urokinase treatment (FIG. 11A). More than 2 million cells could be isolated and harvested per 100 mg of tissue after 2 weeks of organ culture, and although it was lower than the number of cells harvested after urokinase treatment, there was no significant difference.

However, the uPAR expression rate of cells harvested after PAI withdrawal was significantly higher than that of cells harvested after urokinase treatment (FIG. 11B). In particular, the yield of selectively isolating uPAR+ cells from tissues with high uPAR expression and plasmin activity was high. This has high hydrogel degradation activity in uPAR+ cells, and as a result, it can be verified that the method is highly efficient in selectively separating uPAR+ cells by removing PAI and washing.

< Example 7> Recovered after 3D organ culture tissue section Repeated organ culture and cell isolation

After embedding fat, bone marrow, myocardium, nerve, skeletal muscle, and synovial tissue sections in fibrin or fibrin/collagen hydrogel, 3D organ culture was performed. The long-term culture solution was added twice as much as the hydrogel capacity, and the culture vessel was placed on an orbital shaker and cultured for 14 days while stirring at a speed of 30 rpm. In order to inhibit hydrogel degradation, PAI was added every day during the culture period to suppress wound repair matrix-mimicking hydrogel degradation according to the plasmin activity of cells that migrated and grew in the hydrogel.

After 14 days of 3D organ culture, the culture medium was removed. After adding DMEM, washing was performed while stirring at 30 rpm for 30 minutes, and the washing solution was removed. The washing process was repeated three times to remove remaining PAI in the culture medium and hydrogel. After adding fresh culture medium, the culture was incubated for 2 hours while stirring at a speed of 30 rpm, and PAI in the culture medium was not added. Cells and tissue fragments separated from the hydrogel were collected using a transfer pipette, transferred to tubes, centrifuged at 3,000 rpm for 10 minutes, and the supernatant was removed. Since tissue fragments precipitate faster than cells as a result of their high specific gravity, the cell and tissue fragment pellets are dispersed in DMEM, left for 30 seconds to induce sedimentation of the tissue fragments, and only the supernatant is transferred to a new tube. This process is repeated three times. The harvested cells and tissue sections were separated.

Tissue sections separated from the hydrogel by the PAI withdrawal method were re-embedded into the hydrogel, followed by 3D organ culture by the method described in Example 2, and 2 weeks later by the method described in Example 7. Cells and tissue sections that had migrated within the gel were separated and harvested. The tissue fragments recovered after PAI withdrawal were subjected to three more consecutive 3D organ cultures to induce migration and growth of cells in the tissue repeatedly, and the cells in the hydrogel were separated and recovered, and the total number of cells separated and recovered was measured using a hemocytometer.

In this example, after PAI withdrawal, tissue sections with preserved structures are recovered, and the recovered tissue sections are subjected to repeated organ culture to isolate cells that have migrated into the hydrogel after organ culture and grown. and provide a method for collection. Treatment with an exogenous protease such as urokinase can degrade the hydrogel and the infiltrated tissue slice, resulting in the loss of the structural and functional microenvironment of the tissue slice.

In this example, cells and tissue fragments that migrated after growth in tissues without exogenous protease treatment were isolated and collected through PAI withdrawal, and tissue sections collected at the same time were also subjected to repetitive organ culture. Three successive organ cultures were performed using tissue sections recovered after organ culture, and it was confirmed that cells could be harvested with a yield similar to that of the first organ culture (Fig. 12). It can be confirmed that the PAI withdrawal method is a method capable of preserving the structural and functional microenvironment of tissue slices infiltrated together with cells that have migrated and grown in the hydrogel after 3D organ culture. It can be confirmed that this is a method for isolating tissue-intrinsic cells with high efficiency and stability.

< Example 8> PAI withdrawal and urokinase after processing from hydrogel Culture of isolated cells

After embedding fat, bone marrow, myocardium, nerve, skeletal muscle, and synovial tissue sections in fibrin or fibrin/collagen hydrogel, 3D organ culture was performed. The long-term culture solution was added twice as much as the hydrogel capacity, and the culture vessel was placed on an orbital shaker and cultured for 14 days while stirring at a speed of 30 rpm. To inhibit hydrogel degradation, tranexamic acid, a PAI, was added to the culture medium every day.

