CN117413050A

CN117413050A - Method for isolating and culturing tissue resident uPAR+/nestin+ stem cells and use thereof

Info

Publication number: CN117413050A
Application number: CN202280039679.8A
Authority: CN
Inventors: 郭命辰
Original assignee: Hanchuang Stem Cell Biotechnology Co
Current assignee: Hanchuang Stem Cell Biotechnology Co
Priority date: 2021-06-03
Filing date: 2022-06-03
Publication date: 2024-01-16

Abstract

The present invention relates to methods for isolating and culturing tissue resident upar+/nestin+ stem cells and uses thereof, and provides methods for isolating tissue resident stem cells present in solid tissue based on uPAR-plasmin activity. It is suggested that uPAR-plasmin activity of stem cells is closely related to growth, migration, physiological activity and differentiation ability of stem cells, and thus can be used for a method of isolating high-efficiency stem cells. The upar+ stem cells of the present invention isolated from solid tissues are suitable for the production of cell therapeutic agents, tissue engineering therapeutic agents, and novel biopharmaceuticals using secretions including exosomes, and thus have high industrial applicability. In addition, the invention is applicable to basic cell biology and molecular biology research related to cell division, migration, growth and differentiation of tissue resident stem cells, and new drug development research.

Description

Method for isolating and culturing tissue resident uPAR+/nestin+ stem cells and use thereof

Technical Field

The present disclosure relates to methods of isolating and culturing tissue resident upar+/nestin+ stem cells that play an important role in tissue homeostasis and tissue regeneration in solid tissues, and uses thereof.

Background

Conventional methods for isolating and culturing stem cells present in solid tissues require the following procedures: tissue dissociation is performed to degrade the tissue by protease treatment and separate each cell constituting the tissue, and then a single cell suspension is prepared. The single cell yield obtained shows a large variation in the tissue dissociation process based on the type of protease used, concentration, reaction time, reaction temperature and the type of tissue used, so that cell damage is an unavoidable problem in the tissue dissociation process. Thus, the tissue dissociation process shows a significant difference in the degree of cell damage and cell yield depending on the tissue type, with the disadvantage of being difficult to standardize. In particular, in the case of stem cells in solid tissues with a frequency lower than 0.1%, it is known that the tissue dissociation step after protease treatment results in extremely low cell yield.

A subsequent step of separating and purifying the tissue resident stem cells from the single cell suspension dissociated from the self-tissues is required. The procedure of isolating and purifying stem cells involves the use of markers (e.g., c-Kit and Sca-1) to isolate cells that are positive or negative for the markers. However, since these markers are expressed not only in tissue resident stem cells but also in hematopoietic cells, unwanted cells may be separated together, and stem cells whose markers are negative exist, thus limiting the methods of isolating and purifying tissue resident stem cells using specific markers.

Disclosure of Invention

Technical object

It is an object of the present disclosure to provide methods of inducing cells of tissue resident upar+/nestin+ stem cells in solid tissue (e.g., adipose tissue, bone marrow tissue, myocardial tissue, peripheral nerve tissue, skeletal muscle tissue, or synovial tissue) into the cell cycle, inducing migration and growth, and isolating cells.

Furthermore, it is another object of the present disclosure to provide tissue resident upar+ and nestin+ stem cells or cultures thereof isolated and cultured according to the methods.

Furthermore, another object of the present disclosure is to provide a pharmaceutical composition for preventing or treating an inflammatory disease or an autoimmune disease, which comprises tissue resident upar+ and nestin+ stem cells or a culture thereof as active ingredients.

Furthermore, it is another object of the present disclosure to provide a pharmaceutical composition for healing wounds or promoting revascularization, comprising tissue resident upar+ and nestin+ stem cells or cultures thereof as active ingredients.

Technical proposal

To achieve the above object, the present disclosure provides a method of inducing tissue resident upar+ and nestin+ stem cells into a cell cycle, the method comprising: (1) preparing a temporary matrix simulated hydrogel; (2) Encapsulating the isolated tissue fragments into the temporary matrix simulated hydrogel; and (3) three-dimensional (3D) culturing the temporary matrix simulated hydrogel in which the tissue fragments are encapsulated in a medium supplemented with a Plasminogen Activator Inhibitor (PAI).

Furthermore, the present disclosure provides methods of isolating and culturing tissue-resident upar+ and nestin+ stem cells, the methods comprising: (1) preparing a temporary matrix simulated hydrogel; (2) Encapsulating the isolated tissue fragments into the temporary matrix simulated hydrogel; (3) 3D culturing a temporary matrix simulated hydrogel in which the tissue fragments are encapsulated in a medium supplemented with PAI; (4) removing the 3D medium and removing PAI by washing; (5) Re-culturing the PAI-removed culture with a PAI-free medium to degrade the temporary matrix simulated hydrogel; and (6) isolating the stem cells released in the re-culture medium.

Furthermore, the present disclosure provides tissue resident upar+ and nestin+ stem cells or cultures thereof isolated and cultured according to the methods.

Furthermore, the present disclosure provides a pharmaceutical composition for preventing or treating an inflammatory disease, which comprises tissue resident upar+ and nestin+ stem cells or a culture thereof as active ingredients.

Furthermore, the present disclosure provides a pharmaceutical composition for preventing or treating autoimmune diseases, which comprises tissue resident upar+ and nestin+ stem cells or a culture thereof as active ingredients.

Furthermore, the present disclosure provides a pharmaceutical composition for healing wounds, comprising tissue resident upar+ and nestin+ stem cells or cultures thereof as active ingredients.

Furthermore, the present disclosure provides a pharmaceutical composition for promoting angiogenesis, comprising tissue resident upar+ and nestin+ stem cells or a culture thereof as active ingredients.

Advantageous effects

The present disclosure relates to methods of isolating and culturing tissue resident upar+/nestin+ stem cells and uses thereof, providing methods of isolating tissue resident stem cells present in solid tissue based on uPAR-plasmin activity. The uPAR-plasmin activity of stem cells is closely related to the growth, migration, biochemical and differentiation ability of stem cells and is therefore suitable as a method for isolating stem cells having a high level of functional activity. The upar+ stem cells isolated from solid tissues of the present disclosure have great industrial potential due to their suitability for the production of cell therapeutic agents, tissue engineering therapeutic agents, and the use of novel biopharmaceuticals including exosomes secretions. Furthermore, in the present disclosure, it can be applied to basic research of cell biology and molecular biology related to cell division, migration, growth and differentiation of tissue-resident stem cells, and new drug development research.

Drawings

FIG. 1 shows the expression rates of pFAK, uPAR, nestin and Ki-67 of constituent cells in tissue fragments of in vitro organ culture. 2D, monolayer organ culture; 3D,3D organ culture; AT, adipose tissue; BM, bone marrow; myocarps, myocardium; PN, peripheral nerve; SM, skeletal muscle; syn, synovial membrane. * P <0.01 compared to 2D.

Figure 2 shows the increased profile of pfak+, upar+ and nestin+ expressing cells in myocardium according to 3D myocardial organ culture.

FIG. 3 shows the increased characteristics of ki-67 positive cells with cell division in myocardium according to 3D organ culture.

FIG. 4 shows that the frequency of pFAK, uPAR, nestin and ki-67 positive cells in myocardium increases in proportion to the time of 3D myocardial organ culture. * P <0.01 compared to 0d (before incubation).

FIG. 5 shows the expression of uPAR, nestin and BrdU by cells that migrate into and grow in hydrogels after 3D myocardial organ culture.

Fig. 6 shows the expression rates of uPAR and nestin of cells that migrated into and grew in the hydrogel after 2 weeks of 3D organ culture. AT, adipose tissue; BM, bone marrow; myocarps, myocardium; PN, peripheral nerve; SM, skeletal muscle; syn, synovial membrane.

FIG. 7 shows uPAR mRNA expression (A) and plasmin activity (B) in tissue fragments before and 3 days after organ culture. AT, adipose tissue; BM, bone marrow; myocarps, myocardium; PN, peripheral nerve; SM, skeletal muscle; syn, synovial membrane. * P < 0.05 compared to before (before) cultivation; * P <0.01 compared to before (before) incubation.

FIG. 8 shows that the hydrogel is degraded by PAI removal and culture washing in the medium after organ culture and cells that migrate into and grow in the hydrogel are released into the medium.

Fig. 9 shows that cells migrating into and growing in the hydrogel are detached from the hydrogel by PAI removal in the medium and culture washing, and the released cells shrink and aggregate (phase contrast microscopy).

FIG. 10 shows the isolation in which uPAR+ cells in hydrogels were released after PAI removal and culture washing (HE staining and uPAR immunohistochemical staining of paraffin sections).

Fig. 11 shows the yield of cells collected after separation from the hydrogel after PAI removal, culture wash (PAI removal) and exogenous Urokinase addition (Urokinase) and the expression rate of uPAR in the collected cells. AT, adipose tissue; BM, bone marrow; myocarps, myocardium; PN, peripheral nerve; SM, skeletal muscle; syn, synovial membrane. * P <0.01 compared to Urokinase.

FIG. 12 shows the yield of cells collected after repeated separations from hydrogels by repeated organ culture of tissue fragments collected after PAI removal and culture washing. AT, adipose tissue; BM, bone marrow; myocarps, myocardium; PN, peripheral nerve; SM, skeletal muscle; syn, synovial membrane.

FIG. 13 shows the adhesion and growth of cells in a monolayer culture environment after seeding PAI-removed and culture washed cells in a culture vessel. A, PAI removes and washes aggregates of cells released from the hydrogel. B, aggregates of cells collected from the hydrogels. C-E, adhesion and growth of cells 30 minutes (C), 1 hour (D) and 2 hours (D) after seeding the collected cells in the culture vessel (phase contrast photomicrographs).

Fig. 14 shows the results (bottom) of cells released from the hydrogel after culturing adipose tissue, bone marrow, cardiac muscle, nerve, skeletal muscle and synovial organ, followed by PAI removal and washing of the culture (top), and collected cells were inoculated, adhered to the culture vessel and grown (phase contrast microscopy).

Figure 15 shows adhesion and growth of cells released from the hydrogel after PAI removal and culture wash following neural and myocardial organ culture in a monolayer culture environment.

Figure 16 shows immunophenotypic characteristics of cells collected from hydrogels. A, immunophenotyping characteristics of cells released after PAI removal and culture wash after withdrawal from hydrogel. B, immunophenotype characteristics of cells released from hydrogels after exogenous urokinase treatment. AT, adipose tissue; BM, bone marrow; myocarps, myocardium; PN, peripheral nerve; SM, skeletal muscle; syn, synovial membrane. * P < 0.01 compared to urokinase (B).

Fig. 17 shows the expression rates of hematopoietic and vascular endothelial markers of cells collected from hydrogels. A, expression rate of cells isolated and collected after PAI removal and culture washing. B, expression rate of cells isolated and collected after exogenous urokinase treatment. AT, adipose tissue; BM, bone marrow; myocarps, myocardium; PN, peripheral nerve; SM, skeletal muscle; syn, synovial membrane. * P < 0.01 compared to urokinase (B).

