KR20200132455A - Method for Quantitative Evaluating of Recruiting Activity of Substances having Progenitor Cell or Stem Cell Recruiting Activity Using Programming Algorithm and Numerical Analysis - Google Patents

Abstract

The present invention relates to a method for quantitatively evaluating recruiting activity of a substance having progenitor cell or stem cell recruiting activity using a programming algorithm and numerical analysis. In the present invention, when stem cells are attached to an elastic substrate composed of a polyacrylamide gel containing fluorescent microparticles, the displacement information of fluorescent microparticles and the deformation observed on the PA gel surface are calculated by physical force through a programming algorithm and numerical analysis, and thus it was confirmed that cell traction force according to the degree of stem cell migration can be measured. Therefore, the method of the present invention not only can be used to accurately evaluate the recruiting activity of a substance having progenitor cell or stem cell recruiting activity in vitro, but also can be usefully applied to the selection of a substance having recruiting activity.

Description

Method for Quantitative Evaluating of Recruiting Activity of Substances having Progenitor Cell or Stem Cell Recruiting Activity Using Programming Algorithm and Numerical Analysis}

The present invention relates to a method for quantitatively evaluating the mobilization activity of a substance having a progenitor cell or stem cell mobilization activity using a programming algorithm and 　 numerical analysis, and in more detail, an elastic substrate composed of a polyacrylamide gel containing fluorescent fine particles It relates to a programming algorithm characterized by measuring the traction force of stem cells by using and a quantitative evaluation method for the mobilization activity of progenitor cells or substances having stem cell mobilization activity using numerical analysis.

Mesenchymal stem cells (MSC) are adult stem cells that have the ability to self-renewal and differentiate into mesenchymal tissues such as bone, fat, and cartilage. In addition, these mesenchymal stem cells have a tropism that can move to damaged tissues and inflamed areas, helping to cure diseases, and exhibiting immunosuppressive properties under various conditions. However, there is a problem in that chemokine receptors and factors for migrating stem cells to disease sites are expressed in a relatively small amount, and stem cell inducing factors are required to solve this problem.

The previously reported substance P (Substance P; SP) is of 11 amino acids (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH ₂ ) that play a role in neurotransmission and neuromodulation. As a neuropeptide, the endogenous receptor for substance P is NK1R (neurokinin 1 receptor), and NK1R is a G-protein coupled receptor, tachykinin receptor subfamily. NK1R is distributed in many cell types (neurons, glial cells, capillaries, lymph glands, fibroblasts, stem cells, and leukocytes) in various tissues and organs, and substance P amplifies or activates most cellular signaling processes and is well studied. It is one of the stem cell recruiting factors.

Accordingly, in the present invention, as a result of trying to perform an accurate predictive evaluation and analysis on the mobilization activity of a substance having progenitor cell or stem cell mobilization activity, polyacrylamide containing fluorescent microparticles integrated with traction force microscopy When the cell image measured using an elastic substrate composed of a gel is analyzed through a programming algorithm and numerical analysis, it was confirmed that the traction force of the stem cell by a progenitor cell or a substance having stem cell mobilization activity can be measured. Completed.

An object of the present invention is to provide a method for quantitatively evaluating the mobilization activity of a substance having a progenitor cell or stem cell mobilization activity using a programming algorithm and 　 numerical analysis.

Another object of the present invention is to provide a method for selecting a substance having progenitor cell or stem cell mobilization activity using the above method.

To achieve the above object,

The present invention includes the steps of: (a) forming a PA (polyacrylamide) gel layer in which fluorescent microbeads are fixed on the surface;

(b) modifying the surface of the PA gel layer so that cells can attach, and then applying a matrix protein;

(c) attaching progenitor cells or stem cells to the PA gel coated with the matrix protein, treating a substance having progenitor cells or stem cell mobilization activity, and then collecting images of cells and fluorescent fine particles;

(d) removing the cells from which the images were collected, and then collecting fluorescent microparticle images in the PA gel in a reference state without cells; And

(e) measuring the traction force of stem cells based on the movement of fluorescent microparticles in the collected image; programming algorithm including, and 　 for the mobilization activity of progenitor cells or substances having stem cell mobilization activity using numerical analysis Provides a quantitative evaluation method.

