KR20170100693A - Technology for depositing a multi-layer film on the cell surface - Google Patents
Technology for depositing a multi-layer film on the cell surface Download PDFInfo
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- KR20170100693A KR20170100693A KR1020160022391A KR20160022391A KR20170100693A KR 20170100693 A KR20170100693 A KR 20170100693A KR 1020160022391 A KR1020160022391 A KR 1020160022391A KR 20160022391 A KR20160022391 A KR 20160022391A KR 20170100693 A KR20170100693 A KR 20170100693A
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- polymer
- polymer layer
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- C12N5/0602—Vertebrate cells
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- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
- C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
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Abstract
In the present invention, a polymer layer made of various materials can be laminated on a cell surface using a layer and a layer deposition method.
The above method is very simple, and there is no limitation on the type of cells that can be applied, and the thickness of the polymer layer can be controlled to several nanometers. In addition, the structure and density of the polymer layer can be adjusted to a desired purpose.
Description
The present invention relates to a method for producing a nano-thick polymer layer on a stem cell surface using a layer-by-layer self-assembly method.
In the present invention, it is possible to provide a technique of producing various combinations of polymer layers based on positively charged polymers, which are extracellular matrix proteins, and inducing the proliferation and differentiation of stem cells without the aid of growth factors or drugs.
Stem cells are multipotent cells capable of differentiating into osteoblasts, chondrocytes, adipocytes or muscle cells, and they have been studied extensively in the field of regenerative medicine and tissue engineering since they can proliferate infinitely without changing the traits in vitro .
In particular, a number of researchers have attempted a variety of approaches in either chemical or physical directions to induce stem cells to differentiate into the cells of the desired tissue. Although the approach to the physical direction has not been actively researched, it has been reported that the degree of tethering of proteins, ligands, and the like is increased from the paper (Non-Patent Document 1) that the differentiation is controlled by the elasticity of the substrate, (Non-Patent Document 2), and control of differentiation by biochemical signals according to the magnitude of the tension of extracellular matrix fibers (Non-Patent Document 3) are presented have. However, active research is still underway to find the factors that have the greatest influence on stem cell differentiation.
In addition, high-level studies are underway to analyze the intracellular substances and signal transduction systems in order to elucidate mechanisms that regulate differentiation of stem cells. In this regard, it has been found that a specific protein TAZ (transcriptional coactivator with PDZ-binding motif) is a transcriptional modulator that inhibits the formation of adipocytes and promotes the formation of osteoblasts (Non-Patent Document 4) (Non-patent document 5) that regulates the differentiation of stem cells. Subsequent studies have been carried out to interpret the signaling system in the stem cell by external mechanical stimulation, but it has not been clarified yet.
In the meantime, various growth factors are used to induce differentiation into specific cells depending on the intended purpose. For example, in order to induce differentiation into osteoblasts, stem cells are treated with bFGF (basic fibroblast growth factor) and BMP-2 (Bone morphogenetic protein 2) (Non-Patent Document 6). However, these growth factors are not only difficult to manage and expensive, but also have side effects such as inflammation reaction, tumor formation and osteolysis in BMPs. In order to control the function of the cells, a multilayer thin film is prepared on the surface of cells. In this case, when the cells are treated with the positive charge substance solution by the cytotoxicity test for the substance, the cell membrane having a negative charge is damaged and the cell viability is very poor Non-Patent Document 7). Therefore, fibronectin and gelatin, which are negatively charged extracellular matrix proteins at pH 7.4, are multilayered on the cell surface using biological recognition (Non-Patent Document 8) . However, since both fibronectin and gelatin are negatively charged, there is a doubt as to whether the multilayer thin film is actually formed because the repulsive force acts more strongly on the materials having the same charge than the attractive force.
Therefore, in order to commercialize stem cells, efforts are needed to control the proliferation and differentiation of stem cells by a simple and simple method.
The present invention relates to a technique for laminating a polymer layer on a cell surface by using a layer and a layer lamination method. The polymer layer based on the extracellular matrix protein can provide an artificial extracellular matrix to the cell, stabilize the cell, and promote cell proliferation. It is also possible to differentiate stem cells in specific directions without the aid of growth factors or drugs.