Figure 18 shows the self-replicating capacity of cells isolated and collected from hydrogels. A, A is as follows; representative pictures of Colony Forming Units (CFU). B, a step of preparing a composite material; CFU frequency of cells isolated and collected from hydrogels according to tissue source. Urokinase, cells isolated and collected after exogenous urokinase treatment; PAI withdrawal, cells isolated and collected after PAI removal and culture wash; AT, adipose tissue; BM, bone marrow; myocarps, myocardium; PN, peripheral nerve; SM, skeletal muscle; syn, synovial membrane. * P < 0.01 compared to urokinase.

Figure 19 shows the in vitro growth capacity of cells isolated and collected from hydrogels. A, A is as follows; population Doubling Time (PDT). B, a step of preparing a composite material; population Doubling Level (PDL). Urokinase, cells isolated and collected after exogenous urokinase treatment; PAI withdrawal, cells isolated and collected after PAI removal and culture wash; AT, adipose tissue; BM, bone marrow; myocarps, myocardium; PN, peripheral nerve; SM, skeletal muscle; syn, synovial membrane. * P < 0.05 compared to urokinase; * P < 0.01 compared to urokinase.

FIG. 20 shows Ki-67 expression rates of cells isolated and collected from hydrogels. Urokinase, cells isolated and collected after exogenous urokinase treatment; PAI withdrawal, cells isolated and collected after PAI removal and culture wash; AT, adipose tissue; BM, bone marrow; myocarps, myocardium; PN, peripheral nerve; SM, skeletal muscle; syn, synovial membrane. * P < 0.01 compared to urokinase.

Fig. 21 shows the ability of cells isolated and collected from the hydrogel to differentiate into osteoblasts (alizarin red) and adipocytes (oil red O). Urokinase, cells isolated and collected after exogenous urokinase treatment; PAI withdrawal, cells isolated and collected after PAI removal and culture wash.

Fig. 22 shows a comparison of the ability of cells isolated and collected from hydrogels to differentiate into osteoblasts (alizarin red) and adipocytes (oil red O). Urokinase, cells isolated and collected after exogenous urokinase treatment; PAI withdrawal, cells isolated and collected after PAI removal and culture wash; AT, adipose tissue; BM, bone marrow; myocarps, myocardium; PN, peripheral nerve; SM, skeletal muscle; syn, synovial membrane. * P < 0.01 compared to urokinase.

FIG. 23 shows the expression levels of mRNA associated with tissue regeneration, stem cell mobilization and angiogenesis of cells isolated and collected from hydrogels. Urokinase, cells isolated and collected after exogenous urokinase treatment; PAI withdrawal, cells isolated and collected after PAI removal and culture wash; CB-MSC, mesenchymal stem cells derived from umbilical cord blood; AT, adipose tissue; BM, bone marrow; myocarps, myocardium; PN, peripheral nerve; SM, skeletal muscle; syn, synovial membrane. * P < 0.05 compared to urokinase; * P < 0.01 compared to urokinase.

FIG. 24 shows plasmin activity and nestin expression rate of cells isolated and collected from hydrogels. uPAR-, isolated and purified uPAR negative cells after exogenous urokinase treatment; upar+, upar+ cells isolated and purified after exogenous urokinase treatment; PAI withdrawal, cells isolated and collected after PAI removal and culture wash; AT, adipose tissue; BM, bone marrow; myocarps, myocardium; PN, peripheral nerve; SM, skeletal muscle; syn, synovial membrane. * P < 0.05 compared to uPAR-; * P is 0.01 relative to uPAR-; * P < 0.05 compared to uPAR-; * P < 0.01 compared to uPAR-.

FIG. 25 shows the self-replication (colony forming units; CFU) and in vitro growth (Ki-67) capacity of cells isolated and collected from hydrogels. uPAR-, isolated and purified uPAR negative cells after exogenous urokinase treatment; upar+, upar+ cells isolated and purified after exogenous urokinase treatment; PAI withdrawal, cells isolated and collected after PAI removal and culture wash; AT, adipose tissue; BM, bone marrow; myocarps, myocardium; PN, peripheral nerve; SM, skeletal muscle; syn, synovial membrane. * P < 0.05 compared to uPAR-; * P is 0.01 relative to uPAR-; * P < 0.05 compared to uPAR-; * P < 0.01 compared to uPAR-.

Figure 26 shows the anti-inflammatory ability of cells isolated and collected from hydrogels. TNF alpha and IL-1 beta secretion inhibitory effects of LPS-sensitized RAW 264.7 cells in conditioned medium. uPAR-, isolated and purified uPAR negative cells after exogenous urokinase treatment; upar+, upar+ cells isolated and purified after exogenous urokinase treatment; PAI withdrawal, PAI removal and culture wash, cells isolated and collected; AT, adipose tissue; BM, bone marrow; myocarps, myocardium; PN, peripheral nerve; SM, skeletal muscle; syn, synovial membrane. * P < 0.05 compared to uPAR-; * P is 0.01 relative to uPAR-; * P < 0.05 compared to uPAR-; * P < 0.01 compared to uPAR-.

FIG. 27 shows vascular endothelial cell (HUVEC) and fibroblast (DF) growth stimulating activity of cells isolated and collected from hydrogels. uPAR-, isolated and purified uPAR negative cells after exogenous urokinase treatment; upar+, upar+ cells isolated and purified after exogenous urokinase treatment; PAI withdrawal, PAI removal and culture wash, cells isolated and collected; AT, adipose tissue; BM, bone marrow; myocarps, myocardium; PN, peripheral nerve; SM, skeletal muscle; syn, synovial membrane. * P < 0.05 compared to uPAR-; * P is 0.01 relative to uPAR-; * P < 0.05 compared to uPAR-; * P < 0.01 compared to uPAR-.

FIG. 28 shows vascular endothelial cell (HUVEC) and fibroblast (DF) cytoprotective activity of cells isolated and collected from hydrogels. uPAR-, isolated and purified uPAR negative cells after exogenous urokinase treatment; upar+, upar+ cells isolated and purified after exogenous urokinase treatment; PAI withdrawal, PAI removal and culture wash, cells isolated and collected; AT, adipose tissue; BM, bone marrow; myocarps, myocardium; PN, peripheral nerve; SM, skeletal muscle; syn, synovial membrane. * P < 0.05 compared to uPAR-; * P is 0.01 relative to uPAR-; * P < 0.05 compared to uPAR-; * P < 0.01 compared to uPAR-.

Fig. 29 shows a schematic diagram of the present disclosure compared to the prior art.

Detailed Description

The present disclosure provides a method of inducing tissue resident upar+ and nestin+ stem cells into a cell cycle, the method comprising: (1) preparing a temporary matrix simulated hydrogel; (2) Encapsulating the isolated tissue fragments into the temporary matrix simulated hydrogel; and (3) 3D culturing the temporary matrix simulated hydrogel in which the tissue fragments are encapsulated in a medium supplemented with a Plasminogen Activator Inhibitor (PAI).

Preferably, the temporary matrix simulated hydrogel may be a fibrin hydrogel, wherein a fibrinogen solution having a concentration of 0.25% to 2.5% is mixed with a thrombin solution having a concentration of 0.5i.u./mL to 5 i.u./mL; a fibrin/collagen mixed hydrogel, wherein a collagen solution having a concentration of 0.1% to 0.5% is mixed in the fibrin hydrogel; or a fibrin/gelatin mixed hydrogel, wherein a gelatin solution having a concentration of 0.1% to 0.5% is mixed in 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, and synovial tissue, but is not limited thereto.

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

Preferably, the method may activate integrin-FAK cell signaling of cells in tissue to induce cell division and cell growth of tissue resident upar+ and nestin+ stem cells, but is not limited thereto.

Preferably, the method can induce tissue resident upar+ and nestin+ stem cells into a temporary matrix to mimic cell migration in the hydrogel and cell growth in the hydrogel, but is not limited thereto.

Tissue-specific temporary matrices are required to induce migration of upar+ and nestin+ tissue-resident stem cells of the present disclosure into the temporary matrices. The preparation can be performed using biopolymers by mimicking the temporary matrix components formed in plasma released from blood vessels after coagulation of tissue lesions. The temporary matrix of the present disclosure may be prepared using fibrin, collagen and gelatin, alone or in combination. The uPA-plasmin activity of the engineered temporary matrix varies from organ to organ, and a temporary matrix resistant to uPA-plasmin activity can be prepared for use. The temporary matrix may be prepared by adjusting the content or composition of the biopolymer. In other words, a temporary matrix obtained by mixing two or more components (for example, fibrin-collagen, fibrin-gelatin, and collagen-gelatin) may be used. The temporary matrix of the present disclosure may contain constituent polymers, e.g., fibrinogen, collagen, and gelatin, in an amount of 1.0mg/mL to 20.0 mg/mL. To enhance the structural properties of the temporary matrix, the degree of crosslinking may be adjusted, and the structural properties may be adjusted by controlling the degree of crosslinking of the temporary matrix using a crosslinking agent (e.g., ca++ or factor XIIIa).

Activation of cell signaling pathways is necessary to induce cell division and growth of stem cells in tissues by in vitro culture. Signals capable of inducing cell growth and migration in combination with integrin ligands of cells may be transmitted and activated through the temporary matrix. Cell division, growth and migration of stem cells in tissue can be induced by supporting the tissue with a temporary matrix capable of transmitting signals into the cells via integrins that bind directly to the cells. The integrin-beta 1-FAK signaling pathway transmits signals to tissue resident stem cells through integrins and induces activation with the support of a temporary matrix, and the tissue resident stem cells can increase expression of uPAR and nestin and induce division, growth and migration of regenerative stem cells in the tissue according to the transmitted signals. The temporary matrix of the present disclosure capable of activating the integrin-FAK cell signaling pathway may accomplish the above object by preparing a temporary matrix mimetic hydrogel composed of polymers having RGD motif (e.g., fibrin, collagen, and gelatin).

The present disclosure provides methods for inducing migration, cell division and growth of tissue resident stem cells by activating the integrin- β1-FAK-uPAR signaling pathway. The efficiency of signaling to stem cells in a tissue is proportional to the density of receptors capable of binding integrin ligands. The density of receptors capable of binding integrin ligands can be achieved by increasing the interface with tissue. Thus, binding of integrin ligands and receptors can be increased by providing a three-dimensional (3D) environment instead of a two-dimensional (2D) environment to increase the area that adheres to cells. Therefore, the efficiency of signal transduction to stem cells in tissues can be improved. The present disclosure provides methods for activating the integrin- β1-FAK-uPAR signaling pathway by providing a 3D support of artificial temporary matrix to tissue. To this end, methods are provided for enhancing signaling pathways by providing 3D binding to tissue fragments using hydrogels capable of sol-gel phase transition as temporary matrices.

uPA-plasmin activity may vary depending on the tissue, extent of injury and cause. Nerve tissue has high uPA-plasmin activity compared to fat, placenta and umbilical cord, and therefore, nerve tissue has high degradation of temporary matrix. Degradation of the temporary matrix can be controlled by adjusting the content of the components constituting the temporary matrix, the molecular weight of the polymer, and the degree of crosslinking density. The present disclosure provides methods for controlling the increased degree of resistance to uPA-plasmin activity by increasing the concentration, molecular weight, and degree of crosslinking of the polymers comprising the temporary matrix. Furthermore, the present disclosure provides methods for modulating uPA-plasmin activity by PAI addition to continuously induce activity, growth, and migration of stem cells in tissue in a temporary matrix. PAI suitable for use in the present disclosure may be selected from the group consisting of aminocaproic acid, tranexamic acid, aprotinin, and aminomethylbenzoic acid. PAI applied to the present disclosure may be used by adding at a concentration of 10 μg to 10mg per mL hydrogel dose. Thus, the present disclosure provides methods of inducing and supporting the growth and migration of tissue-resident stem cells during organ culture by maintaining the addition of PAI and structural stabilization of the temporary matrix.