According to a preferred embodiment of the present invention, the substance having the progenitor cell or stem cell mobilization activity may be a substance P (Substance P) or an analog of substance P (Substance P analog).

According to another preferred embodiment of the present invention, the progenitor cells or stem cells may be endogenous progenitor cells or endogenous stem cells.

According to another preferred embodiment of the present invention, the endogenous stem cells may be at least one selected from the group consisting of mesenchymal stem cells, corneal stem cells, myocardial stem cells, and neural stem cells.

According to another preferred embodiment of the present invention, the PA (polyacrylamide) gel of step (a) may have an elastic modulus of 1 to 100 kPa, and the diameter of the fluorescent fine particles contained in the PA gel is 0.2 to 1 May be μm.

According to another preferred embodiment of the present invention, the PA (polyacrylamide) gel layer of step (a) is

(a1) Acrylamide, 2% N,N′ methylenebis(acrylamide) bis solution [N,N′-Methylenebis(acrylamide) BIS solution], tetramethylethylenediamine, and fluorescent micro-bead ) Preparing a PA (polyacrylamide) gel solution consisting of;

(a2) putting the PA gel solution in a glass chip having a rectangular groove with a depth of 100 μm to which a silane group is attached and covering the cover glass on the PA gel;

(a3) centrifuging the fluorescent microparticles so that the top faces downward in order to align the fluorescent microparticles on the top surface of the PA gel, and then curing at room temperature and removing the cover glass.

According to another preferred embodiment of the present invention, the matrix protein in step (b) may be collagen (collagen type I).

According to another preferred embodiment of the present invention, the cell and fluorescent microparticle images of step (c) may be photographed and collected using real-time imaging equipment for 12 hours every 10 minutes.

According to another preferred embodiment of the present invention, in step (e), the movement displacement of the microparticles is determined by cross-correlation between the reference state fluorescent microparticle image and the fluorescent microparticle image when cells are attached. Cells by converting from (pixel) unit to µm unit and calculating the deformation observed on the PA gel surface as a physical force through finite element method using the elastic modulus, gel thickness, and Poisson's ratio of the PA gel. The traction force by can be measured.

According to another preferred embodiment of the present invention, in step (e), the stem cell traction force may be measured by analyzing the collected cell images and fluorescent microparticle images with MATLAB software.

In addition, the present invention provides a method for selecting a substance having a progenitor cell or stem cell mobilization activity using a quantitative evaluation method for the mobilization activity of the substance having the progenitor cell or stem cell mobilization activity.

In the present invention, when stem cells are attached to an elastic substrate made of a polyacrylamide gel containing fluorescent microparticles, the displacement information of the fluorescent microparticles and the deformation observed on the PA gel surface are measured by a programming algorithm and numerical analysis. Since it was confirmed that the cell traction force according to the degree of migration of stem cells can be measured by calculating as, the method of the present invention can be utilized not only for accurate evaluation of the mobilization activity of a substance having progenitor cells or stem cell mobilization activity in vitro. In addition, it can be usefully applied to the selection of substances having mobilization activity.