After 14 days of 3D organ culture, the culture medium was removed. After adding DMEM, the mixture was washed for 30 minutes while stirring at a speed of 30 rpm, and this washing process was repeated three times. Cells that migrated and grew in the hydrogel after washing were collected after separating the cells by the PAI withdrawal or urokinase method presented in Example 6. After 14 days of long-term culture, the culture medium was removed and washed three times using DMEM to remove the remaining PAI in the culture medium and hydrogel. After adding fresh culture medium, the culture was incubated for 1 hour while stirring at a speed of 30 rpm, and PAI in the culture medium was not added. Cells separated from the hydrogel were collected using a transfer pipette, transferred to tubes, centrifuged at 3,000 rpm for 10 minutes, and the supernatant was removed. The collected cells and tissue fragment pellet were suspended in a culture medium.

Urokinase treatment is performed after washing three times, adding 1,000 unit/mL urokinase to the culture medium, dissolving the hydrogel for 2 hours, collecting cells and tissue fragments freed from the disintegrated hydrogel, transferring them to a tube at 3,000 rpm for 10 minutes After centrifugation, the supernatant was removed, and the collected cell and tissue section pellets were suspended in the culture medium.

The cells separated and collected from the hydrogel were seeded at 5,000 cells per cm2 in a polystyrene culture container, and then the culture medium was added. The adhesion and growth characteristics of the cells separated and collected from the hydrogel in a monolayer culture environment were evaluated using a microscope.

After PAI withdrawal, it was confirmed that cell aggregates were formed after being released from the hydrogel (FIG. 13A), and the collected cells were suspended in a culture medium and composed of small circular aggregates (FIG. 13B), and seeded in a PS culture container to form a monolayer. cultured in the environment. Cells harvested in an aggregated state adhered to the culture vessel 30 minutes after seeding (FIG. 13C), the cytoplasm of the cells seeded 1 hour after seeding expanded (FIG. 13D), and the cells were stably attached after 2 hours of culture. and growth of cells around the cell aggregates was confirmed (FIG. 13E).

After long-term culture of all tissue sections, such as fat, bone marrow, myocardium, nerve, skeletal muscle, and synovial membrane, cells could be stably separated and collected through PAI withdrawal. It was confirmed that all the cells collected within 30 minutes adhered to the culture vessel and stably expanded the cytoplasm, and there was no significant difference according to the tissue (FIG. 14).

Cells seeded and cultured in a monolayer environment showed a high growth rate, and after 2 days of culture, they grew to the periphery of the seeded cells and expanded to the periphery. It was confirmed that the growth rate was maintained (FIG. 15).

< Example 9> PAI Immunoexpression characteristics of cells isolated after withdrawal

After embedding fat, bone marrow, myocardium, nerve, skeletal muscle, and synovial tissue sections in fibrin or fibrin/collagen hydrogel, 3D organ culture was performed. The long-term culture solution was added twice as much as the hydrogel capacity, and the culture vessel was placed on an orbital shaker and cultured for 14 days while stirring at a speed of 30 rpm. In order to inhibit hydrogel degradation, tranexamic acid, a PAI, was added to the culture solution at a concentration and cultured.

After 14 days of 3D organ culture, the culture medium was removed. After adding DMEM, the mixture was washed for 30 minutes while stirring at a speed of 30 rpm, and this washing process was repeated three times. After washing, the cells that migrated and grew in the hydrogel were collected after separating the cells by the PAI withdrawal method. After 14 days of long-term culture, the culture medium was removed and washed three times using DMEM to remove the remaining PAI in the culture medium and hydrogel. After adding fresh culture medium, the culture was incubated for 1 hour while stirring at a speed of 30 rpm, and PAI in the culture medium was not added. Cells separated from the hydrogel were collected using a transfer pipette, transferred to tubes, centrifuged at 3,000 rpm for 10 minutes, and the supernatant was removed. The collected cells and tissue fragment pellet were suspended in a culture medium.

Cells isolated and harvested after PAI withdrawal method or urokinase treatment were suspended in PBS and immunoexpressed characteristics were analyzed using flow cytometry. Mesenchymal stem cell (MSC) markers CD29, CD44, CD105, CD140b, along with uPAR and nestin expression rates were evaluated. Using CD34 and CD45 as hematopoietic cell markers and CD31 as a vascular endothelial cell marker, expression rates of the corresponding markers were evaluated.

Cells grown by migration from the hydrogel as a result of urokinase treatment or PAI withdrawal can be separated and collected. Regardless of the cell collection method, the cells grown in the hydrogel from all cultured tissues showed the same immunorepresentation characteristics as mesenchymal stem cells, with CD29, CD73, CD105, and CD140b expression rates of 90% or more (FIG. 16A). There was no difference in the expression rate of mesenchymal stem cell markers in the cells separated and collected from the hydrogel according to the separation method.