In the present invention, the method includes repeatedly laminating a positive charge polymer solution and a negative charge polymer solution on the surface of a stem cell, and laminating the surface of the stem cell with two or more polymer layers,
Wherein the positively charged polymer is collagen, elastin or decolin,
Wherein the negative charge polymer is selected from the group consisting of poly-L-glutamic acid, lignin-alkali, tannic acid, chondroitin sulfate and alginic acid. The present invention provides a method for inducing differentiation of at least one stem cell into osteoblast.
In addition, in the present invention, positively charged polymer layers and negatively charged polymer layers are alternately laminated on the stem cell surface,
Wherein the positively charged polymer layer is composed of collagen, elastin or decolin,
The negative charge polymer layer may be formed from a group consisting of poly-L-glutamic acid, lignin-alkali, tannic acid, chondroitin sulfate and alginic acid. One or more selected ones,
The polymer layer provides two or more layers of stem cells.
In the present invention, it is possible to induce stem cell proliferation and osteoblast differentiation by forming a polymer layer on the surface of a stem cell in a very simple manner.
Since the polymer layer is based on an extracellular matrix protein that is not only produced under the same conditions as the growth medium of the cell but also does not damage the cell membrane, unlike the existing multilayer thin film on the cell surface, It is advantageous.
This makes it possible to manufacture a polymer layer on the cell surface at a low cost without using a growth promoter or a drug and to apply it to various fields.
FIG. 1 is a graph (A) in which the thickness of a polymer layer laminated on a silicon substrate is measured, and a graph (B) in which a polymer layer laminated on a gold electrode is analyzed by QCM (Quartz Crystal Microbalance).
FIG. 2 is a graph showing a hardness (A), a Young's modulus (B), and a morphology image (C) of the mechanical properties of the polymer layer.
3 is a graph of degradation curves of the polymer layer under physiological conditions (cell isotonic solution conditions, 37 ° C).
FIG. 4 (A) is a schematic view illustrating a process of promoting stem cell proliferation and osteoblast differentiation by coating a polymer layer on the surface of mesenchymal stem cells, and FIG. 4 (B) It is a table showing the combination of name and polymer layer.
FIG. 5A is a graph showing the degree of survival of cells in which polymer layers are stacked. FIG. 5B is a graph showing the TAZ protein target (FIG. 5A) through qRT-PCR (Quantitative Real Time Polymerase Chain Reaction) (C) is a graph showing the proliferation of stem cells at intervals of two days for 6 days under the adherent condition and the suspended condition.
FIG. 6 is a graph showing quantitative analysis of osteoblast differentiation markers through qRT-PCR (Quantitative Real Time Polymerase Chain Reaction) 6 days after the start of differentiation.
Figure 7 shows the results of Western blot analysis of MAPK (Mitogen Activated Protein Kinases) pathway (A) and DAPI (4 ', 6-diamidino-2-phenylindole) An image showing immunofluorescence (B) taken using a confocal microscope to confirm localization.
The present invention includes a step of repeatedly laminating a positive charge polymer solution and a negative charge polymer solution on the surface of stem cells and laminating the surface of the stem cells with two or more polymer layers,
Wherein the positively charged polymer is collagen, elastin or decolin,
Wherein the negative charge polymer is selected from the group consisting of poly-L-glutamic acid, lignin-alkali, tannic acid, chondroitin sulfate and alginic acid. The present invention relates to a method for inducing differentiation of at least one stem cell into osteoblast.
Hereinafter, a method for inducing the differentiation of stem cells into osteoblasts will be described in more detail.
In the present invention, 'stem cell' refers to a cell capable of cell division by itself and capable of differentiating into a very specific type of specific cell type. The type of the stem cells is not particularly limited, and in one embodiment, mesenchymal stem cells can be used as the stem cells.
In the present invention, the kind of mesenchymal stem cells is not particularly limited. The mesenchymal stem cells can be used irrespective of where they originate from. In one embodiment, the mesenchymal stem cells can be obtained from known mesenchymal stem cell sources, such as fats, bone marrow, embryos, cord blood, blood or body fluids. The differentiation pattern may be different depending on the tissue origin of the stem cells. In addition, the subject animal such as bone marrow or tissue may be a mammal, and may be specifically a human. Methods for obtaining mesenchymal stem cells from such known mesenchymal stem cell sources are well known in the art.
In the present invention, mesenchymal stem cells can proliferate or differentiate. In one embodiment, the mesenchymal stem cells can differentiate into osteoblasts.
As described above, the method of inducing differentiation of stem cells according to the present invention into osteoblasts involves repeatedly laminating a positive charge polymer solution and a negative charge polymer solution on the surface of stem cells, and coating the surface of the stem cells with two or more polymer layers .
