TW201219572A - A method to producing a spheroid population of adult stem cells - Google Patents

Abstract

This invention relates to a method to producing a spheroid cell population of adult stem cells, comprising culturing adult stem cells on a thin film made by biocompatibility polymer materials. The biocompatibility polymer materials is chitosan, alginate, hyaluronic acid, tremella polysaccharide, polycaprolactone or any combination thereof. The adult stem cells are selected from the group consisting of neural stem cells, neural progenitor cells, adipose-derived adult stem cells, human gingival fibroblasts, bone mesenchymal stem cells, lung stem cell and placenta-derived mesenchymal stem cells. The method can sustain the self-renew and stemness of adult stem cells.

Description

201219572, sixth, invention description: [Technical field of the invention] The present invention relates to a method for culturing adult stem cells, and more particularly to a method for using a biocompatible polymer to aggregate adult stem cells into spheres and to improve differentiation ability . [Prior Art] Stem cells are cells that have not yet fully differentiated, have self-renew and differentiated plasticity, and have great potential in tissue engineering and regenerative medicine applications; stem cell roots are divided into omnipotents based on their potential for differentiation. Totipotent stem cells, three-potential pluripotent stem cells, pluripotent stem cells, and unipotent stem cells 'eg each cell before morula All have versatility, that is, any cell can be developed into a complete individual in the uterus, and cells with this potential are called pluripotent stem cells. Cells with only inner cell mass alone, although unable to develop into the entire individual (because of the lack of ability to differentiate into trophectoderm, will differentiate into amniotic membrane and placenta after trophoblast development), but have evolved into individual processes The ability to differentiate into various cells, called the three germ layer pluripotent stem cells. Mesenchymal stem cells (MSCs) can differentiate into hard bone, cartilage, Φ fat and other connective tissues or transdifferentiate into nerve cells, liver cells, etc., called single germ layer pluripotent stem cells. Neural stem cells can only differentiate into neurons or neuroglial cells, called pluripotent stem cells. Stem cells can be further classified into embryonic stem cells (ESCs) and adult stem cells according to sources. Embryonic stem cells generally refer to cells derived from the inner cell mass of the blastocyst, and adult stem cells generally refer to stem cells taken from the baby's birth. Adult stem cells include, but are not limited to, hemat〇P〇ietic stem cells, mesenchymal stem cells, neural stem cells (NSCs), hepatic stem cells (oval cdl), and the like. Among them, mesenchymal stem cells include but are not limited to bone marrow mesenchymal stem cells (BMSCs), placenta stem cells 4 201219572 (placenta-derived mesenchymal stem cells (PDMSCs), adipodderd adult stem cells (ADASs), and gingival stem cells (Human gingivai). Fibroblasts, HGF) and more. Embryonic stem cells have unlimited replication ability and pluripotency differentiation potential. However, there are many problems to be overcome in clinical application, such as moral controversy and immune rejection during transplantation. Adult stem cells can avoid the problem of immune rejection and the moral controversy during transplantation because of the clinical application of autologous transplantation. However, adult stem cells are usually only able to differentiate into a few due to limited number of divisions. Seed cells, such as neural stem cells, can be differentiated into neurons (neur〇ns), astrocytes (astrocytes) and dendritic cells (〇endigCytes,) and adult stem cells in vitro under appropriate conditions. In the process of weight-increasing culture, it is often difficult to maintain the differentiation activity and functionality. On the other hand, 'the general materials suitable for culturing stem cells are polystyrene, gelatin, poly-α-hydroxy acid, Polylactic acid glycolic acid, etc., has the advantages of stable biocompatibility, but these materials are used for the proliferation and culture process of adult stem cells, have the disadvantage of being difficult to differentiate or lose function, and affect the effect of adult stem cell proliferation and differentiation. It has been found that cell morphology can regulate the differentiation of stem cells before treatment, and the cell morphology of the sphere has been confirmed and the differentiation potential has For example, the colony of embryonic stem cells can self-renew from tight cell-cell contact; Embryoid bodies (EB) are three-dimensional spatial cell aggregates of embryonic stem cells, which can