However, in the present Example, the expression rates of uPAR and nestin in cells separated and collected by PAI withdrawal treatment after long-term culture of all tissue sections were higher than 90%. On the other hand, cells harvested after urokinase treatment showed an expression rate ranging from 30 to 67%. According to the high plasmin activity characteristics of uPAR+ and nestin+ cells in the hydrogel, it was possible to confirm the results of selective separation and collection through PAI withdrawal. On the other hand, it was confirmed that urokinase treatment resulted in mixed collection of cells with various immunorepresentational characteristics in the hydrogel (FIG. 16B).

Hematopoietic cells and vascular endothelial cells present in tissues may be mixed during the process of separating cells from tissues, and the degree of mixing of these cells may be confirmed by identifying CD31+, CD34+, and CD45+ cells. In the case of cells separated and collected from the hydrogel by the PAI withdrawal method, the expression rate of CD31, CD34, and CD45 was less than 1%, but the cells separated and collected after treatment with Urokinase were 2.8 ~ 7.0%, confirming a high mixing of hematopoietic cells and vascular endothelial cells. could (FIG. 17).

Through this example, it was confirmed that the method selectively separates and collects uPAR+ and nestin+ cells that have migrated from the tissue into the hydrogel through PAI withdrawal, and at the same time, mixing of hematopoietic cells and vascular endothelial cells during the separation process can be minimized. .

< Example 10> PAI Separately collected after withdrawal uPAR +/ nestin + Self-renewal ability of cells

After embedding fat, bone marrow, myocardium, nerve, skeletal muscle, and synovial tissue slices into fibrin or fibrin/collagen hydrogel, 3D organ culture was performed for 2 weeks, and then PAI withdrawal and urokinawe methods described in Example 6 were performed. Cells that had migrated and grown in the hydrogel were separated and harvested.

The cells separated and collected from the hydrogel were transferred to tubes, centrifuged at 3,000 rpm for 10 minutes, and the supernatant was removed. The cell precipitate was resuspended in the culture medium and then adjusted to a density of 1.0E+06 cells per mL. In order to verify the self-renewal ability of the cells separated and collected from the hydrogel, colony forming assay (CFU) was evaluated. After seeding 6,000 cells in a 100-mm culture vessel, they were cultured for 2 weeks and stained with 1% crystal violet. After washing, the number of colonies with a diameter of 2 mm or more was measured.

Although the frequency of CFU formation was different depending on the tissue type, in all tissue sections, the cells separated and collected from the hydrogel through PAI withdrawal were significantly higher than those collected after treatment with urokinase (FIG. 18). In particular, cells derived from cardiac muscle, nerve and skeletal muscle showed high colony formation ability compared to cells derived from other tissues. In the case of cells isolated after urokinase treatment, the CFU frequency was 3.1 ~ 8.4%, but cells isolated by the PAI withdrawal method had a high CFU frequency of 8.8 ~ 37.9%.

Self-renewal ability is one of the main characteristics of stem cells, and 3D organ culture can activate stem cells in tissues to induce migration and growth into hydrogels. Stem cells with high self-replication titer can be selectively separated and collected, which is a result proving that uPAR+/nestin+ cells with high plasmin activity are stem cells with high titer (FIG. 18).

< Example 11> PAI In vitro growth ability of cells isolated after withdrawal

After embedding fat, bone marrow, myocardium, nerve, skeletal muscle, and synovial tissue slices into fibrin or fibrin/collagen hydrogel, 3D organ culture was performed for 2 weeks, and then PAI withdrawal and urokinase methods described in Example 6 were used. Cells that had migrated and grown in the hydrogel were separated and harvested.

The cells separated and collected from the hydrogel were transferred to tubes, centrifuged at 3,000 rpm for 10 minutes, and the supernatant was removed. The cell precipitate was resuspended in the culture medium and then adjusted to a density of 1.0E+06 cells per mL. In order to verify the in vitro growth ability of the cells separated and collected from the hydrogel, cell doubling time (population doubling time, PDT) and cell doubling level (population doubling level, PDL) were analyzed and evaluated. 3,000 cells per cm ² were seeded in a T75 flask, cultured for 7 days, and cells were collected after treatment with trypsin/EDTA, and the total number of cells was calculated using a hemocytometer. PDT and PDL were calculated and compared through the number of disseminated cells, the total number of harvested cells, and the culture period. PDT was calculated according to the following formula. It was calculated using the formula PDT = [(cultivation time)/((logN2-logN1)/log2)], where N1 is the number of cells at the time of seeding, and N2 is the number of cells harvested after culturing. PDL was calculated according to the following formula. The formula PDL = [PDL0 + 3.322 (logN2-logN1)] was used, where N1 is the number of cells at the time of seeding, and N2 is the number of cells harvested after cell culture.