The coating can be carried out by a self-assembly method. The self-assembly means that the thin film of the polymer solution is laminated by repeating the process of sequentially spraying the polymer solution having a relatively high electric charge on the substrate. Since the polymer thin film can be sequentially produced, it is also called a 'layer by layer' (LBL). Since the solution is not mixed with each other but a substrate is used, a thin film (polymer layer) in which phase separation does not occur can be produced.
In the present invention, two or more polymer layers can be prepared by sequentially laminating a positively charged polymer solution on a surface of a stem cell and a thin film (layer) of a negative charge polymer solution.
In the present invention, a positive charge polymer and a negative charge polymer refer to a polymer capable of forming a positive charge and a negative charge, respectively, when the polymer is dissolved in a solvent.
The type of the positive charge polymer is not particularly limited, and extracellular matrix proteins can be used. The extracellular matrix protein binds to receptors on the cell surface to enhance the stability felt by the stem cells, thereby promoting cell proliferation. In addition, stem cells can be induced to differentiate without the aid of active ingredients such as growth factors or drugs. These growth factors have side effects such as inflammation reaction, tumor formation and osteolysis, and have a risk of recombination depending on the origin of the growth factors. As such extracellular matrix proteins, collagen, elastin or decolin can be used.
The type of the negative charge polymer is not particularly limited, and poly-L-glutamic acid, lignin-alkali, tannic acid, chondroitin sulfate and alginic acid alginic acid) may be used. The differentiation of stem cells can be controlled depending on the kind of the polymer constituting the multilayer thin film.
The positive charge polymer solution means the solution containing the positive charge polymer, and the negative charge polymer solution means the solution including the negative charge polymer. The positive charge polymer and the negative charge polymer may form a bond through electrostatic attraction and / or biological recognition. The electrostatic attraction refers to attraction between particles charged with opposite charges, and the negatively charged cell membrane forms a bond with a positive charge polymer by an electrostatic attraction, and the positive charge polymer forms a bond with a negative charge polymer by an electrostatic attraction do. In addition, the biological recognition may be performed by binding a cell, a cell adhesion protein, or an extracellular matrix protein with a cell specific receptor, or by a specific domain of each cell adhesion protein or extracellular matrix protein or each other . In the present invention, the electrostatic attractive force and the biological recognition function together to form a bond, whereby the polymer layer can be formed in multiple layers on the cells.
In one embodiment, the laminating of the polymer solution comprises: (a) floating the stem cells in a positive charge polymer solution;
(b) centrifuging the stem cell suspension prepared in (a) to obtain a cell pellet;
(c) suspending the cell pellet obtained in (b) in the negative charge polymer solution; And
(d) centrifuging the suspension prepared in (c) to obtain cell pellets.
In one embodiment, a further step of pre-treating the cells to harvest the cells prior to performing step (a) may be performed. The cell pretreatment may be a trypsin treatment. The trypsin treatment is carried out when the cells are removed from the plate on which the cells were cultured and harvested. By this treatment, the adherent proteins can be removed and the cells can be harvested in a single cell state.
In one embodiment, the positively or negatively charged polymer in the positive or negative charge polymer solution may be any of the polymers described above. As a solvent for the polymer solution, PBS, Tris-buffer or growth medium can be used.
In one embodiment, the amount of the positively charged or negatively charged polymer solution in steps (a) and / or (c) may be 0.3 to 3 ml or 0.5 to 1 ml. In addition, the suspension of stem cells or cell pellets in the polymer solution may be performed by pipetting for 5 to 50 times.
In one embodiment, in step (b) and / or (d), centrifugation can be performed at 1000 to 1300 rpm for 1 to 10 minutes. Cell pellets can be easily obtained in the above range.
In addition, after performing steps (b) and / or (d) in one embodiment, the step of washing the obtained cell pellets may be further performed.
For example, a weakly bonded polymer can be removed using a solvent (hereinafter referred to as a cleaning solution) of the polymer solution used in the previous step. The washing may be performed by placing the cell pellet in a washing solution, pipetting and centrifuging. The washing may be performed two or more times.
In the present invention, steps (a) and (c) may be repeatedly performed in accordance with the desired number of polymer layers.
The differentiation pattern of stem cells into osteoblasts according to the present invention may vary depending on, for example, the type of the material constituting the multilayered membrane, the thickness and structure of the polymer layer, and the like.