For excessive activity of uPAR-uPA-plasmin of stem cells, due to rapid degradation of the temporary matrix mimicking hydrogel during the in vitro culture procedure, tissue fragments and temporary matrix surrounding the cells are degraded and lost, resulting in loss of temporary matrix that may adhere and migrate to the stem cells, resulting in loss, reduction, or loss of cells migrating from the tissue into the hydrogel. To control excessive degradation and loss of the temporary matrix mimetic hydrogels, the present disclosure provides methods for maintaining and maintaining the structural function as a matrix by controlling excessive plasmin activity of stem cells and tissue fragments via the addition of PAIs such that the temporary matrix mimetic hydrogels are maintained to function as a physical support matrix for the tissue fragments during organ culture while enabling migration and growth of activated tissue resident stem cells.

Furthermore, the present disclosure provides methods of isolating and culturing tissue-resident upar+ and nestin+ stem cells, the methods comprising: (1) preparing a temporary matrix simulated hydrogel; (2) Encapsulating the isolated tissue fragments into the temporary matrix simulated hydrogel; (3) 3D culturing a temporary matrix simulated hydrogel having tissue fragments encapsulated therein in a medium supplemented with PAI; (4) removing the 3D medium and removing PAI by washing; (5) Re-culturing the PAI-removed culture with a PAI-free medium to degrade the temporary matrix simulated hydrogel; and (6) isolating the stem cells released in the re-culture medium.

Preferably, the temporary matrix simulated hydrogel is a fibrin hydrogel, wherein a fibrinogen solution having a concentration of 0.25% to 2.5% is mixed with a thrombin solution having a concentration of 0.5i.u./mL to 5 i.u./mL; a fibrin/collagen mixed hydrogel, wherein a collagen solution having a concentration of 0.1% to 0.5% is mixed in the fibrin hydrogel; or a fibrin/gelatin mixed hydrogel, wherein a gelatin solution having a concentration of 0.1% to 0.5% is mixed in the fibrin hydrogel, but is not limited thereto.

Preferably, step (5) may induce an increase in uPAR expression in the tissue and degrade the temporary matrix mimetic hydrogel by an increase in plasmin activity, but is not limited thereto.

Preferably, the tissue resident upar+ and nestin+ stem cells may have increased capacity in terms of self replication, in vitro growth, differentiation, or induction of tissue regeneration, but are not limited thereto.

Preferably, the tissue fragments collected from the reculture medium in step (5) may further include a process of repeating steps (2) to (5) 1 to 10 times, but the present disclosure is not limited thereto.

The present disclosure provides methods of selectively isolating upar+ stem cells that migrate into and grow in a temporary matrix. Although the degradation of the temporary matrix by excessive uPAR-plasmin activity can be controlled by the addition of PAI, methods are provided to collect cells from the temporary matrix after migration and growth of the target stem cells are achieved. By taking advantage of the inherent uPAR-plasmin activity of stem cells, a method is provided for harvesting released stem cells that migrate into and grow in the matrix as the temporary matrix degrades by removing PAI after washing. The present disclosure provides methods for isolating upar+ stem cells from solid tissue without the use of any exogenous protease and without subsequent purification procedures, degrading a temporary matrix by uPAR-plasmin according to uPAR expression rate and plasmin activity in the stem cells.

The present disclosure provides methods for harvesting tissue fragments used in organ culture without the use of any exogenous proteases in a state that maintains the structural and functional integrity. Methods are provided for inducing migration into a temporary matrix by repeating organ culture to collect tissue resident upar+ stem cells and preserve the structure of tissue fragments collected after organ culture, as follows.

The present disclosure provides compositions of temporary matrices in which the uPAR-uPA-plasmin activity of tissue-resident stem cells varies according to tissue, which are not degraded and lost due to the activity of uPAR-uPA-plasmin according to tissue, and also provides types and concentrations of PAI according to tissue, wherein excessive degradation is controllable.

Methods of isolating high purity stem cells are provided, without the need for subsequent purification processes, by PAI flushing and removal (PAI withdrawal) of tissue resident uPAR+/nestin+ stem cells that migrate into and grow in the temporary matrix mimetic hydrogels. The tissue resident upar+/nestin + stem cells are useful for the production of stem cell-derived biopharmaceuticals and stem cell therapeutics with high self-replication, in vitro growth, differentiation capacity, tissue regeneration-inducing gene expression, vascular regeneration and wound healing effects.

Furthermore, the present disclosure provides tissue resident upar+ and nestin+ stem cells, or cultures thereof, isolated and cultured according to the methods.

In the present disclosure, the term "culture medium" as used herein refers to a medium capable of supporting the growth and survival of stem cells in vitro, and includes secretions of the cultured stem cells contained in the medium. The medium used for the culture includes all conventional media used in the art suitable for stem cell culture. Depending on the type of cell, the medium and culture conditions may be selected. The medium used for the culture is preferably a cell culture basal medium (CCMM), typically comprising a carbon source, a nitrogen source and a trace element component. Such cell culture basal media include, but are not limited to, for example, dulbecco's Modified Eagle Medium (DMEM), minimal Essential Medium (MEM), eagle Basal Medium (BME), RPMI1640, F-10, F-12, alpha minimal essential Medium (alpha MEM), glasgow Minimal Essential Medium (GMEM), and Iscove's modified Dulbecco's Medium.

In another aspect, in the present disclosure, useful forms include: all forms, including stem cells, their secretions and media components; including only the form of the secretion and the medium components; the isolated secretions are used alone or in combination with stem cells; or in vivo by administration to stem cells alone.

Stem cells may be obtained using any method known in the art.

The tissue resident upar+ and nestin+ stem cells of the present disclosure are useful as cell therapeutics for treating certain diseases, the treatment of which may be a pretreatment of the molecule or direct treatment.

The term "cell therapeutic" as used herein refers to a drug that uses living autologous cells, allogeneic cells, and xenogeneic cells in vitro through a series of actions (e.g., proliferation, selection, or modification of biological properties of the cells by other means) to restore cell and tissue function for therapeutic, diagnostic, and prophylactic purposes.

The cytotherapeutic agent may be administered to the human body by any general route as long as it reaches the target tissue.

In addition to the active ingredient, the pharmaceutical compositions of the present disclosure may also be prepared using pharmaceutically suitable and physiologically acceptable excipients, wherein solubilizers (e.g., excipients), disintegrants, sweeteners, binders, coatings, friction reducers, lubricants, glidants, or flavoring agents may be used as the excipients. The pharmaceutical compositions of the present disclosure may preferably be prepared as pharmaceutical compositions comprising the active ingredient together with one or more types of pharmaceutically acceptable carriers for administration. In compositions prepared in liquid solutions, acceptable pharmaceutical carriers including saline, sterile water, ringer's solution, buffered saline, albumin injection solutions, dextrose solutions, maltodextrin solutions, glycerol, ethanol, and one or more of the sterile and suitable components for use in the body may be mixed and used, as well as other conventional additives (e.g., antioxidants, buffers, and bacteriostats) may be added as desired. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to prepare injectable formulations, for example, aqueous solutions, suspensions and emulsions, pills, capsules, granules or tablets.

The pharmaceutical formulation of the pharmaceutical composition of the present disclosure may be in the form of a sustained release formulation, granules, acids, coated tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injection solutions of the active compound. The pharmaceutical compositions of the present disclosure may be administered in a conventional manner by intravenous, intra-arterial, intraperitoneal, intramuscular, intra-arterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular, or intradermal routes. An effective dose of an active ingredient in the pharmaceutical compositions of the present disclosure refers to the amount required to prevent or treat a disease. Thus, it may be adjusted according to various factors including the type of disease, the severity of the disease, the type and amount of active ingredient and other components contained in the composition, the type of formulation, and the age, weight, general health, sex and diet of the patient, the time of administration, the route of administration and secretion rate of the composition, the duration of treatment, and the drugs used together.

The present disclosure provides methods of isolating stem cells that do not use any exogenous proteases and that do not have tissue dissociation in the process of isolating solid tissue-resident stem cells. Since exogenous proteases are not used, the tissue can retain its function and structure after isolation of stem cells, thereby providing a method that can be used as a tissue source for repeated isolation of stem cells from tissue.

It is well known that the proportion of tissue resident stem cells in the cells constituting the tissue is small, usually less than 0.01%. After tissue damage, the number of somatic cells is reduced and stem cells that replace or regenerate these cells are activated, such that the frequency of stem cells is increased compared to before the damage. After injury, stem cells in the tissue self-replicate by cell division, generating progeny progenitor cells, and the progeny stem cells migrate to the site of injury. The present patent provides methods for inducing self-replication and cell division of stem cells in tissue fragments by activating tissue homeostasis mechanisms by in vitro culture and recruiting self-replicating and dividing stem cells to the site of injury in vitro.

After tissue injury, a temporary matrix is formed around the injured tissue, through which the matrix-cytokinin pathway is activated, and stem cells acting as tissue injury migrate to the injured site after cell division, causing initiation of tissue regeneration mechanisms. The present disclosure provides methods of activating stem cells in tissue, inducing cell division, growth, and migration into a temporary matrix-mimicking hydrogel, and isolating stem cells that migrate into the hydrogel by maximizing matrix-cell integrin interactions through a 3D support of the temporary matrix-mimicking hydrogel around tissue fragments.

Tissue resident stem cells are reportedly activated in response to tissue damage, and the activated stem cells have increased uPAR or nestin expression. uPAR and nestin expression have been revealed to play an important role in stem cell growth and migration, and upar+ and nestin+ cells are known to play a key role in tissue regeneration. Due to the increased expression of uPAR and nestin in reparative stem cells, uPAR and nestin can serve as key target markers in stem cell isolation. The present disclosure provides methods for selectively isolating and collecting upar+ stem cells by inducing cell division, growth and migration of tissue resident upar+ and nestin+ stem cells as a result of activation of the matrix-cytokinin pathway by in vitro culture.

In addition, the present disclosure provides methods for isolating and collecting cells from hydrogels based on uPAR-plasmin-MMP activity as a result of physiological responses to activation and stimulation of stem cells, without the need for exogenous proteases, based on plasmin activity and uPAR expression of stem cells that migrate into and grow in hydrogels after induction of temporary matrix provided upon migration into 3D culture mimics hydrogels and grows therein.