be initially differentiated under simulated normal embryo growth environment, but There is still no effective method for aggregating adult stem cells into spheres. SUMMARY OF THE INVENTION In order to solve the problem that conventional adult stem cells are difficult to maintain sternness and differentiation in vitro, the object of the present invention is to provide an adult stem cell The method of a spheroid cell population comprises the in vitro culture of adult stem cells in a thin layer of 201219572 formed on a biocompatible polymer, wherein the biocompatible polymer is chitosan, alginate, transparent Acidic acid, polycaprolactone, tremella polysaccharide or any combination thereof; and the ability to collect a population of spheroid cells aggregated by adult stem cells, wherein the spheroid cell population expands and has the ability to self-renew and differentiate into one cell; Adult stem cells are selected from the group consisting of neural stem cells, neural precursor cells, adipose stem cells, and dried gingiva a group consisting of cells, mesenchymal stem cells, lung stem cells, and placental stem cells, and the biocompatible polymer can be a combination of chitosan and hyaluronic acid, and the ratio of chitosan to hyaluronic acid ( w/w) is about 3/0.177 to 3/4.425, since the present invention uses l.lmg/cm2, 5.5mg/cm2, 2_5 mg/cm2, because a slide is 1.77 cm2, so it is 3 mg (chitosan): 177.177mg (HA), 3 mg (chitosan): 0.885 mg (HA), 3 ru mg (chitosan): 4.425 mg (HA) are effective, using hyaluronic acid negatively adsorbed on the surface of chitosan (positive) The double-layer material, otherwise the hyaluronic acid can not grasp the surface will dissolve into the water, the somatic cell can be a nerve cell. On the other hand, the population of spheroid cells produced by adult stem cells further has the ability to transdifferentiation into a cardiomyocyte or a chondrocyte, wherein when the spheroid cell population is adipose stem cells, the adipose stem cells are incubated with 5-azacytidine. The contact is transdifferentiated into cardiomyocytes; and when the spheroid cell population is gingival stem cells, adipose stem cells, or placental stem cells, the cells are transdifferentiated into chondrocytes by contact with a transforming growth factor P3 (TGF-p3). • Another object of the present invention is to provide an adult stem cell having differentiation ability, which is produced by contacting a thin stem cell with a biocompatible polymer for an effective period of time, wherein The biocompatible polymer is chitinase, alginate, hyaluronic acid or any combination thereof; wherein the adult stem cells form a spheroid cell population, and the spheroid cell population expands and has a transdifferentiation to The ability of a chondrocyte or a cardiomyocyte; the adult stem cell is selected from the group consisting of a neural stem cell, a neural precursor cell, a fat stem cell, a gingival stem cell, and a placental stem cell, and the biocompatible polymer may further comprise a The cellulose binding domain-RGD ( CBD-RGD) of the SEQ ID ΝΟ:1 amino acid sequence can enhance the transdifferentiation ability of the spheroids of the spheroids; biocompatible The polymer may be a combination of chitosan and hyaluronic acid, and the ratio of chitosan to hyaluronic acid is about 3: 〇i. 4.425 (W/W); The combination of polyglycoside polysaccharide, and the ratio of chitosan to tremella polysaccharide is about 3. G. 4 to 3: 4.5 (w / w); can also be a combination of poly- and transparent f acid. Can be a nerve cell. σ, Still another object of the present invention is to provide a pharmaceutical composition comprising the aforementioned adult stem, field cell and freshly acceptable mineral. Newcomers are _ 彳 (such as water), filling (such as fun or Lin), adhesives (such as cellulose derivatives), thinners, disintegrants, absorption enhancers or sweet supplements, but not in the ship. The money of the money-making group can be produced according to the preparation method of the conventional stem cell therapeutic agent. The adult stem cells of the present invention are mixed with the body of the above-mentioned body to prepare a desired dosage form, and the dosage form may include a tablet, Powder, granules, capsules or other liquids (4) 'but not limited to this. The present invention can be used for clinical stem cell therapy to promote the regeneration of nerve cells and sputum cells, and the pharmaceutical composition containing secret cells can be used to avoid the difficulty and ethical consideration of embryonic stem cells. . The invention provides a marine animal stem cell, a pharmaceutical composition thereof, and a method for growing an adult stem cell, which is self-renewing and maintaining dryness by contact with a biocompatible polymer (stemness) ) The outstanding effect. In the