That is, in the present invention, the above-described positively charged and negatively charged polymers are used, and the thickness and structure of the polymer layer are optimally limited, and the cells can be differentiated into osteoblasts without the use of growth factors and the like.
In one embodiment, the total thickness of the polymer layer formed of two or more layers is not particularly limited, and may be, for example, 50 to 80 nm or 60 to 70 nm. Within this range, stem cells can differentiate into osteoblasts.
In addition, in one embodiment, the number of polymer layers is not particularly limited, and may be, for example, 2 to 9 or 2 to 5 layers.
Further, the structure of the polymer layer may be a flat structure, a porous structure, a net structure, a hierarchical structure, or a fibrous structure. In addition, the roughness of the polymer layer formed on the surface of the stem cells may be 30 nm to 100 nm.
As described above, stem cells can be easily differentiated into osteoblasts through control of the type of material constituting the multilayered film, the thickness and structure of the polymer layer, and the like.
In the present invention, the stem cells are located inside the polymer layer and can be differentiated into osteoblasts.
In the stem cell differentiation process, the polymer layer may be completely decomposed or partially separated depending on the constituent components of the polymer layer.
The present invention also relates to a method for producing stem cells comprising repeatedly laminating a collagen solution and an alginic acid solution on the surface of mesenchymal stem cells and laminating the surface of the stem cells with two or more polymer layers, .
In the present invention, the efficiency of differentiation of the stem cells into osteoblasts can be improved by using collagen as a positively charged polymer and alginic acid as a negative charge polymer.
In addition, the present invention is characterized in that a positively charged polymer layer and a negatively charged polymer layer are alternately laminated on the stem cell surface,
Wherein the positively charged polymer is collagen, elastin or decolin,
Wherein the negative charge polymer is selected from the group consisting of poly-L-glutamic acid, lignin-alkali, tannic acid, chondroitin sulfate and alginic acid. More than one stem cell.
In the present invention, the positive charge polymer layer is formed of a positive charge polymer, and the negative charge polymer layer is formed of a negative charge polymer.
The stem cell has a polymer layer composed of two or more layers, and the positively charged polymer layer can be bonded to the neighboring negatively charged polymer layer through an electrostatic attractive force.
As described above, the stem cells can be prepared by repeatedly laminating a positive charge polymer solution and a negative charge polymer solution on the surface of stem cells and laminating the surface of the stem cells with two or more polymer layers.
In the present invention, the type of the material constituting the multilayer thin film, the thickness and structure of the polymer layer, and the degree of differentiation of stem cells can be controlled to determine the timing of injecting the stem cells into the living body according to the type of disease.
Hereinafter, the present invention will be described in detail with reference to examples. The following examples illustrate the invention and are not intended to limit the scope of the invention. These embodiments are provided so that the disclosure of the present invention is not limited thereto and that those skilled in the art will fully understand the scope of the present invention and that the present invention is not limited by the scope of the claims Only.
Example
Example 1. Preparation of a polymer layer on a variety of substrates
The positively charged polymers include collagen-type 1-from rat tail and the negatively charged polymers include poly-L-glutamic acid, lignin-alkali, tannic acid, chondroitin sulfate chondroitin sulfate and alginic acid were used.
The polymer was prepared as a solution with PBS (phosphate buffer saline) as a solvent at a concentration of 1 mg / mL at pH 7.4. The solution contains 0.15M salt, and the solvent has the same conditions as the cell isotonic solution.
(1) Production of a polymer layer on a silicon substrate
Prior to laminating the polymer layer on the silicon substrate, the silicon substrate was subjected to O 2 plasma treatment to modify it to a negative charge, and then to carry it on a collagen solution as a positive charge polymer solution for 10 minutes. In order to remove the collagen stacked with a weak bond, it was carried in the washing solution for 2 minutes, 1 minute and 1 minute. Thereafter, the above procedure was repeated using a negative charge solution to laminate the negative charge polymer.
(2) Production of polymer layer on gold electrode
Gold electrode was cleaned in piranha solution (sulfuric acid: hydrogen peroxide = 3: 2) for 5 minutes, washed in distilled water, and then negatively charged through O2 plasma treatment. Then, it was carried in a collagen solution, which is a positive charge polymer solution, for 10 minutes, and then carried in a washing solution for 2 minutes, 1 minute, and 1 minute to remove collagen stacked with a weak bond. Thereafter, the above procedure was repeated using a negative charge solution to laminate the negative charge polymer.