The present disclosure provides a general method for isolating tissue resident upar+ and nestin+ stem cells from representative solid tissues (e.g., fat, bone marrow, myocardium, nerves, skeletal muscle, and synovium). Tissue resident upar+ and nestin+ stem cells have the ability to self-replicate, grow highly in vitro, multipotency, and highly regenerate tissue, making stem cells useful as regenerative therapeutics.

Examples

Hereinafter, the present disclosure will be described in more detail by way of example embodiments. These example embodiments are only for the purpose of describing the present disclosure in more detail, and it will be apparent to those skilled in the art that the scope of the present disclosure is not limited by these example embodiments according to the gist of the present disclosure.

< example 1> preparation of temporary matrix simulated hydrogel

Temporary matrix simulated hydrogels are prepared by mixing fibrinogen, collagen, gelatin, or these polymers. Dissolving plasma-derived fibrinogen at a concentration of 2.5mg/mL to 10mg/mL in a solution comprising 10mM to 50mM CaCl ₂ To prepare a fibrinogen solution. Thrombin was dissolved in PBS at a concentration of 1 unit/mL to 10units/mL to prepare a thrombin solution.

Collagen (Matrix BioScience, germany) or Gelatin (befMatrix Collagen, nitta Gelatin, japan) of dermal origin was dissolved in 0.1% (wt./vol.) acetic acid to use a collagen solution at a concentration of 1.0 mg/mL-20.0 mg/mL. With 50mM NaHCO ₃ 10 Xreconstitution buffer for preparing neutral gelatin or collagen solution was prepared, 40mM HEPES and 0.01N NaOH, and mixed with collagen or gelatin solution in a ratio of 9:1 to prepare neutral collagen or gelatin solution.

The temporary matrix simulated hydrogel was prepared by: a fibrinogen gel is produced using a fibrinogen solution having a concentration of 0.25% to 2.5% and a thrombin solution having a concentration of 0.5i.u./mL to 5i.u./mL, and a collagen or gelatin solution having a concentration of 0.1% to 0.5% is mixed in the preparation of the fibrin hydrogel, thereby preparing a fibrin/collagen or fibrin/gelatin mixed hydrogel.

< example 2> two-dimensional (2D) and three-dimensional (3D) organ culture

Organs used for culture include Adipose Tissue (AT), bone Marrow (BM), cardiac muscle (myoar), peripheral Nerve (PN), skeletal Muscle (SM), and synovium (Syn). This study using tissue derived from brain death donors was approved by the institutional ethical review board and the tissue was donated by brain death patients. After removing fibrous tissue and hematoma attached to surrounding tissue with scissors, the donated tissue was finely cut into 0.2mm using a surgical knife ³ To 2mm ³ Small slices of size. Thereafter, the tissue fragments were suspended with PBS, centrifuged at 1,000rpm, and the washing process was repeated three times to remove the supernatant.

After washing, the tissue fragments were suspended in culture medium and then cultured by two organ culture methods. In the first method, defined as a monolayer (2D) organ culture method, is a case where tissue fragments suspended in a culture medium are inoculated into a culture vessel and cultured on the surface of the culture vessel.

In the second method, defined as a 3D organ culture method, tissue fragments are embedded in fibrin, fibrin/collagen or fibrin/gelatin temporary matrix mimic hydrogels and cultured in a three-dimensional (3D) environment. In the 3D organ culture method, tissue fragments are mixed with 0.25% -2.5% fibrinogen solution, then mixed with an equivalent amount of 0.5 units/mL-5 units/mL thrombin solution at a dose of 1:1, and the mixture is polymerized at 37℃for 1 hour to embed the tissue fragments in fibrin hydrogel. For fat, bone marrow, heart and muscle, tissue fragments are embedded in a temporary matrix simulated fibrin hydrogel having a final fibrinogen solution of 0.25% to 1.25% and a thrombin solution of 0.25 units/mL to 2.5 units/mL. For nerve and synovial tissue fragments, the tissue fragments are embedded in a temporary matrix simulated fibrin/collagen or gelatin hydrogel consisting of 0.25% -2.5% fibrinogen, 0.1% -0.5% neutral collagen or gelatin solution and 0.25 units/mL to 2.5 units/mL thrombin solution.

The tissue fragments mixed with the temporary matrix simulated hydrogel solution were transferred to a 100-mm to 150-mm culture vessel, then placed in an incubator at 37 ℃ for 1 hour, and converted into gel after the polymerization process. The composition of the organ culture medium contained 45% v/v DMEM, 45% v/v Ham's F, 10% fetal bovine serum (FBS, invitrogen), 20ng/mL EGF, 2ng/mL bFGF, 10ng/mL IGF, and 10 μg/mL gentamicin (Invitrogn). The medium was added in an amount equal to twice the amount of gel, and after the addition of the medium, the culture vessel was placed on an orbital shaker for 14 days while stirring at a rate of 30 rpm. The medium was changed twice a week.

In order to inhibit the degradation of the hydrogel, PAI such as tranexamic acid or aminomethylbenzoic acid is added to the medium at a concentration of 10. Mu.g/mL to 500. Mu.g/mL per day during the culture. For the culture of fat, bone marrow, cardiac muscle and skeletal muscle organs, tranexamic acid or aminomethylbenzoic acid at a concentration of 100 μg/mL to 250 μg/mL is added to the medium to inhibit degradation of the temporary matrix mimetic hydrogel. In the case of peripheral nerves and synovium, tranexamic acid or aminomethylbenzoic acid is added to a medium at a concentration of 250. Mu.g/mL to 500. Mu.g/mL, followed by culturing.

< example 3> expression of pFAK, uPAR, nestin and Ki-67 in tissue fragments according to organ culture

After 7 days of 2D and 3D organ culture, cultured adipose tissue, bone marrow, cardiac muscle, nerve, skeletal muscle and synovial tissue fragments were collected from the culture vessel or hydrogel. Paraffin blocks were prepared after fixation for 2 hours using 4% neutral formalin solution. Sections with a thickness of 4 μm were peeled from paraffin blocks and subjected to immunohistochemical staining.

To assess the extent of activity of integrin-FAK cell signaling pathways in cultured tissues, primary antibodies (e.g., anti-phosphorylated FAK (3283,Cell Signaling Technology,USA;pFAK), anti-uPAR (MAB 807, R & D Systems, USA) and anti-Ki-67 (M7240, DAKO, USA)) were used to assess expression. After reaction with primary antibody, positive cells were assessed for localization and expression rate after undergoing three washes, reaction with HRP-conjugated secondary antibody (ImmPRESS One-Step Polymer Systems, vector Laboratories, USA), development using DAB as substrate, and then counterstaining with hematoxylin (H-3401-500,Vector Laboratories).

According to in vitro organ culture, constituent cells in tissue fragments are activated, and the activated cells proliferate after cell division to operate tissue homeostasis mechanisms to replace or regenerate damaged or lost cells. The integrin cell signaling pathway is one of the main mechanisms that induce tissue regeneration, and integrin cell signaling is activated by the binding of cell ligands, extracellular matrix and receptors. In this example embodiment, the expression of pFAK, uPAR and nestin proteins, which are target factors activated by integrin cell signaling in constituent cells within tissue fragments cultured in monolayer culture (two-dimensional, 2D), was analyzed, wherein the tissue fragments were cultured on the surface of the culture vessel after direct inoculation into the culture vessel, and were cultured in a three-dimensional (3D) environment after embedding in hydrogels.

After 3D organ culture, pFAK expression rates in tissue fragments were found to be significantly higher than in 2D organ culture. Differences in pFAK expression rates after organ culture were identified according to tissue type, and pFAK expression after 3D organ culture was significantly higher in myocardium, nerve and skeletal muscle (fig. 1A).

In the 3D organ culture environment, the expression of the downstream factor uPAR of the integrin signaling pathway was also significantly high, with a trend similar to pFAK, and high expression was observed in myocardium, nerve and skeletal muscle (fig. 1B).

Cell division and growth of constituent cells in an organ can be induced by integrin signaling. Cell regeneration and cell growth capacity was assessed by expression of nestin and Ki-67 specifically expressed in cells undergoing regeneration or cell division. The expression rate of nestin in constituent cells in tissue fragments after 3D organ culture was significantly high compared to nestin expression in tissues after 2D organ culture (fig. 1C). The expression rate of Ki-67 (a marker of cell division) was also significantly high in tissue fragments after 3D organ culture (fig. 1D).

Cell division and growth of cells constituting tissue fragments can be induced by in vitro organ culture, integrin signaling is maximized by 3D culture environment using ligands capable of binding integrins, and expression of pFAK and uPAR as downstream target factors is enhanced, so it was found that 3D organ culture induces significantly higher cell division and cell growth of constituent cells in tissue fragments compared to 2D organ culture.

The 3D physical stimulation of the temporary matrix mimicking hydrogels can provide a method to significantly increase cell division and growth by activating signaling pathways of constituent cells in an organ.

< example 4> localization and characterization of pFAK+, uPAR+ and nestin+ cells in tissue fragments after 3D organ culture

Myocardial tissue fragments cultured before 3D organ culture (0D) or 3 days, 5 days, 7 days, and 14 days after culture were collected from the hydrogel. Paraffin blocks were prepared after fixation for 2 hours using 4% neutral formalin solution. Sections with a thickness of 4 μm were obtained from paraffin blocks and subjected to immunohistochemical staining. To assess the extent of activity of integrin-FAK cell signaling pathways in post-culture tissues, expression was assessed using primary antibodies (e.g., anti-phosphorylated FAK (3283,Cell Signaling Technology,USA;pFAK), anti-uPAR (MAB 807, R & D Systems, USA), anti-nestin (MAB 5326, millipore, USA) and anti-Ki-67 (M7240, DAKO, USA)). After reaction with primary antibody, positive cells were assessed for localization and expression rate after undergoing three washes, reaction with HRP-conjugated secondary antibody (ImmPRESS One-Step Polymer Systems, vector Laboratories, USA), color development using DAB as substrate, and counterstaining with hematoxylin (H-3401-500,Vector Laboratories).

After encapsulation of the myocardium in the temporary matrix-mimicking hydrogel, 3D organ culture was performed and the frequency of pfak+, upar+ and nestin+ cells in the myocardium was determined. The expression rates of pFAK, uPAR and nestin were lower than 6% before culture. However, pFAK, uPAR and nestin-positive cell counts increased significantly in proportion to the culture period following 3D organ culture. After 2 weeks of culture, the expression rates of pFAK, uPAR and nestin in tissue fragments were greater than or equal to 30% (fig. 2 and 3). pFAK, uPAR and nestin are expressed in cells in the matrix between mature somatic cells, pFAK, uPAR and nestin are expressed in pericapillary cells but not capillary endothelial cells.