The expression rate of Ki-67, a cell cycle marker, is high in cultured cells due to its high in vitro growth ability. In vitro cell growth ability was compared and analyzed by analyzing the anti-Ki67 expression rate by flow cytometry.

After treatment with urokinase or PAI withdrawal, cells grown by migration were separated from the hydrogel, and the in vitro growth ability of the collected cells was compared and analyzed. PDT was significantly lower and PDL was significantly higher in cells isolated and harvested through PAI withdrawal compared to cells obtained after urokinase treatment. In the same tendency as the self-replication ability, it was confirmed that the cells isolated and collected through PAI withdrawal had excellent in vitro growth ability with low PDT and high PDL (FIG. 19).

Ki-67 expression was analyzed by flow cytometry to verify the high growth ability of the cells separated and collected from the hydrogel after PAI withdrawal treatment. Since the Ki-67 marker refers to cells in the cell cycle, the higher the Ki-67 expression rate, the higher the cell growth ability. Cells obtained through PAI withdrawal showed a high Ki-67 expression rate of 58.5 to 75.4%, which was significantly higher than 33.6 to 48.7% in cells obtained after urokinase treatment (FIG. 20).

As a result, it can be confirmed that PAI withdrawal is a method for selectively isolating cells with high in vitro growth potential, which supports that uPAR+/nestin+ cells with high plasmin activity are cells with high growth potential.

< Example 12> PAI Differentiation capacity of cells isolated after withdrawal

After embedding fat, bone marrow, myocardium, nerve, skeletal muscle, and synovial tissue slices into fibrin or fibrin/collagen hydrogel, 3D organ culture was performed for 2 weeks, and then PAI withdrawal and urokinawe methods described in Example 6 were performed. Cells that had migrated and grown in the hydrogel were separated and harvested.

The cells separated and collected from the hydrogel were transferred to tubes, centrifuged at 3,000 rpm for 10 minutes, and the supernatant was removed. The cell precipitate was resuspended in the culture medium and then adjusted to a density of 1.0E+06 cells per mL. The ability of the cells in the hydrogel to differentiate into adipocytes and osteoblasts was evaluated. After seeding 2.0E+05 cells in a 24-well culture dish, 10% CS, 0.5 mM 3-isobutyl-1-methylxanthine (Sigma), 80 μM indomethacin (Sigma), 1 μM DEX, 5 μg/ml in 90% DMEM After adding mL insulin-added differentiation medium, it was cultured for 14 days. The degree of differentiation of stem cells into adipocytes was examined by staining with 0.5% Oil Red O (Sigma) solution, which is an indicator of fat accumulation in the cytoplasm, for 1 hour at room temperature 2 weeks after differentiation induction, and the triglyceride content in the cytoplasm was measured using the Triglyceride-Glo kit (Promega). evaluated using In order to evaluate the ability to differentiate into osteoblasts, 2.0E+05 cells were seeded in a 24-well culture container, and then α containing 1 μM DEX, 50 μM Ascorbic Acid, 10 mM β-glycerol phosphate, and 10% calf serum. After adding -MEM, differentiation was induced by culturing for 2 weeks. Whether or not to differentiate into osteoblasts was stained after 2 weeks of induction of differentiation by adding Alizarin Red (Sigma), and the degree of mineral deposition was measured by optical density by measuring absorbance at 520 nm.

21 shows that cells separated and collected from the bone marrow were differentiated into osteoblasts (left) and adipocytes (right), and then calcium phosphate crytal accumulation and deposition were confirmed by alizarin red staining, and differentiation into adipocytes was induced. The accumulation of lipids in the cytoplasm of cells can be confirmed by oil red O staining. It was confirmed that all cells derived from all tissue sections had the ability to differentiate into osteoblasts and adipocytes.

The osteoblast and adipocyte differentiation abilities were compared and evaluated through the OD values measured after dissolving deposited or accumulated mineral cryostal and fat vacuole. In all tissue sections, it was confirmed that the ability of cells separated and collected from the hydrogel by PAI withdrawal to differentiate into osteoblasts and adipocytes was significantly higher than that of cells isolated and collected after treatment with urokinase (FIG. 22). Differentiation into osteoblasts showed high ability in bone marrow, cardiac muscle, and skeletal muscle-derived cells, and differentiation into adipocytes showed high ability in fat, skeletal muscle, and synovial membrane. However, regardless of the origin of the tissue section, as the cells separated and recovered from the hydrogel by the PAI withdrawal method were confirmed to have a significantly high ability to differentiate into osteoblasts and adipocytes, uPAR+/nestin+ cells with high plasmin activity were identified as stem cells with high pluripotency. cells could be identified.