following, the embodiment will be described in conjunction with the embodiment of the present invention, and the following examples are given to the present invention, and the present invention can be used without departing from the spirit and scope of the present invention. In the meantime, # may be modified and retouched, and the scope of protection of the present invention is defined by the scope of the appended claims. [Embodiment] The present invention makes Lai Tianling's phase heteropolymers a few impurities, alginic acid 201219572 • salt, hyaluronic acid or any combination thereof, wherein chitosan (coffee) is composed of aminopolysaccharides (amin〇_p〇lySaccharide) The composition of chitosan contains glyCosamino-glycan which is the same component as the extracellular matrix, so it is biocompatible and biodegradable. Alginate is a natural soft polymer which can promote the regeneration of axons. Alginate can be cross-linked with physiological ions under physiological conditions (at room temperature or body temperature, physiological pH and isotonic pressure); Calcium alginate is a biocompatible and non-immunogenic one, so it is a surface stability and two biocompatible materials. Hyaluronic acid (HA) is a material that promotes cell migration, proliferation, and interstitial secretion, and does not form a film for water solubility. In the present invention, it is combined with chitosan to form a film. Therefore, the present invention discloses a novel method for applying the aforementioned biocompatible polymer to adult stem cell culture. The adult stem cells used in the embodiments of the present invention are neural stem cells, neural precursor cells, adipose stem cells, gingival stem cells, and placental stem cells, which are brought into contact with a film formed by the aforementioned biocompatible polymer to cause aggregation of the adult stem cells. Forming a spheroid cell population' and treating the spheroid cell population of the present invention with trypsin is the detachment of the globular globule from the membrane. If only the stem cell aggregation phenomenon mentioned in the prior literature is used, treatment with trypsin is one. One cell separates. In each of the embodiments of the present invention, Examples 1 and 2 are two-dimensional (2D) films made of chitosan having different molecular weights and alginate having different Μ/G ratios, and the neural stem cells are cultured thereon. Compare with the morphology and differentiation results of other conventional materials (ex.TCPS). Example 3-5 is to culture adipose stem cells, gingival stem cells, foreskin fibroblasts or placenta by using chitosan of different molecular weight, modified chitosan, or chitosan membrane combined with hyaluronic acid. Stem cells were compared with the morphology of cultured other known materials (ex. TCPS) and differentiation results. Example 6 was to culture adipose stem cells and neural stem cells using chitosan-white fungus polysaccharide, and observe the morphology thereof. One embodiment of the present invention determines the self-renewal and dryness maintenance of neural stem cells and neural precursor cells on different 201219572 materials; the different materials are de-acetylated, surface potential And water contact angle analysis of each material produces a spherical cell population effect. Further confirm the cell type and differentiation effect of the spheroid cell population. 'F1B-fluorescent chloroplast (F1B-GFP) will show green fluorescence in undifferentiated neural stem cells, so the analysis of neural stem cell differentiation detects F1B. _ Fluorescent green protein performance, combined with immunofluorescence to detect neural stem cells labeled nestin and undeveloped neurons labeled β3-tubulin. In the present invention, a human neural stem cell, or "a neural precursor cell" refers to an undifferentiated middle phrenic nerve (CNS) pluripotent stem cell. "An adult stem cell with differentiation ability means that an adult stem cell is easily differentiated into a plurality of types of somatic cells. The treatment of adult stem cells according to the method of the present invention enables the adult stem cells to have the ability to self-renew and differentiate, and enhance Plasticity of adult stem cells. ''A set of effective time' refers to the period of time necessary for the biocompatible polymer to form a spheroid cell population with the adult stem cells of the present invention, for example, the neural stem cells are in contact with chitosan for a period of time. It aggregates to form a sphere. The effective time for contact with the biocompatible polymer may range from 4 hours to 14 Torr or more, preferably from 曰 to 4 本 in the present specification. "Pharmaceutically acceptable carrier" means a substance used to achieve or enhance the successful delivery of a