1 is a schematic diagram showing the movement of fluorescent fine particles by an elastic substrate made of polyacrylamide gel containing fluorescent fine particles and attached cells.
2 is data showing a bright field cell image (A) and a fluorescent fine particle image (B) collected in a state in which stem cells are attached to the surface of a PA gel and in a reference state from which stem cells are removed.
3A is a cross-correlation-based PIV (particle image velocimetry) between a cell image (t) and a cell image (t-1) using MATLAB for the collected bright field stem cell image. This is the data obtained by spatially plotting the cell migration displacement through color mapping after calculating the movement displacement numerically.
3B is a cross-correlation-based PIV between the collected fluorescence fine particle image and the fluorescence fine particle image in a reference state after removal of cells using MATLAB. This is the data obtained by spatially plotting the movement displacement of fine particles by color mapping after calculating the movement displacement numerically. The color of each position shown in the drawing is a moving displacement of a pixel, and an arrow indicates a moving direction of the pixel.
4 is data obtained by spatially plotting the cell movement displacement by color mapping after converting the stem cell movement displacement from pixels to µm units. The color of each location in FIG. 4A is the magnitude of the cell movement speed in radial coordinates, the arrow indicates the direction of movement of the cell, and the color of each location in FIG. 4B is the magnitude of the cell traction force in the radial coordinates. Arrows indicate the direction of traction.
Figure 5 shows the data (A) quantifying the deviation of the speed, which is a scalar value for the stem cell movement speed, and the median value in a box plot (A) and the magnitude of the traction force, which is the absolute value of the calculated cell traction. This is data (B) obtained by quantifying the deviation (midquartile) and median value in a box plot.
6 is data showing an average value (mean or average) and a distribution diagram (histogram or probability) over all time for the magnitudes of the calculated moving speed (A) and cell traction force (B).
7 is data showing a bright field cell image according to the presence and concentration of a substance P (Substance P).
8 is data obtained by spatially plotting the cell movement displacement by color mapping after converting the stem cell movement displacement according to the presence or absence of a substance P (substance P) and concentration in µm units from pixels.
9 is a data obtained by quantifying the deviation (median quartile, midquartile) and median value of the speed, which is a scalar value for the stem cell movement speed according to the presence and concentration of a substance P (Substance P), and calculated cells. This is the data obtained by quantifying the deviation of the magnitude of the traction force, which is the absolute value of the traction force (midquartile) and the median value, in a box plot.
10 is data showing the average value and distribution over all time for the magnitude of stem cell movement speed and cell traction force according to the presence and absence of substance P (Substance P) and concentration.

Hereinafter, the present invention will be described in detail.

In a consistent aspect, the present invention comprises the steps of: (a) forming a PA (polyacrylamide) gel layer in which fluorescent micro-beads are fixed on the surface;

(b) modifying the surface of the PA gel layer so that cells can attach, and then applying a matrix protein;

(c) attaching progenitor cells or stem cells to the PA gel coated with the matrix protein, treating a substance having progenitor cells or stem cell mobilization activity, and then collecting images of cells and fluorescent fine particles;

(d) removing the cells from which the images were collected, and then collecting fluorescent microparticle images in the PA gel in a reference state without cells; And

(e) measuring the traction force of stem cells based on the movement of fluorescent microparticles in the collected image; programming algorithm including, and 　 for the mobilization activity of progenitor cells or substances having stem cell mobilization activity using numerical analysis It relates to a quantitative evaluation method.

In the present invention, the substance having the progenitor cell or stem cell mobilization activity may be a substance P (Substance P) or an analog of substance P (Substance P analog). Substance P (Substance P, hereinafter referred to as "SP") is one of mammalian tachykinins consisting of 11 amino acid sequences of RPKPQQFFGLM, and is secreted excessively when wounds in tissues occur, which are all for wound recovery. It is a substance that has the ability to induce the mobilization (or'capture','mobility promotion') of stem cells with differentiation ability.

In the present invention, the progenitor cells or stem cells are endogenous cells, and may be at least one selected from the group consisting of mesenchymal stem cells, corneal stem cells, myocardial stem cells, neural stem cells, and vascular endothelial progenitor cells, and preferably May be characterized as mesenchymal stem cells.