< Example 13> PAI Ability to induce tissue regeneration of cells isolated after withdrawal

After embedding fat, bone marrow, myocardium, nerve, skeletal muscle, and synovial tissue slices into fibrin or fibrin/collagen hydrogel, 3D organ culture was performed for 2 weeks, and then PAI withdrawal and urokinawe methods described in Example 6 were performed. Cells that had migrated and grown in the hydrogel were separated and harvested.

The cells separated and collected from the hydrogel were transferred to tubes, centrifuged at 3,000 rpm for 10 minutes, and the supernatant was removed. The cell precipitate was resuspended in the culture medium and then adjusted to a density of 1.0E+06 cells per mL. After seeding 5.0E+06 cells in a T175 flask and culturing for 3 days, 1 mL of TRIzol was added, cell lysate was collected, and stored frozen at -20°C until total RNA was isolated. Total RNA was extracted and purified using the PureLink RNA kit (Thermo Fisher), and cDNA was synthesized using reverse transcriptase. Basic FGF (bFGF), HGF, IGF, SDF-1 mRNA expression, which plays a major role in tissue protection and regeneration, was calculated as the expression rate compared to cord blodd-derived mesenchymal stem cells (CB-MSC). comparative evaluation. The target mRNA gene was amplified through real-time gene amplification using a target mRNA specific primer and SYBR Green Real-Time PCR kit, and the mRNA expression rate compared to cord blood-derived mesenchymal stem cells (CB-MSC) was evaluated by the 2-ΔΔCt method.

After treatment with urokinase or PAI withdrawal, cells grown by migration were separated from the hydrogel, and the tissue regeneration inducing ability of the collected cells was compared and analyzed through the expression of related mRNAs. Depending on the tissue of origin, gene expression inducing tissue regeneration was different. Expression of these genes was high in cardiomyocyte, nerve and skeletal muscle-derived cells. HGF and SDF-1 mRNA were high in cardiomyocyte-derived cells, but IGF in nerve-derived cells. mRNA expression was high [Fig. 23]. The expression of all tissue regeneration genes was significantly higher in the cells isolated and collected by PAI withdrawal compared to the cells isolated and collected after urokinase treatment. These results confirm that uPAR expression has a proportional relationship with high tissue regeneration regulatory gene expression.

< Example 14> uPAR + Cellular plasmin activity and nestin expression rate

After embedding fat, bone marrow, myocardium, nerve, skeletal muscle, and synovial tissue slices into fibrin or fibrin/collagen hydrogel, 3D organ culture was performed for 2 weeks, and then the urokinase method described in Example 6 was performed in the hydrogel. Cells grown by migration were separated and harvested. Subsequently, uPAR+ (UK+uPAR+) and uPAR- (UK+uPAR-) cells were separated using FACSAria (BD Bioscience), cultured in a monolayer environment for 7 days, and plasmin activity and nestin expression were evaluated. Since the cells separated and collected from the hydrogel by the PAI withdrawal method had a uPAR expression rate of more than 90%, secondary uPAR+ cells were not isolated. Plasmin activity was performed according to the method described in Example 6, and nestin expression was evaluated using flow cytometry.

Significantly high plasmin activity was confirmed in uPAR+ cells (Fig. 24, left). UK+uPAR+ cells isolated and cultured after urokinase treatment and cells isolated and cultured after PAI withdrawal showed significantly higher plasmin activity compared to UK+uPAR- cells. Cellular plasmin activity was different depending on the tissue of origin, and was high in myocardium, nerve, and skeletal muscle, and the plasmin activity of endogenous cells was determined by the tissue of origin.

These results confirmed a close correlation between plasmin activity and nestin expression in uPAR+ cells.