Ki-67 is a marker of cell division and is not expressed in mature somatic cells but is expressed less than 1% prior to culture. However, the expression rate of Ki-67 increased in proportion to the culture period after in vitro 3D organ culture, and the expression rate of Ki-67 was 45.7% in cells residing in the myocardium after 2 weeks of organ culture, with the result that division and growth of intramyocardial cells was induced by 3D organ culture while Ki-67 was also expressed mainly in perivascular cells (FIG. 4).

In this example embodiment, it was found that cell division and growth in the myocardium can be induced by 3D temporary matrix mimicking hydrogels, and division and growth of pfak+, upar+ and nestin+ perivascular cells can be induced by organ culture.

< example 5> migration of uPAR+ and nestin+ cells in tissue to a temporary matrix mimetic hydrogel by 3D organ culture and growth therein

Fat, bone marrow, cardiac muscle, nerve, skeletal muscle and synovial tissue fragments were encapsulated in fibrin or fibrin/collagen hydrogels and 3D organ cultures were performed. The culture medium was added in an amount twice that of the hydrogel, and the culture vessel was placed on an orbital shaker for 14 days while stirring at a rate of 30 rpm. PAI is added to the culture medium to inhibit hydrogel degradation. To the medium was added 5. Mu.M bromodeoxyuridine (B5002, sigma-Aldrich, USA) daily for 7 days to label cells that underwent cell division during in vitro culture. The culture was performed for 2 weeks with medium changes twice a week.

After 3 days, 7 days and 14 days of 3D organ culture, the cultured tissue and hydrogel were collected simultaneously. Paraffin blocks were prepared after fixation for 2 hours using 4% neutral formalin solution. Sections with a thickness of 4 μm were peeled from paraffin blocks and subjected to immunohistochemical staining.

To assess the expression of uPAR, nestin and BrdU in cells that migrate into and grow in hydrogels, anti-uPAR (MAB 807, R & D Systems, USA), anti-nestin (MAB 5326, millipore, USA) and anti-BrdU (347580,BD Biosciences,USA) primary antibodies were used. After reaction with primary antibody, expression rate was assessed by three washes, reaction with HRP-conjugated secondary antibody (ImmPRESS One-Step Polymer Systems, vector Laboratories, USA), color development using DAB as substrate, and counterstaining with hematoxylin (H-3401-500,Vector Laboratories).

The temporary matrix mimicking hydrogels function as extracellular matrices capable of supporting cell migration and growth, which can be achieved by providing fibronectin, collagen, and fibrin, which are cell adhesion factors necessary for cell migration. Activation of integrin-pFAK cell signaling pathways via receptor-ligand binding via 3D temporary matrix hydrogels can induce cell division and growth of upar+ and nestin+ cells in organs, and induce recruitment, migration and growth of upar+ or nestin+ cells into temporary matrix mimetic hydrogels.

The number of cells migrating into and growing in the hydrogel was observed to increase in proportion to the incubation period of the 3D myocardial organ (fig. 5). Cells migrating into and growing in the hydrogel can be observed to take on a spindle shape, binding to the hydrogel resulting in cytoplasmic swelling.

Cells migrating from the myocardium into the hydrogel and growing in the hydrogel have uPAR and nestin expression rates of 88.5% and 95.4% or higher, exhibiting the same upar+ and nestin+ characteristics as cells undergoing division and growth after culturing organs in tissues. More than 89.4% of the cells migrating into the hydrogel are BrdU positive, which is cells that synthesize DNA by absorbing BrdU added during organ culture, such that the majority of the cells in the hydrogel are cells that migrate after cell division and growth after organ culture. Brdu+ cells were not detected in the myocardium prior to 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 fragments, the cells in the temporary matrix mimicking hydrogel were about 90% upar positive and over 90% BrdU positive, and by organ culture, tissue resident cells were found to be cells dividing, growing and migrating into the hydrogel, no significant differences according to tissue were observed (fig. 6).

< example 6> isolation of cells migrating into and growing in hydrogels after 3D organ culture

Fat, bone marrow, cardiac muscle, nerve, skeletal muscle and synovial tissue fragments were embedded in fibrin or fibrin/collagen hydrogels and 3D organ cultures were performed. The culture medium was added in an amount twice that of the hydrogel, and the culture vessel was placed on an orbital shaker, and culturing was performed for 14 days while stirring at a rate of 30 rpm. To inhibit hydrogel degradation, PAI is added daily during the incubation period to inhibit degradation of the temporary matrix mimetic hydrogel based on PA activity of cells that migrate into and grow in the hydrogel.

The uPAR mRNA and plasmin activity of tissue fragments collected after 3D organ culture were evaluated. After preparing tissue lysates from 10mg tissue fragments, plasmin activity was analyzed using plasmin activity assay kit (ab 204728, abcam). After adding a fluorogenic substrate to the tissue lysate, plasmin activity was measured by measuring fluorescence intensity after 20 minutes of reaction at 37 ℃. Plasmin activity was calculated by the following formula, Δrfu360/450 nm= (RFU) ₂ -RFU _2BG )-(RFU ₁ -RFU _1BG ). The expression rate of uPAR mRNA in tissue fragments was analyzed by real-time qPCR by extracting total RNA from 10mg tissue fragments and performing reverse transcriptase reaction to construct cDNA.

After 14 days of 3D organ culture, the medium was removed. After the DMEM addition, washing was performed by shaking at 30rpm for 30 minutes on an orbital shaker, and the washing solution was removed. This washing procedure was repeated three times. After washing, the cells that migrate into and grow in the hydrogel are separated and collected in two ways. In the first method, 1,000 units/mL urokinase is added to a culture medium, which is then added to a culture vessel, the hydrogel is degraded for 2 hours, cells and tissue fragments released from the degraded hydrogel are transferred to a tube, the supernatant is removed after centrifugation at 3,000rpm for 10 minutes, and the collected cell and tissue fragment pellet is suspended in the culture medium.

Cells can be isolated and collected from hydrogels by PAI removal. After 14 days of organ culture, the medium was removed, and then three washes were performed using DMEM to remove PAI remaining in the medium and hydrogel. After addition of fresh medium, the culture was performed on an orbital shaker with shaking at 30rpm for 1 hour, and no PAI was added to the medium. Cells separated and released from the hydrogel were collected using a pipette, transferred into a tube, and centrifuged at 3,000rpm for 10 minutes, the supernatant was removed, and the collected cells and tissue debris pellet were suspended with a medium.

Cells isolated and collected from the hydrogel were suspended in a medium, the total cell number was calculated by a hemocytometer, and the uPAR expression rate of the collected cells was analyzed using flow cytometry.

In this exemplary embodiment, a method is provided for selectively isolating and collecting upar+/nestin+ cells by inducing migration and growth of upar+/nestin+ cells in hydrogels grown after cell division in tissues after 3D organ culture and degrading hydrogels according to plasmin activity of upar+/nestin+ cells themselves in the absence of exogenous proteases with respect to cells migrating into and growing in hydrogels.

Expression and plasmin activity of uPAR mRNA in tissue fragments before and after organ culture was assessed. Even before in vitro culture, uPAR mRNA expression and plasmin activity varied depending on the organ, and uPAR mRNA expression and plasmin activity were high in cardiac, neural and skeletal muscle compared to other tissues (fig. 7). In all tissue fragments assessed after organ culture, uPAR mRNA expression and plasmin activity were significantly increased compared to before culture. In particular, mRNA expression and plasmin activity were measured highest in myocardium, neurons and skeletal muscle. Since plasmin activity is proportional to uPAR expression, an increase in uPAR expression in a tissue can be induced by organ culture and methods for increasing plasmin activity in a tissue by an increase in uPAR after organ culture are provided.

During 3D organ culture, a Plasminogen Activator Inhibitor (PAI) is added to the culture medium, and the added PAI can inhibit hydrogel degradation by controlling excessive plasmin and protease activity of organs and cells. When cells undergoing cell division and growth within a tissue by organ culture are induced to migrate into and grow in the hydrogel, and then PAI is removed from the culture medium and the hydrogel by a rinsing and washing process, degradation of the hydrogel can be induced only by plasmin and protease activity in which the organ and cells reside, thereby releasing the cells that migrate into and grow in the hydrogel, and thus enabling separation and collection of the released cells.

For PAI removal, the PAI-containing medium was removed, the PAI remaining in the hydrogel and organs was removed by washing three times with DMEM, and the culture was performed by adding two times the amount of the medium containing no PAI to the hydrogel. After 30 minutes of incubation, the hydrogel began to degrade due to PAI removal; as the hydrogel degrades, cells migrating into and growing in the hydrogel are released from the hydrogel and aggregate between the cells; and after 2 hours of incubation, the hydrogel around the migrating and growing cells and tissue fragments was completely degraded, allowing the cells in the hydrogel to be released into the culture medium (fig. 8). With this exemplary embodiment, the temporary matrix mimics the degradation of the hydrogel only with withdrawal of PAI due to plasmin and protease activity where the cells reside, and the cells in the hydrogel are released into the culture medium, thus, the released cells in the culture medium can be isolated and collected in a form of cytoplasmic contraction and aggregation.

During organ culture, the hydrogel is prevented from degradation by the addition of PAI, and the cells in the hydrogel exhibit spindle shape due to cytoplasmic unfolding. However, by removing PAI in the medium, the hydrogel around the cells starts to degrade to shrink the cytoplasm, the cells induce the cells to form aggregates as a result of cell-cell attachment, and the aggregated cells are released into the medium (fig. 9).

In the histological method, it was clearly shown that although the cells in the hydrogel showed complete expansion of the cytoplasm before PAI removal, when the hydrogel began to degrade after PAI removal, the hydrogel to which the cells could attach was lost, and the cells were aggregated, separated, and released from the hydrogel due to cell-cell connection (fig. 10). All aggregated cells released from the hydrogel showed the characteristic of expressing uPAR, so the higher uPAR expression, the higher plasmin activity, providing the basis for isolating upar+ cells in the hydrogel with only PAI removal (fig. 10).

Following PAI withdrawal or urokinase treatment, cells that migrate into and grow in the hydrogel may be isolated and collected. In the present disclosure, the utility of methods for isolating cells that migrate into and grow in hydrogels, even in the absence of urokinase treatment with PAI removal, was compared and evaluated.

By way of example, the result of isolating and collecting cells corresponding to the number of cells treated by urokinase by the PAI removal method can be determined (FIG. 11A). After 2 weeks of organ culture, more than 200 ten thousand cells per 100mg of tissue could be isolated and collected, which is lower than the number of cells collected after urokinase treatment, but without significant differences.

However, uPAR expression rates were significantly higher for cells collected after PAI withdrawal than for urokinase treatment (fig. 11B). In particular, the yield of selectively isolating upar+ cells from tissues with high uPAR expression rate and plasmin activity is high. This suggests that the hydrogel has high degradation activity in upar+ cells, and thus, it can be verified that it is a highly efficient method for selectively isolating upar+ cells by PAI removal and washing.