Test Example 1. Quantitative Analysis of Polymer Layer
1 is a graph (A) showing a total thickness of a polymer layer laminated on a silicon substrate and a graph (B) showing a QCM (Quartz Crystal Microbalance) analysis of a polymer layer laminated on a gold electrode.
In order to easily distinguish the polymer layer prepared in Example 1, the polymer layer was named as the negative charge polymer used. That is, when poly-L-glutamic acid is used, lignin is used when PGA, lignin-alkali is used, TA is used when tannic acid is used, chondroitin sulfate is used When used, CS and alginic acid were named AA.
As shown in Fig. 1 (A), it can be seen that the thickness of the polymer layer adsorbed on the silicon substrate increases with the increase in the number of polymer layers. As a result, it can be confirmed that all kinds of polymer layers have been successfully manufactured. However, the thickness of the polymer layer varies depending on the type of the negative charge polymer, because the charge intensity and structure of the negative charge are different from each other in the cell isotonic solution condition. This can be confirmed from the QCM results in (B).
All types of polymer layers show an overall increase in thickness as the layer increases, while the thicknesses of the TA and PGA in the even layers decrease. This is because a large amount of collagen is deposited on the substrate in the odd-numbered layer, but when the substrate is supported on the negative-charged polymer solution, a part of the collagen layer on the substrate surface is separated and the negative charge polymer is laminated. That is, the value of the even layer in which the negative charge polymer layer is stacked is decreased, but the value of the odd layer in which the positive charge polymer layer is stacked increases and thus the value increases as a whole. As a result, the negative charge polymer layer is sufficiently stacked, It can be stacked.
The SAuerbrey equation can be used to calculate the change in the frequency of the QCM by mass change. The density of the polymer layer can be calculated from the thickness and mass of the polymer layer. As a result, the density of PGA was much larger than that of other polymer layers.
Experimental Example 2. Analysis of Physical Properties of Polymer Layer
In the present invention, FIG. 2 is a graph and photographs showing analysis of mechanical properties and morphology of a polymer layer, showing hardness (A), Young's modulus (B), and polymer layer morphology image (C).
The physical properties were measured using a polymer layer laminated on the silicon substrate of Example 1, wherein the thickness of the polymer layer was 400 nm. The hardness and Young's modulus were measured by nano-indentation and the morphology images were measured by AFM (Atomic Force Microscope).
The hardness (A) and the Young's modulus (B) represent the comparison values through modeling rather than actual values. The hardness and the Young's modulus do not vary greatly depending on the kind of the polymer layer. The polymer layer has a large amount of collagen in all the polymer layers and has a similar mechanical property because it is a nano-thick polymer layer composed only of a protein and a polymer.
This can also be confirmed by morphology AFM image (C), where the appearance of collagen fibers in all types of polymer layers is predominant and also has a similar morphology.
3 is a graph of a degradation curve of a polymer layer at physiological conditions (cell isotonic solution conditions, 37 DEG C).
Specifically, the polymer layer (thickness: 100 nm) laminated on the silicon substrate of Example 1 was used to confirm a change in the thickness of the polymer layer for 6 days required for stem cell differentiation. The thickness variation of the 100 nm-thick polymer layer was divided by the initial value and normalized and plotted.
As shown in FIG. 3, the thickness of the polymer layer was decreased within 1 h in most cases, although the thickness of the polymer was varied depending on the type of the negative charge polymer. Thereafter, the thickness of the polymer layer was maintained constant. Since the polymer layer has the same conditions as the conditions for producing the polymer layer and the conditions for confirming the degradation, the thickness of the polymer layer is slightly reduced but remains constant. Thus, it can be predicted that the polymer layer on the cell surface can be maintained during cell differentiation have.
Example 2. Preparation of Stem Cell-Based Polymer Layer
The positively charged polymers include collagen-type 1-from rat tail and the negatively charged polymers include poly-L-glutamic acid, lignin-alkali, tannic acid, chondroitin sulfate chondroitin sulfate and alginic acid were used.
In addition, human bone marrow-derived mesenchymal stem cells (hMSCs) were used as the cells.
All procedures were carried out under sterile conditions (disinfected bench).
The positively and negatively charged polymers described above were prepared as solutions at a concentration of 1 mg / mL in hMSC growth medium (DMEM medium containing 10% Fetal bovine serum) as a solvent at pH 7.4. Sterilization was performed using a 0.2 μm filter for disinfection such as removal of bacteria.