pharmaceutical composition of the invention, and may be an excipient (such as water), a filler (such as sucrose or starch), a binder (such as a cellulose derivative). , diluent, disintegrant, absorption enhancer or sweetener, but not limited thereto. "About" or "approximately" generally means 20%, preferably 1%, preferably Within 5% of the range. Numerical values herein are approximate and may imply the meaning of "about" or "approximately" if not explicitly defined. "一" means one or more (ie, at least one) grammatical vocabulary. Example 201219572 - "One cell" refers to one cell or more than one cell. In the embodiment of the present invention, a method for measuring the surface potential of a biocompatible polymer was prepared, and a film of 4 cm M. 5 cm was prepared, and its surface potential (mV, Surface zeta potential) was measured by a surface potential analyzer (Beckman Coulter). The neural stem cell immunofluorescence staining method used in the embodiments of the present invention is for measuring the differentiation state of the neural stem cell sphere on the chitosan membrane, and the cell marker (nestin, labeled with neural stem cells; p3-tubulin, labeled with neurons) Immunofluorescence staining. It was fixed in 4% paraformaldehyde at different cycle times in cell culture. Rinse three times with acid buffer. 0.5% Triton X-100 (t-octylphenoxypolyethoxyethanol, Sigma, USA) was added for 50 minutes for 10 minutes to destroy the cell membrane and nuclear membrane. The sample was rinsed with phosphate buffer several times, and 20 μl of the grade antibody was added to the dark chamber for 37 hours at 37 ° C for 1 hour. To stain the cells, add 0.1% of 4,6-biindole-3-bendobate dihydrochloride (4,6-out 3111丨£1丨11〇-2-卩11611>4丨1^ 〇16 dihydrochloride) (purchased from DAPI, Sigma, D9542, USA) 200 pl in the dark room for 30 minutes. Finally, the sample was rinsed and placed on a microscope slide and sealed with glycerin gel. The sample was observed under an upright fluorescent microscope (purchased from Nikon, 80i). The dilution ratio of the primary antibody was as follows: anti-nestin (R-20) goat-poly IgG antibody (purchased from Santa Cruz, USA; 1:1000) and anti-p3-tublin mouse-mono IgG (purchased from Santa Cruz, USA; :1000). The dilution ratio of the two reference antibodies was FITC-conjugated goat anti-mouse IgG (diluted with horse serum albumin; 1:100; purchased from Chemicon, CA) and goat anti-rabbit IgG-horeseradish peroxidase (HRP) (purchased from Santa Cruz, USA; 1:100).

The analysis of the myocardial differentiation ability of the examples of the present invention is performed by using a Orthogonal Fluorescence Microscope (NIKON). The sample is rinsed with a buffer buffer. Adding 4% paraformaldehyde for 20 minutes at room temperature, and washing 3 times with phosphate buffer. Each time 1 minute, add 3% bsa / citrate buffer for 30 minutes at room temperature, aspirate, add 3% BSA prepared primary antibody, the dilution ratio is the following table 'sealed in parafilm and placed in 4t refrigerator Overnight; the next day, the primary antibody was aspirated and rinsed three times with phosphate buffer (the following steps were protected from light), and the primary antibody prepared by adding 3% BSA 201219572 was covered with ITO and placed in π. . The incubator was incubated for 2 hours; the secondary antibody was aspirated and washed three times with the discate buffer, and the DApi dye of 3% BSA was added for 3 minutes. After sucking out the salt buffer for three times, the acid buffer was added. The material was taken out from the 24-well culture plate using a needle and an inflammatory tract, and placed on a glass slide and observed by a Orthodox light microscope (purchased from NIKON). The cell surface self-characteristic of the cell surface analyzed by the present invention is a flow cytometry method using a flow cytometer (purchased in & fat, that is, said). First, remove the cells from the culture plate, take 5x1〇5 acid salt solution, remove 1~77 knots, repeat the second time, collect the cells, add ι〇〇μ1 phosphate buffer to reconstitute the cells. After adding '1 μμl primary antibody to the cell solution, the reaction was 3 〇~6 〇 minutes, and the acid buffer was rinsed three times, and 10 μl of the secondary antibody was added in the dark for 3 〇~6 〇 minutes to cut off the salt buffer. The liquid was washed by centrifugation at 1000 rpm for 5 minutes, repeated three times to remove unreacted antibody, and finally added to 5 μl of phosphate buffer to reconstitute the cells, and then transferred to a flow cytometer for analysis. The above analysis results are expressed as mean ± standard deviation, and t-test is used for statistical analysis. When the P value is less than 0.05, there is a statistically significant difference. Hereinafter, the present invention will be described