< Example 15> uPAR + Self-renewal and in vitro growth ability of cells

After embedding fat, bone marrow, myocardium, nerve, skeletal muscle, and synovial tissue slices into fibrin or fibrin/collagen hydrogel, 3D organ culture was performed for 2 weeks, and then the urokinase method described in Example 6 was performed in the hydrogel. Cells grown by migration were separated and harvested. Thereafter, uPAR+ (UK+uPAR+) and uPAR- (UK+uPAR-) cells were separated using FACSAria (BD Bioscience), cultured in a monolayer environment for 7 days, and self-replication and growth capabilities were evaluated. Since the cells separated and collected from the hydrogel by the PAI withdrawal method had a uPAR expression rate of more than 90%, secondary uPAR+ cells were not isolated. Self-replication ability was performed according to the method described in Example 10, and growth ability was evaluated using flow cytometry for Ki-67 expression.

A significantly high self-replication ability was confirmed in uPAR+ cells. The CFU frequency was 12.5 to 37.5% in UK + uPAR + cells and PAI withdrawal cells, but it was 3.1 to 8.8% in UK + uPAR- cells, and it was confirmed that uPAR + cells had significantly high self-renewal ability (Fig. 25 left ).

Self-replication ability and in vitro growth ability are closely related, and it can be confirmed that uPAR+ cells have Ki-67 expression of 40.8 ~ 82.4%, which is significantly higher than that of uPAR- cells, Ki-67 expression of 28.1 ~ 45.7%. (Fig. 25, right). Cardiac, nerve, and skeletal muscle-derived uPAR+ cells showed higher CFU formation ability and Ki-67 expression compared to other tissues.

< Example 16> uPAR + anti-inflammatory ability of cells

After embedding fat, bone marrow, myocardium, nerve, skeletal muscle, and synovial tissue slices into fibrin or fibrin/collagen hydrogel, 3D organ culture was performed for 2 weeks, and then the urokinase method described in Example 6 was performed in the hydrogel. Cells grown by migration were separated and harvested. Then, uPAR+ (UK+uPAR+) and uPAR- (UK+uPAR-) cells were separated using FACSAria (BD Bioscience), cultured in a monolayer environment for 7 days, and anti-inflammatory ability was evaluated. Since the cells separated and collected from the hydrogel by the PAI withdrawal method had a uPAR expression rate of more than 90%, secondary uPAR+ cells were not isolated.

Anti-inflammatory ability was evaluated using conditioned media containing factors secreted from cells. To prepare the conditioned media, UK+uPAR+, UK+uPAR- and PAI withdrawal cells were cultured for 1 week after adding DMEM/F12 serum-free medium seeded with 40,000 cells per cm ² in a T175 flask.

RAW 264.7 cells were suspended in 90% RPMI 1640/10% calf serum, and 100,000 cells per cm ² were seeded in a 12-multiwell plate and cultured for 1 day. RAW 264.7 cells were sensitized by adding 100 μg/mL LPS to the culture. At the time of LPS sensitization, the anti-inflammatory ability was evaluated by adding the conditioned medium to the culture medium at a ratio of 1:10. Anti-inflammatory evaluation indicators were compared and evaluated by measuring the levels of TNFα and IL-1β secreted from RAW 264.7 cells by ELISA method.

RAW 264.7 cells showed a 5-fold increase in TNFα and IL-1β secretion levels after LPS stimulation. Substances secreted from tissue endogenous stem cells separated and collected from the hydrogels were confirmed to have the ability to inhibit inflammatory cytokine separation of RAW 264.7 cells by 30 to 70% (FIG. 26). The conditioned medium prepared from UK+uPAR+ cells and PAI withdrawal cells showed significantly higher TNFα and IL-1β secretion inhibition ability than UK+uPAR− cells. Regardless of the tissue of origin, uPAR+ cells showed higher anti-inflammatory effects compared to uPAR- cells.

< Example 17> uPAR + Effect of inducing growth of vascular endothelial cells and fibroblasts

After embedding fat, bone marrow, myocardium, nerve, skeletal muscle, and synovial tissue slices into fibrin or fibrin/collagen hydrogel, 3D organ culture was performed for 2 weeks, and then the urokinase method described in Example 6 was performed in the hydrogel. Cells grown by migration were separated and harvested. Then, uPAR+ (UK+uPAR+) and uPAR- (UK+uPAR-) cells were separated using FACSAria (BD Bioscience), cultured in a monolayer environment for 7 days, and anti-inflammatory ability was evaluated. Since the cells separated and collected from the hydrogel by the PAI withdrawal method had a uPAR expression rate of more than 90%, secondary uPAR+ cells were not isolated.