Intrinsic progenitor cells or intrinsic stem cells are inherently present in a specific organ and/or tissue, contributing to the regeneration of the tissue and/or organ when the organ and/or tissue is damaged, and self-replicating ability and It refers to cells with pluripotency. Specific examples include mesenchymal stem cells, corneal stem cells, myocardial stem cells, neural stem cells, and vascular endothelial progenitor cells.

The progenitor cell or stem cell mobilization activity promotes the migration of the progenitor cell or stem cell, and the migration is circulated by the progenitor cell or stem cell from the bone marrow, organ and/or tissue to the circulating blood; Accumulates in damaged organs and/or tissues from circulating blood; Or it refers to moving inside an organ and/or tissue.

1 is a schematic diagram showing the movement of fluorescent microparticles by an elastic substrate made of a polyacrylamide gel containing fluorescent microparticles and attached cells. When a single or multiple cells attach and move to a two-dimensional surface, the cell-substrate Physical interactions can be quantified. The PA gel may have an elastic modulus of 1 to 100 kPa, preferably 1 to 10 kPa, more preferably 6 kPa, and the diameter of the fluorescent fine particles included in the PA gel is 0.2 to 1 µm. can do.

The elastic substrate composed of the polyacrylamide gel containing the fluorescent fine particles of the present invention is specifically,

(a1) Acrylamide, N,N' methylenebis (acrylamide) bis solution [N,N'- Methylenebis (acrylamide) BIS solution], tetramethylethylenediamine (tetramethylethylenediamine) and fluorescent micro-bead (fluorescent micro-bead) Preparing a composed PA (polyacrylamide) gel solution;

(a2) putting the PA gel solution in a glass chip having a rectangular groove with a depth of 100 μm to which a silane group is attached and covering the cover glass on the PA gel; And

(a3) It can be characterized in that it is produced by a method comprising the step of removing the cover glass after centrifuging the fluorescent fine particles with the top facing down in order to align the fluorescent fine particles on the top surface of the PA gel, and then curing at room temperature. .

In a specific embodiment of the present invention, a glass chip having a rectangular groove having a depth of 100 μm was prepared and used to fill the PA gel with a constant height, and acetic acid and silane A174 (3- (trimethoxysilyl) A bond-silane solution composed of propyl methacrylate [silane A174(3-(trimethoxysilyl)propyl methacrylate]) was treated to attach a silane group to a glass chip, and PA gel was added to the rectangular groove of the glass chip to which the silane group was attached. Then, in order to align the fluorescent microparticles on the top surface of the PA gel, the glass chip filled with the PA gel solution was turned upside down and centrifuged at 700 rpm for 10 minutes, and then the top was placed face down for polymerization of the PA gel. After curing at, the cover glass was removed to finally produce an elastic substrate composed of polyacrylamide gel containing fluorescent fine particles.

In the present invention, the substrate protein of step (b) is characterized in that collagen (collagen type I), in a specific embodiment of the present invention SANPAH [sulphosuccin-imidyl-6-(4-azido-2-nitrophenylamino ) The PA gel surface was modified with a hexanoate] solution, and then collagen type I was applied.

In the present invention, the cell and fluorescent microparticle images of step (c) are photographed and collected every 10 minutes for 12 hours using real-time imaging equipment, and cells are imaged using a bright field, and green Fluorescent fine particles were imaged and analyzed using a green fluorescent protein (GFP) filter (FIG. 2).

In the present invention, the step (e) is characterized in that the stem cell traction is measured by analyzing the collected cell image and the fluorescent fine particle image with MATLAB software, and specifically, the reference state fluorescent fine particle image and the fluorescence upon cell attachment The movement displacement of the fine particles is converted from the pixel unit to the µm unit through cross-correlation of the fine particle image, and the finite element analysis method is used using the elastic modulus, gel thickness, and Poisson ratio of the PA gel ( finite element method) to measure the traction force by cells by calculating the deformation observed on the PA gel surface as a physical force through a programming algorithm and numerical analysis.