< example 7> cell separation of tissue fragments collected after 3D organ culture and repeated organ culture

Fat, bone marrow, cardiac muscle, nerve, skeletal muscle and synovial tissue fragments were encapsulated in fibrin or fibrin/collagen hydrogels and 3D organ cultures were performed. Organ culture medium was added in twice the amount of hydrogel and the culture vessel was placed on an orbital shaker and cultured for 14 days while stirring at a rate of 30 rpm. To inhibit hydrogel degradation, PAI is added daily during the incubation period to inhibit temporary matrix mimicking hydrogel degradation due to plasmin activity of cells migrating into and growing in the hydrogel.

After 14 days of 3D organ culture, the medium was removed. After the DMEM addition, washing was performed with stirring at 30rpm for 30 minutes on an orbital shaker, and the washing solution was removed. The washing procedure was repeated three times to remove the PAI remaining in the medium and hydrogel. After addition of fresh medium, the culture was performed with shaking at 30rpm for 2 hours, and no PAI was added to the medium. Cell and tissue fragments separated and released from the hydrogel were collected using a pipette, transferred into a tube, and centrifuged at 3,000rpm for 10 minutes, and then the supernatant was removed. Due to the high specific gravity, the tissue debris was precipitated faster than the cells, so the cells and tissue debris precipitate were dispersed in DMEM, and then left for 30 seconds to induce the precipitation of tissue debris, and then the supernatant was transferred only to a new tube, and repeated three times to separate the collected cells and tissue debris.

The tissue fragments separated from the hydrogel were re-encapsulated in the hydrogel by the PAI removal method, and after two weeks of 3D organ culture by the method described in example 2, the cells and tissue fragments that migrated into and grew in the hydrogel by the method described in example 7 were isolated and collected. The tissue fragments collected after PAI withdrawal were repeatedly induced to undergo cell migration and growth in the tissue through three consecutive 3D organ cultures, thereby separating and collecting cells in the hydrogel, and the total number of the separated and collected cells was measured using a cytometer.

In the present exemplary embodiment, the following method is provided: cells grown after migration into the hydrogel after organ culture were isolated and collected by collecting tissue fragments that remained in the structure after PAI withdrawal and performing repeated organ culture using the collected tissue fragments. Exogenous protease treatment (e.g., urokinase) may degrade tissue fragments embedded with hydrogels, resulting in loss of structural, functional microenvironment of the tissue fragments.

In this example embodiment, cells and tissue fragments that migrate after growth in tissue that has been removed by PAI and that has not been treated with exogenous protease are isolated and collected, while the collected tissue fragments are also subjected to repeated organ cultures. Three consecutive organ cultures were performed using tissue fragments collected after organ culture, with the following results: cells can be harvested in similar yields to the initial organ culture (fig. 12). It is confirmed that the PAI removal method is a method capable of preserving the structure, functional microenvironment of tissue fragments embedded with cells migrating into and growing in a hydrogel after 3D organ culture, and a method of isolating tissue resident cells in an efficient and stable manner by repeating organ culture.

< example 8> culture of cells isolated from hydrogels after removal of PAI and urokinase treatment

Fat, bone marrow, cardiac muscle, nerve, skeletal muscle and synovial tissue fragments were embedded in fibrin or fibrin/collagen hydrogels and 3D organ cultures were performed. Organ culture medium was added in twice the amount of hydrogel and the culture vessel was placed on an orbital shaker and cultured for 14 days while stirring at a rate of 30 rpm. To inhibit hydrogel degradation, tranexamic acid, i.e., PAI, was added to the medium daily.

After 14 days of 3D organ culture, the medium was removed. After DMEM was added, the medium was washed for 30 minutes while stirring at a rate of 30rpm, and the washing process was repeated three times. After washing, cells migrating into and growing in the hydrogel were collected after isolation of the cells by PAI removal or urokinase method presented in example 6. After 14 days of organ culture, the medium was removed and washed three times with DMEM to remove PAI remaining in the medium and hydrogel. After addition of fresh medium, the culture was continued for 1 hour with stirring at 30rpm, and no PAI was added to the medium. Cells separated and released from the hydrogel were collected using a pipette, transferred into a tube, and centrifuged at 3,000rpm for 10 minutes, and then the supernatant was removed, and then the collected cells and tissue debris pellet was suspended with a medium.

For urokinase treatment, after washing three times, 1,000 units/mL of urokinase was added to the medium, the hydrogel was degraded for 2 hours, then cell and tissue fragments released from the degraded hydrogel were collected, transferred to a tube, and centrifuged at 3,000rpm for 10 minutes to remove the supernatant, followed by suspending the collected cell and tissue fragment pellet with the medium.

The cells isolated and collected from the hydrogel were isolated at 5,000 cells per cm ² The polystyrene culture vessel was inoculated, and then the medium was added. The adhesion and growth characteristics of cells isolated and collected from hydrogels in a monolayer culture environment were evaluated using a microscope.

After PAI withdrawal, cell aggregates were observed to form as cells were released from the hydrogel (fig. 13A); suspending the collected cells in a culture medium to form small circular aggregates (fig. 13B); and inoculated in a PS culture vessel for culture in a monolayer environment. Cells collected in an intercellular aggregated state adhered to the culture container 30 minutes after inoculation (fig. 13C); the cytoplasm of the inoculated cells swelled 1 hour after inoculation (fig. 13D); and after 2 hours of culture, cells were found to adhere stably while cells around the cell aggregate grew (fig. 13E).

After organ culture of all tissue fragments (e.g., fat, bone marrow, myocardium, nerve, skeletal muscle, and synovium), cells could be safely isolated and collected by PAI withdrawal, and when the collected cells were mixed with the culture medium and inoculated in the culture vessel, it was found that all the collected cells adhered to the culture vessel within 30 minutes while the cytoplasm stably swelled, without significant differences according to tissues (fig. 14).

Cells seeded and cultured in a monolayer environment showed a high growth rate, grew around the seeded cells after 2 days of culture and spread to the periphery, and cells isolated and collected by PAI withdrawal were found to maintain a high growth rate even when cultured in a monolayer environment (fig. 15).

< example 9> immunophenotyping characteristics of cells isolated after PAI removal

Fat, bone marrow, cardiac muscle, nerve, skeletal muscle and synovial tissue fragments were embedded in fibrin or fibrin/collagen hydrogels and 3D organ cultures were performed. Organ culture medium was added in twice the amount of hydrogel and the culture vessel was placed on an orbital shaker and cultured for 14 days while stirring at a rate of 30 rpm. In order to inhibit hydrogel degradation, tranexamic acid (PAI) is added to the culture medium at a certain concentration while culturing.

After 14 days of 3D organ culture, the medium was removed. After the DMEM was added, washing was performed for 30 minutes while stirring at a rate of 30rpm, and the washing process was repeated three times. After washing, cells that migrate into and grow in the hydrogel are collected after separation of the cells by the PAI removal method. After 14 days of organ culture, the medium was removed and washed three times with DMEM to remove PAI remaining in the medium and hydrogel. After addition of fresh medium, the culture was performed for 1 hour while stirring at a rate of 30rpm, and no PAI was added to the medium. Cells separated and released from the hydrogel were collected using a pipette, transferred to a tube, and centrifuged at 3,000rpm for 10 minutes, the supernatant was removed, and the collected cell and tissue debris pellet was suspended in the medium.

Cells isolated and collected after PAI withdrawal or urokinase treatment were suspended in PBS and analyzed for immunophenotyping characteristics using flow cytometry. Mesenchymal Stem Cell (MSC) markers CD29, CD44, CD105 and CD140b were evaluated for expression rates of uPAR and nestin. CD34 and CD45 (as hematopoietic cell markers) and CD31 (as vascular endothelial cell markers) were used to evaluate the expression rates of these markers.

The migrated and grown cells can be collected and isolated from the hydrogel due to urokinase treatment or PAI withdrawal. Regardless of the method of harvesting the cells, cells migrating and growing in the hydrogel from all cultured tissues showed the same immunophenotyping characteristics as mesenchymal stem cells, with expression rates of CD29, CD73, CD105 and CD140b greater than 90% (fig. 16A). There was no difference in the expression rate of the mesenchymal stem cell markers isolated and collected from the hydrogel according to the isolation method.

However, in this example, the expression rate of uPAR and nestin in cells isolated and collected by PAI withdrawal after organ culture of all tissue fragments exceeded 90%. On the other hand, the expression rate of the cells collected after urokinase treatment was between 30% and 67%. Based on the high plasmin activity of upar+ and nestin+ cells in the hydrogel, results can be obtained that enable selective isolation and collection by PAI removal. On the other hand, cells with various immunophenotype characteristics in hydrogels were determined, mixed and collected in urokinase treatment (fig. 16B).

Hematopoietic cells and vascular endothelial cells present in the tissue may be mixed in the step of isolating the cells from the tissue, and the degree of mixing of these cells may be determined by detecting cd31+, cd34+ and cd45+ cells. In the case of cells isolated and collected from the hydrogel by the PAI removal method, the expression rates of CD31, CD34 and CD45 were less than 1%, but the cells isolated and collected after urokinase treatment were 2.8% to 7.0%, so that the degree of mixing of hematopoietic cells and vascular endothelial cells was found to be high (fig. 17).

By this example, it was found that this is a method of removing selectively separating and collecting upar+ and nestin+ cells migrating from the tissue into the hydrogel by PAI while minimizing the mixing of hematopoietic cells and vascular endothelial cells in the separation process.

< example 10> self-replication ability of uPAR+/nestin+ cells collected and isolated after removal of PAI

Fat, bone marrow, cardiac muscle, nerve, skeletal muscle and synovial tissue fragments were embedded in fibrin or fibrin/collagen hydrogels, 3D organ culture was performed for 2 weeks, and then cells migrating into and growing in the hydrogels were isolated and collected by the PAI removal and urokinase method described in example 6.

Cells separated and collected from the hydrogel were transferred to a tube and centrifuged at 3,000rpm for 10 minutes to remove the supernatant. Suspending the cell pellet in a culture medium and adjusting the cell density to 1.0X10 ⁶ Per mL. To verify the self-replication ability of cells isolated and collected from hydrogels, evaluation was performed by colony formation assay (CFU). After 6,000 cells were inoculated in a 100-mm culture vessel, culture was performed for 2 weeks, and then stained with 1% crystal violet after the culture. After washing, the number of colonies with a diameter of 2mm or more was measured.

The frequency of CFU formation was different depending on the tissue type, but the removal of cells isolated and collected from the hydrogel by PAI was significantly higher in all tissue fragments compared to cells collected after urokinase treatment (fig. 18). In particular, cells derived from cardiac muscle, nerves and skeletal muscle show a high colony forming ability compared to cells derived from other tissues. Among the cells isolated after urokinase treatment, CFU frequency was 3.1% to 8.4%, but the cells isolated by PAI withdrawal method were found to have high CFU frequency of 8.8% to 37.9%.

Self-replication ability is one of the main characteristics of stem cells, 3D organ culture can activate stem cells in tissues to induce migration into and growth in hydrogels, and stem cells with high self-replication titer in hydrogels can be selectively isolated and collected by PAI withdrawal method, demonstrating that upar+/nestin+ cells with high plasmin activity are stem cells with high titer (fig. 18).