For vascular endothelial cells, human umbilical vein endothelial cells (HUVEC) were used, and human dermal fibroblasts (DF) were used. The growth-inducing effect of the conditioned medium on the cells was tested. A conditioned medium prepared by the method described in Example 16 was prepared and the efficacy was tested. HUVEC cells and DFs were suspended in 99% DMEM/1% calf serum culture medium, and 4,000 cells per cm2 were seeded in a 24-multiwell plate. The conditioned medium was added at a ratio of 1:9 to the culture medium and cultured for 3 days, and the number of cells after culture was measured using the PicoGreen dsDNA quantitation kit (Invitrogen). The cell growth effect was compared and analyzed by calculating the increase in the number of cells seeded first.

The conditioned medium prepared from UK+uPAR+ and PAI withdrawal cells was confirmed to have an effect on promoting HUVEC and DF growth, and showed a significantly higher potency than the conditioned medium prepared from UK+uPAR- cells by more than 30%, but the UK+uPAR+ There was no difference in potency between PAI withdrawal cells (FIG. 27). In particular, the conditioned media obtained from myocardial, nerve, and skeletal muscle-derived stem cells were confirmed to have a high growth inducing effect compared to other tissue-derived stem cells.

< Example 18> uPAR + Protective effect on vascular endothelial cells and fibroblasts

After embedding fat, bone marrow, myocardium, nerve, skeletal muscle, and synovial tissue slices into fibrin or fibrin/collagen hydrogel, 3D organ culture was performed for 2 weeks, and then the urokinase method described in Example 6 was performed in the hydrogel. Cells grown by migration were separated and harvested. Then, uPAR+ (UK+uPAR+) and uPAR- (UK+uPAR-) cells were separated using FACSAria (BD Bioscience), cultured in a monolayer environment for 7 days, and anti-inflammatory ability was evaluated. Since the cells separated and collected from the hydrogel by the PAI withdrawal method had a uPAR expression rate of more than 90%, secondary uPAR+ cells were not isolated.

The cytoprotective effect was tested using conditioned media prepared from UK+uPAR- cells, UK+uPAR+ cells, and cells separated and harvested after PAI withdrawal. After suspending HUVECs and DFs in 99% DMEM/1% calf serum culture medium, 5,000 cells per cm ² were seeded in a 24-multiwell plate. One hour before the induction of H ₂ O ₂ -mediated cell damage, the conditioned medium was added at a ratio of 1:9 to the culture medium. Cell damage was induced by adding 0.01% H ₂ O ₂ , and after 6 hours of damage induction, the culture medium was removed and washed twice with PBS. The degree of cell damage was evaluated by annexin V expression rate, and after reacting FITC-conjugated annexin V (BD Biosciences) with the cells, the apoptotic cells were analyzed by flow cytometry.

Conditioned medium prepared from UK + uPAR + cells and collected cells after PAI withdrawal treatment was compared with UK + uPAR- cell-derived conditioned medium, and it was confirmed that H2O2 mediated HUVEC and DF cell death can be significantly protected (FIG. 28). . Differences in cytoprotective effect were observed depending on the tissue of origin, and HUVEC and DF cytoprotective effects were high in cells derived from myocardium, nerve, and skeletal muscle. The expression rate of annexin V in HUVECs and DFs treated with UK+uPAR+ cell conditioned medium was 28.7 ~ 45.1%, which showed a significantly higher cytoprotective effect than 42.1 ~ 71.3% in cells treated with UK+uPAR- cell conditioned medium. A significant difference from the PAI withdrawal cell-derived conditioned medium could not be confirmed. These results confirmed the association between uPAR expression and high cytoprotective effect, and confirmed the basis for isolating and culturing cells with high cytoprotective effect only with PAI withdrawal.

The present invention is achieved through long-term culture supported by artificial wound repair matrix (engineered provisional matrix). Tissue slices are embedded in an artificial provisional matrix and the integrin-FAK cell signaling pathway is activated through the provisional matrix to induce cell division and growth of tissue endogenous stem cells. Through the long-term culture process, the stem cells in the tissue move to the provisional matrix and the growth increases in proportion to the activity of uPAR-uPA-plasmin. Excessive provisional matrix decomposition can be controlled through the addition of PAI to control the result of inhibition and cessation of stem cell migration due to decomposition and loss of provisional matrix due to excessive uPAR-uPA-plasmin activity. Provides a technology to isolate uPAR+ stem cells by stopping the addition of PAI after the migration and growth of stem cells into the provisional matrix reaches the target level. Upon stopping the addition of PAI, uPAR-uPA-plasmin and MMP activation result in uPAR+ stem cells being dissociated from the provisional matrix and liberated into the culture medium. The present invention provides a technology for isolating and culturing uPAR+ stem cells without the use of any tissue degradation protein and stem cell purification process using markers by using the migration and degradation characteristics of provisional matrix according to uPAR expression.