In the present invention, the obtained brightfield cell image and fluorescent fine particle image were subjected to numerical conversion and quantitative analysis using a customized source code developed based on MATLAB (MathWorks Inc.). Specifically, particle image velocimetry (PIV) based on a normalized cross-correlation between a cell image and a reference state image from which cells are removed, or a fluorescence microparticle image and a reference state fluorescent microparticle image. Cell and fluorescence microparticle displacements were analyzed.

In a specific embodiment of the present invention, as shown in FIG. 3, the collected bright field stem cell image is moved using MATLAB through cross-correlation-based PIV between the cell image (t) and the cell image (t-1). After calculating the displacement numerically, the cell movement displacement was spatially plotted by color mapping (Fig. 3A), and the cross-correlation-based PIV between the microparticle image (t) and the reference state microparticle image (r) After calculating the movement displacement numerically, the movement displacement of the fine particles was spatially plotted by color mapping (FIG. 3B).

In addition, FIG. 4A is a spatial schematic diagram of the cell movement displacement by color mapping after converting the stem cell movement displacement from pixels to µm units, and the deformation observed on the PA gel surface using the above analysis method. The traction force by the cells was measured by calculating the physical force, and then the cell traction force was spatially plotted using color mapping (FIG. 4B).

In order to quantify the traction force, as shown in Fig. 5, the deviation of the speed and the median value (Fig. 5A), which is a scalar value for the stem cell movement speed, and the deviation of the size of the traction force, which is the absolute value of the cell traction force, are boxed. It was quantified by a box plot, and as shown in FIG. 6, the calculated moving speed (A) and cell traction force (B) were averaged over all time periods (mean or average) and distribution (histogram or probability) Was analyzed.

In a specific embodiment of the present invention, as a result of measuring the traction force of stem cells according to the treatment of the substance P, as shown in FIGS. 7 to 10, the average speed of the cells relative to the mobilization activity of a progenitor cell or a substance having stem cell mobilization activity And it was confirmed that the change over time of the magnitude of the traction force can be quantified.

That is, in the present invention, when stem cells are attached to an elastic substrate made of polyacrylamide gel containing fluorescent microparticles, information on displacement of fluorescent microparticles and deformation observed on the PA gel surface are physically analyzed through programming algorithms and numerical analysis. Since it was confirmed that the cell traction force according to the degree of migration of stem cells can be measured by calculating with phosphorus force, the method of the present invention can quantitatively analyze the mobilization activity of a substance having progenitor cells or stem cell mobilization activity in vitro. Was confirmed, and furthermore, it was confirmed that it can be used for selection of substances having mobilization activity.

Accordingly, the present invention relates to a method for selecting a substance having a progenitor cell or stem cell mobilization activity using the method of quantitative evaluation of the mobilization activity of the substance having the progenitor cell or stem cell mobilization activity of the present invention from another point of view.

Hereinafter, the present invention will be described in more detail through examples.

These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.

Containing fluorescent fine particles With polyacrylamide gel Composed Elastic substrate making

1-1: PDMS stencil component fabrication

PDMS stencil components for cell patterning were fabricated using general soft lithography using PDMS (Sylgard 184, Dow Corning, USA) using the method reported in the previous study (H. Jang, et al . , Sci. Rep ., 8:45844, 2017).

Briefly, SU-8 master molds (Outsourced; MicroFIT) consisting of 200 μm thick and 700 μm diameter cylinders of a PDMS solution in which the ratio of prepolymer and curing agent is 10: 1 are mixed at regular intervals. , Korea), and then cured in a drying oven at 85° C. for 2 hours.

After curing, a thin PDMS film in which holes having a diameter of 700 μm are aligned was trimmed with a punch having a diameter of 14 mm, and then prepared by autoclaving.