The single cell suspension obtained by trypsin treatment was centrifuged at 1000 to 1300 rpm for 3 minutes to obtain a cell pellet. 0.5 mL of collagen solution, which is a positive charge solution, was added to the cell pellet from which the supernatant was removed, and the cells were resuspended by pipetting 30 times. Then, the cell pellet was obtained again by centrifugation at 1000 to 1300 rpm for 3 minutes. The supernatant was removed, and 1 mL of growth medium, growth medium, was added and the cells were resuspended by pipetting 10 times. The cells were further centrifuged at 1000 to 1300 rpm for 3 minutes to obtain cell pellets, and the washing procedure was further performed. Thereafter, the above procedure was repeated using a negative charge solution to laminate the negative charge polymer.
The above procedure was repeated by the number of layers of the polymer layer.
As a control group, cells without lamination of a polymer layer were used.
4 (A) is a schematic view showing a process of laminating a polymer layer on the surface of mesenchymal stem cells to promote the proliferation of stem cells and the differentiation into osteoblasts. Fig. 4 (B) It is a table showing the combination of the name of the negative charge polymer and the polymer layer.
EXPERIMENTAL EXAMPLE 3 Cell survival experiment
96 is a stacked cell of a polymer layer prepared in Example 2 in well culture plate (mesenchymal stem cells) and the control group respectively, 1 × 10 4 / seeding (seeding) in cm 2 density and 37 ℃, CO 2 at 5% Conditions Lt; / RTI > and incubated. Twenty-four hours later, 10 μL of EZ-Cytox was added and incubated for 30 minutes. All wells were carefully shaken to obtain uniform results, and absorbance was measured at 450 nm wavelength using a bio-radar plate reader.
As a result, the absorbance of the control group and the absorbance of the cell laminated with the polymer layer prepared in Example 2 were obtained.
5A is a graph showing the degree of survival of cells in which a polymer layer is stacked.
As the cells, mesenchymal stem cells in which a polymer layer having a thickness of 60 nm was laminated were used according to the manufacturing method of Example 2, and as a control group, mesenchymal stem cells without a polymer layer were used.
As shown in FIG. 5 (A), when the degree of survival of the control (control), which is an uncoated cell, was set at 100%, the degree of survival of the polymer layer-coated cells was 90% or more . This indicates that cell survival is not significantly affected even after laminating the polymer layer on the cell surface.
In the present invention, high cell viability can be obtained by laminating a collagen-based polymer layer which is an extracellular matrix protein having no cytotoxicity and a weak positive charge at pH 7.4.
Example 4. Measurement of cell proliferation in cell growth conditions
Cell proliferation was measured in adherent condition. The cells were seeded at a concentration of 1 × 10 4 / cm 2 in a 6-well culture plate and incubated at 37 ° C and 5% CO 2 . Cells were trypsinized at 2-day intervals for 6 days to obtain single cells. The number of cells was counted using a hemacytometer, and seeded into a 6-well culture plate.
In suspension conditions, cells were seeded on poly-HEMA (poly-2-hydroxyethyl methacrylate) coated on 6-well culture plates. Similarly, the number of cells was counted at intervals of two days for 6 days. Suspended cells were centrifuged at 1000 rpm for 3 minutes to obtain cell pellet, and then resuspended in growth medium to make single cells. The number of cells was counted using a hemocytometer and seeded in a poly-HEMA coated 6 well culture plate.
FIG. 5 (B) is a graph showing the result of quantitative analysis of the TAZ protein target gene through qRT-PCR (Quantitative Real Time Polymerase Chain Reaction) two days after seeding the cells.
The TAZ protein target gene was quantitatively analyzed for mesenchymal stem cells (
The results of FIG. 5 (B) show that TAZ protein is induced more than control (control), which is an uncoated cell of all kinds of polymer layers under cell growth conditions, and cell proliferation is better by TAZ promoting cell proliferation It shows the possibility to happen.
FIG. 5 (C) is a graph showing the proliferation of stem cells at intervals of two days for six days under the adherent condition and the suspended condition.
The proliferation of the mesenchymal stem cells in which the polymer layer was laminated with the 60 nm-thick polymer layer of Example 2 was confirmed.
The degree of cell proliferation of the cells coated with the polymer layer and the control (control), which is a cell not coated with the polymer layer under the adhering condition, is different depending on the type of the material forming the polymer layer. However, Which promotes cell proliferation. This result is consistent with the result of (B), which is progressed in the cell growth condition and the attachment condition, because the polymer layer laminated on the cell surface acts as an artificial extracellular matrix and the cell is more stable.