The present invention can be applied to the isolation and culture of uPAR+ stem cells present in solid tissues such as bone marrow, fat, skeletal muscle, heart, peripheral nerves, spinal cord, brain, lung, liver, joint membrane, umbilical cord, placenta, and periodontium. The present invention provides a method for isolating and culturing stem cells through repeated long-term culture by isolating stem cells without any tissue degradation enzyme treatment so that the tissue fragments used for organ culture can preserve their structure intact.

The present invention shows the characteristic of positively expressing common stem cell markers along with uPAR among uPAR-positive tissue endogenous stem cells. In the case of bone marrow, fat, muscle, heart, joint membrane, genitalia, and placenta, mesenchymal stem cell markers CD29, CD44, CD73, CD90, and CD105 are expressed positively, but hematopoietic stem cell or vascular endothelial cell markers are negative. According to the present invention, uPAR-positive stem cells derived from peripheral nerve, spinal cord, and brain tissue show the characteristics of simultaneously expressing markers such as nestin, p75, Sox10, and Sox2.

The uPAR-positive stem cells of the present invention show biological characteristics of high secretion of physiologically active factors acting on migration, growth factors, anti-inflammatory factors, stem cell mobilization factors, and tissue regeneration.

The uPAR-positive stem cells of the present invention have a high multipotential ability to differentiate into tissue-constituting cells.

Claims (18)

delete delete delete delete delete delete (1) a fibrin hydrogel in which a 0.25 to 2.5% concentration of a fibrinogen solution and a 0.5 to 5 IU/mL concentration of a thrombin solution are mixed, and a fibrin/collagen mixture hydrogel in which a 0.1 to 0.5% collagen solution is mixed with the fibrin hydrogel Preparing a wound repair matrix mimicking hydrogel composed of a gel or a fibrin/gelatin mixed hydrogel in which a 0.1 to 0.5% gelatin solution is mixed with the fibrin hydrogel;
(2) incorporating the separated tissue sections into the wound repair matrix mimicking hydrogel;
(3) three-dimensionally culturing the wound repair matrix-mimicking hydrogel into which the tissue slice is incorporated in a culture medium to which a plasminogen activator inhibitor (PAI) is added;
(4) removing and washing the three-dimensional culture medium to remove PAI;
(5) degrading the wound repair matrix mimicking hydrogel by re-cultivating the PAI-free culture medium with a PAI-free culture medium; and
(6) A method for separating and culturing endogenous tissue uPAR+ and nestin+ stem cells, including the step of separating free stem cells from the culture medium, without treatment with an exogenous protease. delete The method of separating and culturing endogenous uPAR+ and nestin+ stem cells according to claim 7, wherein the tissue is adipose tissue, bone marrow tissue, myocardial tissue, peripheral nerve tissue, skeletal muscle tissue or synovial tissue. [Claim 8] The method according to claim 7, wherein the PAI is tranexamic acid or aminomethyl benzoic acid. The method of claim 7, wherein step (5) induces an increase in uPAR expression in the tissue and degrades the wound repair matrix mimicking hydrogel through an increase in plasmin activity. and a method for separating and culturing nestin+ stem cells. [Claim 8] The method of separating and culturing tissue-intrinsic uPAR+ and nestin+ stem cells according to claim 7, wherein the tissue-intrinsic uPAR+ and nestin+ stem cells have enhanced self-renewal, in vitro growth, differentiation, and tissue regeneration inducing abilities. The tissue endogenous uPAR+ according to claim 7, wherein the tissue slices recovered from the culture medium in step (5) further comprise repeating steps (2) to (5) 1 to 10 times. and a method for separating and culturing nestin+ stem cells. Tissue endogenous uPAR+ and nestin+ stem cells or a culture medium thereof separated and cultured according to the method of any one of claims 7 and 9 to 13. A pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising the tissue-endogenous uPAR+ and nestin+ stem cells according to claim 14 or their culture medium as an active ingredient. A pharmaceutical composition for preventing or treating autoimmune diseases, comprising the tissue-endogenous uPAR+ and nestin+ stem cells according to claim 14 or their culture medium as an active ingredient. A pharmaceutical composition for treating wounds comprising tissue endogenous uPAR+ and nestin+ stem cells or a culture medium thereof according to claim 14 as an active ingredient. A pharmaceutical composition for promoting blood vessel regeneration comprising the tissue-endogenous uPAR+ and nestin+ stem cells or their culture medium according to claim 14 as an active ingredient.

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