1-2: Preparation of polyacrylamide (PA) gel substrate

Based on a known method (H. Jang, et al ., Sci . Rep ., 8:45844, 2017), 135 μl of 40% acrylamide (Bio-Rad, USA), 2% N,N′-methylenebis (acrylic Amide) bis solution [N,N′-Methylenebis(acrylamide) BIS solution; Bio-Rad, USA] 101 μl, purified water 658 μl, green fluorescent beads (diameter = 500 nm; FluoSpheres; Invitrogen, USA) 100 μl and tetramethylethylenediamine (TEMED; Bio-Rad, USA) ) 1 ml of PA gel solution (6 kPa) consisting of 1 μl was prepared.

To fill the elastic substrate of a certain height, a glass chip with a rectangular groove with a depth of 100 μm was manufactured to order (MicroFIT). After autoclaving the glass chips, 0.04% (v/v) acetic acid and 0.025% (v/v) silane A174 (3- (trimethoxysilyl) propyl methacrylate diluted with high-purity water in the grooves A bond-silane solution consisting of [silane A174 (3-(trimethoxysilyl)propyl methacrylate; Sigma-Aldrich, USA] was treated and reacted for 1 hour at room temperature. Then, the glass chips were washed three times with tertiary distilled water. 10 [mu]l of the prepared PA gel solution was filled into the groove and flattened with a cover glass (diameter = 18 mm, Marienfeld, Germany).

In order to align the fluorescent fine particles along the top of the gel, the glass chip filled with the PA gel solution was turned upside down and centrifuged at 700 rpm for 10 minutes. For polymerization of the PA gel, it was cured for 40 minutes at room temperature with the top facing down, and then purified water was poured onto the top of the glass chip to remove the cover glass.

Measurement of traction force of stem cells using an elastic substrate composed of polyacrylamide gel containing fluorescent fine particles

2-1: cell patterning of PA gel

Sulfo-SANPAH (sulphosuccin-imidyl-6-(4-azido-2-nitrophenylamino) hexanoate on the PA gel surface; 1 mg/ml in 50 mM HEPES buffer; Thermo Scientific Pierce, USA] solution was applied and the surface of the PA gel was modified by irradiation with ultraviolet light (365 nm wavelength) for 10 minutes. The gel surface was washed twice with 0.1 M HEPES (Sigma-Aldrich, USA) solution, and then washed once with PBS (phosphate-buffered salin; Welgene, Korea).

The functionalized PA gel was coated with a substrate protein of collagen type I (rat tail; Corning, USA) diluted at a concentration of 100 μg/ml in a PBS solution, and then reacted at 4° C. for 24 hours. Then, the collagen solution was removed, and the glass chips including the PA gel were washed three times with PBS.

The autoclaved PDMS stencil was coated with 2% Pluronic F-127 solution (Sigma-Aldrich) diluted with PBS, and then reacted at 37° C. for 1 hour and washed 3 times with PBS. After removing the liquid from the stencil and the PA gel, the stencil was placed on the collagen-encoded PA gel surface. After filling the hole of the PDMS stencil with PBS without air bubbles, 200 µl drops of human-derived mesenchymal stem cells (density = 2 × 10 ⁶ cells/ml) suspended in the medium were placed. The sample was incubated at 37° C. and 5% CO ₂ for 1 hour to adhere to the PA gel surface, and then the PDMS stencil was gently washed twice with DMEM and then carefully removed. Thereafter, the medium was exchanged with new DMEM to remove cell debris, and patterned mesenchymal stem cell monolayer islands were formed.

2-2: Time-lapse microscopy

All experiments were performed using a JuLI stage live cell imaging system (NanoEnTek, Korea) with a 4x objective lens (Olympus, Japan) in a cell incubator. The cells were imaged using a bright field, and fluorescent fine particles were imaged using a green fluorescent protein (GFP) filter. Images were taken and collected every 10 minutes for 12 hours.