< example 11> in vitro growth Capacity of isolated cells after PAI removal

Cells separated and collected from the hydrogel were transferred to a tube and centrifuged at 3,000rpm for 10 minutes to remove the supernatant. Suspending the cell pellet in a culture medium and adjusting the cell density to 1.0X10 ⁶ Per mL. To verify the in vitro growth capacity of cells isolated and collected from hydrogels, population Doubling Time (PDT) and Population Doubling Level (PDL) were analyzed and evaluated. Per cm in a T75 flask ² After 3,000 cells were seeded, culture was performed for 7 days, cells were collected after trypsin/EDTA treatment, and the total cell number was calculated by a hemocytometer. PDT and PDL are calculated and compared by the number of seeded cells, the total number of cells collected and the incubation period. PDT is calculated according to the following formula. PDT= [ (incubation time)/((logN 2-logN 1)/log 2) by the formula]Calculations were performed where N1 is the cell count at the time of inoculation and N2 is the cell count collected after culture. PDL is calculated according to the following formula. By the formula pdl= [ pdl0+3.322 (log n2-log n 1)]Calculations were performed where N1 is the cell count at the time of inoculation and N2 is the cell count collected after culture.

The higher the in vitro growth capacity, the higher the expression rate of Ki-67, a cell cycle marker, in cultured cells. The expression rate of anti-Ki 67 was analyzed by flow cytometry, and the in vitro cell growth capacity was analyzed by comparison.

After urokinase or PAI withdrawal treatment, the migrated and grown cells were isolated from the hydrogel and the in vitro growth capacity of the harvested cells was compared and analyzed. PDT was significantly lower in cells isolated and collected by PAI withdrawal and significantly higher in PDL compared to cells obtained after urokinase treatment. The same trend as for self-replication capacity, cells were found to have excellent in vitro growth capacity, with low PDT and high PDL of cells isolated and collected by PAI withdrawal (fig. 19).

To verify the high growth capacity of cells isolated and collected from the hydrogel after PAI removal treatment, the expression rate of Ki-67 was analyzed by flow cytometry. The Ki-67 marker refers to cells in the cell cycle, so a higher Ki-67 expression rate is an indication of higher cell growth capacity. Cells obtained by PAI withdrawal showed high Ki-67 expression rates of 58.5% to 75.4% significantly higher than 33.6% to 48.7% in cells obtained after urokinase treatment (fig. 20).

As a result of the above, PAI withdrawal can be found as a method for selectively isolating cells with high in vitro growth capacity, which supports upar+/nestin+ cells with high plasmin activity as cells with high growth capacity.

< example 12> differentiation ability of isolated cells after PAI removal

Cells separated and collected from the hydrogel were transferred to a tube and centrifuged at 3,000rpm for 10 minutes to remove the supernatant. Suspending the cell pellet in a culture medium and adjusting the cell density to 1.0X10 ⁶ Per mL. The ability of cells in the hydrogels to differentiate into adipocytes and osteoblasts was assessed. Will be 2.0X10 ⁵ The individual cells were inoculated into a 24-well culture vessel, and cultured for 14 days after adding differentiation medium comprising 10% CS, 0.5mM 3-isobutyl-1-methylxanthine (Sigma), 80. Mu.M indomethacin (Sigma), 1. Mu.M DEX and 5. Mu.g/mL insulin to 90% DMEM. The extent of stem cell differentiation into adipocytes was assessed using Triglyceride-Glo kit (Promega) by staining with 0.5% oil red O (Sigma) solution, which is an indicator of fat accumulation in the cytoplasm, at room temperature for 1 hour 2 weeks after differentiation induction, and then measuring triglycerides in the cytoplasm. To assess the ability to differentiate into osteoblasts, 2.0X10 were used ⁵ The individual cells were inoculated in a 24-well culture vessel, and alpha-MEM containing 1. Mu.M DEX, 50. Mu.M ascorbic acid, 10mM beta-glycerophosphate and 10% calf serum was added and cultured for 2 weeks to induce differentiation. Comparative analysis of whether differentiation into osteoblasts occurred by optical density was performed by staining with the addition of alizarin red (Sigma) 2 weeks after differentiation inductionAnd the degree of mineral deposition was analyzed by comparison by measuring absorbance at 520 nm.

In fig. 21, after cells isolated and collected from bone marrow are induced to differentiate into osteoblasts (left) and adipocytes (right), accumulation and deposition of calcium phosphate crystals can be observed by alizarin red staining, and accumulation of lipids in cytoplasm of cells differentiated into adipocytes can be observed by oil red O staining. It was found that all cells derived from all tissue fragments have the ability to differentiate into osteoblasts and adipocytes.

The ability to differentiate into osteoblasts and adipocytes was compared and assessed by the OD value measured after dissolving the deposited or accumulated mineral crystals and lipid vesicles. The ability to differentiate into osteoblasts and adipocytes from the cells isolated and collected from the hydrogels by PAI withdrawal was found to be significantly higher in all tissue fragments compared to the cells isolated and collected after urokinase treatment (fig. 22). High capacity to differentiate into osteoblasts is shown in bone marrow, cardiac and skeletal muscle-derived cells, and high capacity to differentiate into adipocytes is shown in fat, skeletal muscle and synovium. However, it was found that upar+/nestin+ cells with high plasmin activity are stem cells with high multipotency by determining that they differentiate into osteoblasts and adipocytes (regardless of the source of tissue fragments) with a significantly high capacity among cells isolated and collected from hydrogels by the PAI withdrawal method.

< example 13> ability of isolated cells to induce tissue regeneration after PAI removal

Cells separated and collected from the hydrogel were transferred to a tube and centrifuged at 3,000rpm for 10 minutes to remove the supernatant. Suspending the cell pellet in a culture medium and adjusting the cell density to 1.0X10 ⁶ Per mL.Inoculating 5.0X10 in T75 flask ⁶ After each cell, culture was performed for 3 days, 1mL TRIzol was added, and then cell lysates were collected and frozen at-20 degrees 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. The expression of basal FGF (bFGF), HGF, IGF and sdf-1mRNA, which play a major role in tissue protection and regeneration, was compared and evaluated by calculation using the expression rate, as compared with umbilical cord blood-derived mesenchymal stem cells (CB-MSC). Target mRNA genes were amplified by real-time gene amplification using target mRNA specific priming and SYBR green real-time PCR kit, and mRNA expression rates compared to umbilical cord blood-derived mesenchymal stem cells (CB-MSCs) were assessed by the 2- Δct method.

After urokinase or PAI withdrawal treatment, the migrating and growing cells are isolated from the hydrogel and the collected cells are compared and analyzed for their ability to induce tissue regeneration by related mRNA expression. The expression of genes inducing tissue regeneration varies depending on the source tissue, and these genes are highly expressed in cells derived from cardiac muscle, nerve and skeletal muscle, and cells derived from cardiac muscle are highly expressed in HGF and SDF-1mRNA, while IGF mRNA is highly expressed in cells derived from nerve [ FIG. 23]. In the cells isolated and collected by PAI withdrawal, the expression of the whole tissue regeneration gene was significantly high compared to the cells isolated and collected after urokinase treatment. These results indicate that uPAR expression is proportionally related to the expression of high tissue regeneration regulatory genes.

< example 14> plasmin Activity and nestin expression Rate in uPAR+ cells

Fat, bone marrow, cardiac muscle, nerve, skeletal muscle and synovial tissue fragments were embedded in fibrin or fibrin/collagen hydrogels, 3D organ culture was performed for 2 weeks, and then cells migrating into and growing in the hydrogels were isolated and collected by urokinase method described in example 6. Subsequently, upar+ (uk+upar+) and uPAR- (uk+upar-) cells were isolated by FACSAria (BD Bioscience) and cultured in a monolayer environment for 7 days to assess plasmin activity and nestin expression rate. Cells isolated and collected from hydrogels by PAI withdrawal have a uPAR expression rate of 90% or more, such that secondary upar+ cells are not isolated. Plasmin activity was performed according to the method described in example 6 and nestin expression was assessed using flow cytometry.

Significantly higher plasmin activity was observed in upar+ cells (fig. 24, left). The UK+uPAR+ cells isolated and cultured after urokinase treatment and the cells isolated and cultured after removal of PAI showed significantly higher plasmin activity compared to the UK+uPAR-cells. The plasmin activity of cells varies with tissue source, being higher in cardiac, neural and skeletal muscles, whereas plasmin activity is determined by the tissue source of the tissue resident cells.

The above results indicate that there is a close correlation between plasmin activity and nestin expression rate in upar+ cells.

< example 15> self-replication and in vitro growth Capacity of uPAR+ cells

Fat, bone marrow, cardiac muscle, nerve, skeletal muscle and synovial tissue fragments were encapsulated in fibrin or fibrin/collagen hydrogels, followed by 3D organ culture for 2 weeks to isolate and collect cells that migrated into and grew in the hydrogels using the urokinase method described in example 6. Subsequently, upar+ (uk+upar+) and uPAR- (uk+upar-) cells were isolated by FACSAria (BD Bioscience) and cultured in a monolayer environment for 7 days before assessing self-replication and growth capacity. Cells isolated and collected from hydrogels by PAI withdrawal have a uPAR expression rate of 90% or more, such that secondary upar+ cells are not isolated. Self-replication ability was assessed according to the method described in example 10, and growth ability was assessed by flow cytometry using Ki-67 expression rate.

A significantly higher self-replicating capacity can be observed in upar+ cells. CFU frequencies were found to be 12.5% to 37.5% in uk+upar+ cells and PAI depleted cells, 3.1% to 8.8% in uk+upar-cells, and significantly higher self-replication capacity in upar+ cells (fig. 25, left).

The ability to self-replicate was found to be closely related to the ability to grow in vitro, with Ki-67 expressed in uPAR+ cells at 40.8% to 82.4% showing significantly higher growth compared to Ki-67 expressed in uPAR-cells at 28.1% to 45.7% (FIG. 25, right). Upar+ cells of myocardial, neural and skeletal muscle origin have a higher CFU forming capacity and Ki-67 expression rate compared to tissues of other origin.

< example 16> anti-inflammatory ability of upar+ cells

Fat, bone marrow, cardiac muscle, nerve, skeletal muscle and synovial tissue fragments were embedded in fibrin or fibrin/collagen hydrogels, 3D organ culture was performed for 2 weeks, and then cells migrating into and growing in the hydrogels were isolated and collected by urokinase method described in example 6. Subsequently, upar+ (uk+upar+) and uPAR- (uk+upar-) cells were isolated by FACSAria (BD Bioscience) and cultured in a monolayer environment for 7 days to assess anti-inflammatory capacity. Cells isolated and collected from hydrogels by PAI withdrawal have a uPAR expression rate of 90% or more, such that secondary upar+ cells are not isolated.

Conditioned medium containing factors secreted by cells was used to assess anti-inflammatory capacity. To prepare conditioned medium, add per cm in T175 flask ² After plating with 40,000 cells of DMEM/F12 serum-free medium, the cells were removed from the uk+upar+, uk+upar-, and PAI for 1 week.