In the suspension condition, it is possible to confirm the degree of stability of the cell when the polymer layer which plays an artificial extracellular matrix for the adherent cell stem cells does not influence the substrate. Most of the polymer layers showed higher degree of proliferation than the control group, but the degree of proliferation of PGA was lower than that of the control group. This is because when the polymer layer of the same thickness is laminated on the cell surface, the polymer layer with the greatest amount of time is separated over time and does not give sufficient stability to the cell.
In particular, AA has both high cell proliferation effect in both conditions, suggesting that AA can be used to stimulate stem cell proliferation and to secure sufficient amount of stem cells in a short time.
Example 5. Measurement of the degree of differentiation into osteoblasts under cell differentiation conditions
Cells were seeded at a concentration of 1 x 10 4 / cm 2 on a 6-well culture plate and incubated at 37 ° C and 5% CO 2 . Two days later, osteogenic differentiation media (DMEM supplemented with 10% Fetal bovine serum, 50 μg / mL ascorbic acid, 0.1 μM dexamethasone and 10 mM β-glucerophosphate) was added to the growth media two days later. gave. Six days after the initiation of differentiation, total RNA was isolated from the cells using Trizol reagent and cDNA was synthesized from RNA using M-MLV (Moloney Murine Leukemia Virus) reverse transcriptase. The osteogenic differentiation markers DLX5, MSX2, Osteocalcin and RUNX2 were quantitatively analyzed using qRT-PCR (Quantitative Real-Time Polymerase Chain Reaction).
6 is a graph showing quantitative analysis of osteoblast differentiation markers through qRT-PCR (Quantitative Real Time Polymerase Chain Reaction) 6 days after the start of differentiation.
In FIG. 6, osteoblast differentiation markers can be quantitatively analyzed by qRT-PCR to confirm the osteoblast differentiation level of the stem cells. In FIG. 6, the result of the control without coating the polymer layer was set to 1, and all the results were normalized. AA induces osteoblast differentiation more than twice as much as that of the control group. This suggests that stem cell differentiation can be controlled by preparing a polymer layer on the cell surface.
In particular, the effect of promoting differentiation into osteoblasts can be clinically applied to applications such as cell therapy.
Example 6. Analysis of TAZ protein
Western blotting was performed to identify upstream signaling of the TAZ protein and signaling of regulated cell signaling. Cells were seeded at a concentration of 1 x 10 4 / cm 2 on a 6-well culture plate and incubated at 37 ° C and 5% CO 2 . After 24 hours, protease inhibitors were added to TNE lysis buffer [20 mM Tris-HCl (pH 7.5), 1% NP-40, 150 mM NaCl, 2 mM EDTA (pH 8.0), 50 mM NaF, 1 mM Na3VO4] To dissolve the cells. Cell lysates were digested with Bradford protein assay and denatured at 95 ° C for 5 minutes, and quantitated using SDS sample buffer. The sample was placed on 8% acrylamide gel, separated by electrophoresis, blotted on PVDF membrane, blocked with 5% non-fat dry milk in TBST, And diluted in TBST containing 5% BSA. And incubated overnight at 4 ° C with rotation. The cells were washed 3 times with TBST for 5 minutes, diluted with TBST containing 5% non-fat dry milk, and incubated with stirring at room temperature for 1 hour. The cells were washed three times with TBST for 5 minutes and a specific band of the target protein was detected using the ECL system.
Immunocytochemistry was performed to confirm localization of TAZ. Cells were seeded at 1 × 10 4 / cm 2 concentration on a cover-slip coated with poly-L-lysine in a 24-well culture plate and incubated at 37 ° C and 5% CO 2 . After 24 hours, cells were fixed with 3.7% formaldehyde and permeable 3% triton X-100 reagent. The cells were blocked by treatment with 5% normal goat serum for 1 hour, and the primary antibody was diluted in PBS containing 1% BSA. After overnight incubation at 4 ° C, the cells were washed 3 times with PBS for 5 minutes. The secondary antibody bound to the fluorescent dye was diluted in PBS containing 1% BSA, added to the cells, and incubated at room temperature for 2 hours. The cells were washed 3 times with PBS for 5 minutes and treated with mounting media containing DAPI to stain nuclei. A confocal microscope was used to identify the stained samples.