At the last time point, the cells were trypsinized to obtain a reference state image of the position of the fluorescent microparticles at which the stress on the substrate is relaxed from the physical force of the cells (Fig. 2).

2-3: Quantification analysis

The obtained bright field cell image and fluorescent fine particle image were subjected to numerical transformation and quantitative analysis using a customized source code based on MATLAB (MathWorks Inc., USA).

The calculation and analysis codes used in the present invention were performed based on a previous study conducted by the Fredberg group of Harvard TH Chan School of Public Health in the United States (X. Trepat, et al. . , Nat. Phys ., 5:426, 2009; DT Tambe, et al. , Nat. Mater., 10:469-475, 2011).

Briefly, particle image velocimetry (PIV) based on normalized cross-correlation between a cell image and a reference state image from which cells are removed, or a fluorescence microparticle image and a reference state fluorescent microparticle image. Cell and fluorescence microparticle displacement was analyzed through (Fig. 3).

As shown in FIG. 3, the collected bright field stem cell image is calculated numerically by calculating the shift displacement of the pixel through the cross-correlation-based PIV between the cell image (t) and the cell image (t-1) using MATLAB, and then color mapping. The cell movement displacement was spatially plotted by (color mapping) (Fig. 3A), and the pixel movement displacement was calculated numerically through PIV based on the cross-correlation between the fine particle image (t) and the reference state fine particle image (r). The displacement of fine particles was spatially plotted by color mapping (Fig. 3B).

4A is a spatially schematic diagram of the cell migration displacement by color mapping after converting the stem cell movement displacement from pixels to µm units. The deformation observed on the PA gel surface using the above analysis method is physically The force was calculated and the traction force by the cells was measured, and then the cell traction force was spatially plotted using color mapping (Fig. 4B).

In order to quantify the traction force, as shown in Fig. 5, the deviation of the speed and the median value (Fig. 5A), which is a scalar value for the stem cell movement speed, and the deviation of the size of the traction force, which is the absolute value of the cell traction force, are boxed. It was quantified by a box plot, and as shown in FIG. 6, the calculated moving speed (A) and cell traction force (B) were averaged over all time (mean or average) and distribution (histogram or probability) Was analyzed.

Measurement of traction force of stem cells according to material P treatment

In the present invention, in order to confirm whether a substance having a progenitor cell or stem cell mobilization activity can be quantitatively evaluated, the method of Example 2 is applied to an elastic substrate made of a polyacrylamide gel containing the fluorescent fine particles prepared in Example 1 above. After forming a patterned mesenchymal stem cell monolayer island on the PA gel, the substance P (Substance P; RPKPQQFFGLM, SEQ ID NO: 1) was 0 µg/ml, 1 µg/ml, and 5 µg/ml The medium was replaced with DMEM contained at the concentration. The obtained brightfield cell image and fluorescent fine particle image were subjected to numerical transformation and quantitative analysis using a MATLAB-based customized source code in the same manner as in Example 2-3.

7 to 10 are data showing the comprehensive results of the quantification index of cell movement according to the presence and concentration of substance P, a cell movement speed map spatially schematically plotting the cell movement displacement by color mapping and a cell image (FIG. 7 ), and The traction force map (FIG. 8), the stem speed movement speed and the traction force were quantified in box plots (FIG. 9), and the average values and distributions (FIG. 10) for the magnitude of movement speed and cell traction force were shown.

Through this, it was confirmed that the change over time in the average speed of cells and the magnitude of the traction force relative to the mobilization activity of a progenitor cell or a substance having stem cell mobilization activity can be quantified.