RAW 264.7 cells were suspended in 90% RPMI 1640/10% calf serum per cm ² 100,000 cells were seeded on 12-well multiwell plates and cultured for 1 day. 100 μg/mL LPS was added to the medium to sensitize RAW 264.7 cells. During LPS sensitization, conditioned medium was added to the medium at a ratio of 1:10 to assess anti-inflammatory capacity. Regarding the anti-inflammatory evaluation index, the isolated levels of tnfα and IL-1β secreted by RAW 264.7 cells were measured by ELISA method for comparison and evaluation.

RAW 264.7 cells showed 5-fold increase in TNFα and IL-1β secretion levels following LPS stimulation. It was determined that the tissue resident stem cell secreted substances isolated and collected from the hydrogel had the ability to inhibit 30% -70% of inflammatory cytokine isolation of RAW 264.7 cells (fig. 26). Conditioned medium prepared from uk+upar+ cells and PAI-depleted cells showed significantly higher tnfα and IL-1β secretion inhibitory capacity compared to uk+upar-cells. Regardless of the tissue from which it is derived, it was found that upar+ cells can have a high anti-inflammatory effect compared to uPAR-cells.

< example 17> effects of inducing vascular endothelial cell and fibroblast growth of uPAR+ cells

Human Umbilical Vein Endothelial Cells (HUVECs) were used as vascular endothelial cells, and human Dermal Fibroblasts (DF) were used. The corresponding cells were tested for growth induction in conditioned medium. Conditioned medium prepared by the method described in example 16 was prepared to test the effect. HUVEC cells and DF were suspended in 99% DMEM/1% calf serum culture and then per cm ² 4,000 cells were seeded in 24-well multiwell plates. Conditioned medium was added to the medium at a ratio of 1:9 and cultured for 3 days, and the number of cells after culture was measured using PicoGreen dsDNA quantification kit (Invitrogen). The effect of cell growth was compared and analyzed by calculating the rate of increase (fold) of the number of cells inoculated for the first time.

It was determined that the conditioned medium prepared from uk+upar+ and PAI-depleted cells showed an effect of promoting growth of HUVEC and DF, which was significantly 30% higher than the conditioned medium prepared in uk+upar-cells, but there was no difference in effect between uk+upar+ and PAI-depleted cells (fig. 27). In particular, conditioned media obtained from stem cells of myocardial, neural and skeletal muscle origin show a high growth induction compared to stem cells of other tissue origin.

< example 18> vascular endothelial cell and fibroblast protective action of upar+ cells

Fat, bone marrow, cardiac muscle, nerve, skeletal muscle and synovial tissue fragments were embedded in fibrin or fibrin/collagen hydrogels and 3D organ culture was performed for 2 weeks to isolate and collect cells migrating into and growing in the hydrogels by urokinase method described in example 6. Subsequently, upar+ (uk+upar+) and uPAR- (uk+upar-) cells were isolated by FACSAria (BD Bioscience) and cultured in a monolayer environment for 7 days to assess anti-inflammatory capacity. Cells isolated and collected from hydrogels by PAI withdrawal have a uPAR expression rate of 90% or more, such that secondary upar+ cells are not isolated.

Cytoprotective effects were tested using conditioned media prepared from uk+upar-cells, uk+upar+ cells, and cells isolated and collected after PAI removal, respectively. HUVEC and DF were suspended in 99% DMEM/1% calf serum medium and each cm ² 5,000 cells were seeded in 24-well multiwell plates. At H ₂ O ₂ Conditioned medium was added with the medium at a ratio of 1:9 1 hour prior to induction of mediated cell damage. 0.01% H is added ₂ O ₂ To induce cell damage, after 6 hours of damage induction, the medium was removed and washed twice with PBS. The extent of cell damage was assessed by the expression rate of annexin V, and apoptotic cells were analyzed by flow cytometry after FITC-conjugated annexin V (BD Biosciences) reacted with the cells.

The conditioned medium prepared from UK+uPAR+ cells and cells collected after removal of PAI showed a high response to H compared to the UK+uPAR-cell-derived conditioned medium ₂ O ₂ Significant protection of mediated HUVEC and DF cell death (fig. 28). Differences in cytoprotective effects were observed depending on the tissue of origin, and the cytoprotective effects of HUVEC and DF were higher in cells of myocardial, neural and skeletal muscle origin. Conditioned Medium with UK+uPAR+ cells The annexin V expression rates in treated HUVECs and DF were 28.7% to 45.1%, showing significantly higher cytoprotective effect than 42.1% to 71.3% in cells treated with uk+upar-cell conditioned medium, but no significant difference was confirmed compared to conditioned medium derived from PAI-depleted cells. These results confirm that uPAR expression is associated with high cytoprotective effects and there is evidence that PAI withdrawal alone is capable of isolating and culturing cells with high cytoprotective effects.

The present disclosure is realized by an engineered temporary matrix-supported organ culture. Tissue fragments are embedded in an artificial temporary matrix and the integrin-FAK cell signaling pathway is activated by the temporary matrix to induce cell division and growth of tissue resident stem cells. Migration and growth of stem cells into the temporary matrix in the tissue is increased in proportion to uPAR-uPA-plasmin activity by the organ culture process. To control the consequences of stopping the inhibition of stem cell migration due to excessive uPAR-uPA-plasmin activity resulting in temporary matrix degradation and loss, the temporary matrix excessive degradation can be controlled by PAI addition. Techniques are provided that enable isolation of upar+ stem cells by stopping PAI addition after the stem cells migrate into the temporary matrix and grow therein to a target level. Following interruption of PAI addition, upar+ stem cells dissociate from the temporary matrix and are released into the medium upon activation of uPAR-uPA-plasmin and MMP. The present disclosure provides techniques that enable isolation and culture of upar+ stem cells without the use of any tissue degrading proteins, as well as stem cell purification processes by using markers according to the migration and degradation characteristics of the temporary matrix of uPAR expression.

The present disclosure is applicable to isolation and culture of upar+ stem cells present in solid tissues (e.g., bone marrow, fat, skeletal muscle, heart, peripheral nerves, spinal cord, brain, lung, liver, joint membranes, umbilical cord, placenta, and periodontal tissue). The present disclosure provides methods for isolating and culturing stem cells by repeating organ culture, as the structure of tissue fragments used for organ culture can be kept intact by isolating stem cells without any protease treatment.

The present disclosure shows the characteristics of positive expression of conventional stem cell markers other than uPAR in uPAR positive tissue resistant stem cells. In the case of bone marrow, fat, muscle, heart, joint membrane, umbilical cord and placenta, mesenchymal stem cell markers such as CD29, CD44, CD73, CD90 and CD105 are positive for expression, but hematopoietic stem cell or vascular endothelial cell markers show negative characteristics. The present disclosure shows that markers such as nestin, p75, sox10, and Sox2 are co-expressed in uPAR positive stem cells derived from peripheral nerve, spinal cord, and brain tissue.

The uPAR positive stem cells of the present disclosure exhibit high biological properties in terms of secretion of physiologically active factors, growth factors, anti-inflammatory factors, stem cell recruitment factors, and ability to regenerate tissues that act on migration.

The uPAR positive stem cells of the present disclosure have a high multipotency to differentiate into tissue constituent cells.

Claims

1. A method of inducing tissue resident upar+ and nestin+ stem cells into a cell cycle, the method comprising:

(1) Preparing temporary matrix simulated hydrogel;

(2) Encapsulating the isolated tissue fragments into the temporary matrix simulated hydrogel; and

(3) The temporary matrix simulated hydrogel, in which tissue fragments are encapsulated, is 3D cultured in a medium supplemented with a Plasminogen Activator Inhibitor (PAI).

2. The method of claim 1, wherein the temporary matrix simulated hydrogel is:

a fibrin hydrogel, wherein a fibrinogen solution having a concentration of 0.25% to 2.5% is mixed with a thrombin solution having a concentration of 0.5i.u./mL to 5 i.u./mL;

a fibrin/collagen mixed hydrogel, wherein a collagen solution having a concentration of 0.1% to 0.5% is mixed in the fibrin hydrogel; or (b)

A fibrin/gelatin mixed hydrogel, wherein a gelatin solution having a concentration of 0.1% to 0.5% is mixed in the fibrin hydrogel.

3. The method of claim 1, wherein the tissue is adipose tissue, bone marrow tissue, myocardial tissue, peripheral nerve tissue, skeletal muscle tissue, or synovial tissue.

4. The method of claim 1, wherein the PAI is tranexamic acid or aminomethylbenzoic acid.

5. The method of claim 1, wherein the method activates integrin-FAK cell signaling of cells in tissue to induce cell division and cell growth of tissue resident upar+ and nestin+ stem cells.

6. The method of claim 1, wherein the method induces cell migration and cell growth of the tissue resident upar+ and nestin+ stem cells into the temporary matrix mimetic hydrogel.

7. A method of isolating and culturing tissue resident upar+ and nestin+ stem cells, the method comprising:

(1) Preparing temporary matrix simulated hydrogel;

(2) Encapsulating the isolated tissue fragments into the temporary matrix simulated hydrogel;

(3) 3D culturing a temporary matrix simulated hydrogel having tissue fragments encapsulated therein in a medium supplemented with PAI;

(4) Removing the 3D medium and removing PAI by washing;

(5) Re-culturing the PAI-removed culture with a PAI-free medium to degrade the temporary matrix simulated hydrogel; and

(6) The stem cells released in the re-culture medium are isolated.

8. The method of claim 7, wherein the temporary matrix simulated hydrogel is:

9. The method of claim 7, wherein the tissue is adipose tissue, bone marrow tissue, myocardial tissue, peripheral nerve tissue, skeletal muscle tissue, or synovial tissue.

10. The method of claim 7, wherein the PAI is tranexamic acid or aminomethylbenzoic acid.

11. The method of claim 7, wherein step (5) comprises inducing an increase in upar+ cells in the tissue and degrading the temporary matrix mimetic hydrogel by an increase in plasmin activity.

12. The method of claim 7, wherein the tissue-resident upar+ and nestin+ stem cells have an increased capacity for self-replication, in vitro growth, differentiation, or induction of tissue regeneration.

13. The method of claim 7, wherein collecting tissue debris from the reculture medium in step (5) further comprises repeating the process of steps (2) through (5) 1-10 times.

14. Tissue resident upar+ and nestin+ stem cells isolated and cultured according to the method of any one of claims 7-13, or a culture thereof.

15. A pharmaceutical composition for preventing or treating inflammatory diseases, comprising the tissue resident upar+ and nestin+ stem cells or culture thereof according to claim 14 as active ingredients.

16. A pharmaceutical composition for preventing or treating autoimmune diseases, comprising the tissue resident upar+ and nestin+ stem cells or cultures thereof according to claim 14 as active ingredients.

17. A pharmaceutical composition for healing wounds, comprising the tissue resident upar+ and nestin+ stem cells or cultures thereof according to claim 14 as active ingredients.

18. A pharmaceutical composition for promoting revascularization, comprising the tissue resident upar+ and nestin+ stem cells or cultures thereof according to claim 14 as active ingredients.