In the present invention, FIG. 7 shows the results of Western blotting (MAPK) pathway-related western blotting (A) and DAPI (4 ', 6-diamidino-2-phenylindole) (B) images taken with a confocal microscope to confirm the localization of the TAZ.
As a result of the western blot in FIG. 7 (A), phosphorylation of ERK was increased in all types of polymer layers, and phosphorylation of JNK was increased only in AA. This suggests that cell proliferation and promotion of differentiation into osteoblasts by the polymer layer on the cell surface can be mediated by the mitogen activated protein kinase (MAPK) pathway. (B) is an immunofluorescence image taken by a confocal microscope. TAZ localization was confirmed using an antibody conjugated with a fluorescent dye of TAZ. In all types of polymer layers, TAZ is activated and is present in DAPI-stained nuclei, suggesting that cell proliferation and promotion of osteoblast differentiation by cell surface polymer are related to TAZ signaling.
Claims (9)
Wherein the positively charged polymer is collagen, elastin or decolin,
Wherein the negative charge polymer is selected from the group consisting of poly-L-glutamic acid, lignin-alkali, tannic acid, chondroitin sulfate and alginic acid. A method for inducing differentiation of at least one stem cell into osteoblast.
The stem cell is a mesenchymal stem cell.
Wherein the total thickness of the polymer layer is 50 to 80 nm.
Wherein the polymer layer is formed of 2 to 9 multi-layered cells.
The lamination of the polymer solution comprises (a) suspending stem cells in a positive charge polymer solution;
(b) centrifuging the stem cell suspension prepared in (a) to obtain a cell pellet;
(c) suspending the cell pellet obtained in (b) in the negative charge polymer solution; And
(d) centrifuging the suspension prepared in (c) to obtain cell pellet.
Performing steps (b) and (d), and then washing the obtained cell pellet.
A method for culturing stem cells, wherein steps (a) and (c) are repeatedly performed.
Wherein the positively charged polymer layer is composed of collagen, elastin or decolin,
The negative charge polymer layer may be formed from a group consisting of poly-L-glutamic acid, lignin-alkali, tannic acid, chondroitin sulfate and alginic acid. One or more selected ones,
Wherein the polymer layer is at least two layers.
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|---|---|---|---|---|
| KR102066258B1 (en) * | 2018-07-31 | 2020-01-14 | 연세대학교 산학협력단 | A method for coating a surface of a stem cell |
| KR20200113769A (en) * | 2019-03-26 | 2020-10-07 | 연세대학교 산학협력단 | Composition for inducing chondrogenesis and use thereof |
| WO2022019688A1 (en) * | 2020-07-22 | 2022-01-27 | 연세대학교 산학협력단 | Method for producing cultured meat on basis of cell sheet coating technique, and cultured meat produced thereby |
| KR20230046024A (en) | 2021-09-29 | 2023-04-05 | 인하대학교 산학협력단 | Thiolated lignin sulfonate composites and method for preparing the same |
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| KR102289206B1 (en) | 2018-07-12 | 2021-08-13 | 순천향대학교 산학협력단 | A chitosan/TEMPO oxidized cellulose nanofiber hydrogel comprising fk506 for bone therapy and a method for manufacturing the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8172831B2 (en) * | 2008-09-02 | 2012-05-08 | Abbott Cardiovascular Systems Inc. | Catheter configured for incremental rotation |
| JP2013233113A (en) * | 2012-05-09 | 2013-11-21 | Osaka Univ | Method for producing cell with improved tolerance to physical load |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR102066258B1 (en) * | 2018-07-31 | 2020-01-14 | 연세대학교 산학협력단 | A method for coating a surface of a stem cell |
| KR20200113769A (en) * | 2019-03-26 | 2020-10-07 | 연세대학교 산학협력단 | Composition for inducing chondrogenesis and use thereof |
| WO2022019688A1 (en) * | 2020-07-22 | 2022-01-27 | 연세대학교 산학협력단 | Method for producing cultured meat on basis of cell sheet coating technique, and cultured meat produced thereby |
| KR20220012205A (en) * | 2020-07-22 | 2022-02-03 | 연세대학교 산학협력단 | Method for manufacturing cultured meat based on cell sheets coating and cultured meat prepared therefrom |
| KR20230046024A (en) | 2021-09-29 | 2023-04-05 | 인하대학교 산학협력단 | Thiolated lignin sulfonate composites and method for preparing the same |
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