<110> AJOU UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION Korea University Research and Business Foundation <120> {Method for Quantitative Evaluating of Recruiting Activity of Substances having Progenitor Cell or Stem Cell Recruiting Activity Using Programming Algorithm and Numerical Analysis <130> 1065775 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Substance P <400> 1 Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu Met 1 5 10

Claims (10)

(a) forming a PA (polyacrylamide) gel layer in which fluorescent microbeads are fixed on the surface;
(b) modifying the surface of the PA gel layer so that cells can attach, and then applying a matrix protein;
(c) attaching progenitor cells or stem cells to the PA gel coated with the matrix protein, treating a substance having progenitor cells or stem cell mobilization activity, and then collecting images of cells and fluorescent fine particles;
(d) removing the cells from which the images were collected, and then collecting fluorescent microparticle images in the PA gel in a reference state without cells; And
(e) measuring the traction of stem cells based on the movement of fluorescent microparticles in the collected images; for the mobilization activity of progenitor cells or substances having stem cell mobilization activity using a programming algorithm and numerical analysis including Method of quantitative evaluation.
The progenitor cell or stem using a programming algorithm and numerical analysis according to claim 1, wherein the substance having the progenitor cell or stem cell mobilization activity is a substance P (Substance P) or an analog of substance P. A method for quantitatively evaluating the mobilization activity of a substance having cell mobilization activity.
The method of claim 1, wherein the progenitor cell or stem cell is an endogenous progenitor cell or an endogenous stem cell, using a programming algorithm and numerical analysis to quantify the mobilization activity of a progenitor cell or a substance having stem cell mobilization activity. Assessment Methods.
The method of claim 1, wherein the PA gel in step (a) has a modulus of elasticity of 1 to 10 kPa, and the diameter of the fluorescent microparticles is 0.2 to 1 µm. A method for quantitatively evaluating the mobilization activity of a substance having cell mobilization activity.
The method of claim 1, wherein the PA gel layer of step (a),
(a1) Acrylamide, N,N' methylenebis (acrylamide) bis solution (N,N'- Methylenebis (acrylamide) BIS solution), tetramethylethylenediamine (tetramethylethylenediamine) and fluorescent micro-bead Preparing a composed PA (polyacrylamide) gel solution;
(a2) putting the PA gel solution in a glass chip having a rectangular groove having a depth of 100 μm to which a silane group is attached and covering the cover glass on the PA gel; And
(a3) centrifuging the fluorescent microparticles to face down in order to align the fluorescent microparticles on the upper surface of the PA gel, and then curing at room temperature and removing the cover glass; programming characterized in that it is produced by a method comprising A quantitative evaluation method for the mobilization activity of a substance having progenitor cell or stem cell mobilization activity using an algorithm and numerical analysis.
The method of claim 1, wherein the matrix protein in the step (b) is collagen, a method for quantitatively evaluating the mobilization activity of a substance having progenitor cells or stem cell mobilization activity using a programming algorithm and numerical analysis.
The progenitor cell or stem using a programming algorithm and numerical analysis according to claim 1, wherein the cell and fluorescent microparticle images of step (c) are photographed and collected using real-time imaging equipment every 10 minutes for 12 hours. A method for quantitatively evaluating the mobilization activity of a substance having cell mobilization activity.
The method of claim 1, wherein the step (e) is performed to determine the movement displacement of the microparticles in pixels through cross-correlation between the reference state fluorescent microparticle image and the fluorescent microparticle image when cells are attached. Converting to a unit, and using the elastic modulus, gel thickness, and Poisson's ratio of the PA gel, the strain observed on the PA gel surface is calculated as a physical force through a finite element method to measure the traction force by the cell. A method for quantitatively evaluating the mobilization activity of a substance having a progenitor cell or stem cell mobilization activity using a programming algorithm and numerical analysis, characterized in that.
The progenitor cell or stem cell according to claim 1, wherein the (e) step is characterized in that the stem cell traction is measured by analyzing the collected cell image and the fluorescent microparticle image with MATLAB software. Quantitative evaluation method for the mobilization activity of substances with mobilization activity.
A method for selecting a substance having progenitor cell or stem cell mobilization activity using the method of any one of claims 1 to 9.

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