in detail in conjunction with various examples. These examples are provided for illustrative purposes only, and are not intended to limit the invention to these examples. In addition, although it is exemplified in the examples that neural stem cells, neural precursor cells, and adipose stem cells in adult stem cells are isolated from mice, dental silver stem cells and placental stem cells are isolated from humans, but the present invention is not limited thereto but may be It is applied to the isolation of mammals such as humans, rats, mice and monkeys. Example 1 Cultivation of adult neural stem cells/neural precursor cells 1.1 chitosan thinner by two molecular weight chitosan films 201219572 Using two chitosan-1 (purchased from Fluka, USA) and several Glycan-2 (purchased from Sigma, USA) was prepared with 1% chitosan at a concentration of 5 g of chitosan powder and dissolved in 49.5 ml of water at room temperature for half an hour. 5 5 ml of acetic acid and lightly mix at room temperature for 12 hours. The filter was used to filter impurities on the next day as a 1% chitosan solution, wherein the molecular weight of chitosan-1 was about 510 kDa. The molecular weight of chitosan-2 was about 400 kDa. The resulting solution was filtered through a mesh of ΙΟΟμηι and applied to a glass coverslip (each 15 mm diameter slide coated with ΙΟΟμΙ solution) and allowed to air dry for two days. The chitosan membrane was immersed in a 〇5Ν NaOH solution for 5 minutes, and then rinsed with distilled deionized water over a large area until the pH of the water near the film reached a neutral state. Finally, the chitosan film was sufficiently dried. 1.2 Extraction and culture of rat brain neural stem cells The neural stem cells were isolated from the brain part of the gene-transforming mouse containing the promoter F1B-fluorescent green protein (F1B-GFP) gene. The two-month-old mouse was smashed throughout the brain and lightly ground into smaller pieces to obtain a single cell suspension. The minced cells were seeded in 35-mm cultured blood without supplemental matrix and adhesion factor in DMEM/F-12 medium (Dulbecco, s modified Eagle, s medium and Ham's F-12, constructed in Giboco, USA) 1 :1 The culture medium contains feta bovine serum (FBS) (purchased from Gibco, USA). After two days, nearly 10-20 cells per cell were subjected to cell division. After trypsin treatment, concentrate them and continue to culture for 2-3 days. A stable cell line was further selected with 200 pg/ml of glycoside antibiotic geneticin (G4l8, purchased from Gibco). The GFP-positive mouse brain cells were repeatedly purified by flow cytometry (FACS Aria, purchased from BD Biosciences) to a purity of 95% or more. 1.3 Morphological analysis and differentiation of neural stem cells/neural precursor cells into chitosan membranes Before the neural stem cells were implanted into the culture material, the chitosan membrane on the glass coverslip was immersed in 75% ethanol, and then Phosphate buffered saline (PBS), washed, and placed in each well of a 24-well culture plate. Neural stem cells were implanted at a density of 5 x 1 〇 4 cells per well. Add 10% fetal bovine clear (purchased from Gibco, USA), 12 201219572 40 (^g/ml glycobiotic antibiotic (G418) and streptomycin-penicillin (lOOU/ml) as culture medium in DMEM/F-12 medium. Incubate the culture medium twice at a temperature of 37 C ' 5 ° / ° CO;? humidification incubator; and add tissue culture polystyrene surface (TCPS) without growth factor Gelatin was used as a control group, and the results are shown in the first and second figures.

The first panel shows the morphology of neural stem cells cultured on TCPS and gelatin for three days. No cell aggregation was observed on TCPS and gelatin, and the cells were attached to TCpS and Ming. On the other hand, the cells cultured on the chitosan film began to aggregate on day 丨_2, and eventually aggregated into a globular shape. At the beginning of 24 hours, most of the cells were round in shape; most cells aggregated to form spheres within 48 hours. . The number and size of the spheres on the chitosan _ 丨 大致 are roughly larger than those of the diced _2 (as shown in the second figure). In order to recognize that the sphere produced by the embodiment of the present invention is a three-dimensional space of a three-dimensional sphere instead of just gathering, the third-scanned electron microscopic image, the sphere_view can clearly show the third-degree structure, and the silk display The size of the county is increased, up to about the lion. In addition, the fourth to sixth graphs show the degree of differentiation of neural stem cells. The light intensity of the neural stem cells' has a green fluorescing color, which indicates that the green county began to subside after 15 days, but it is still clear (as shown in the fourth figure 6d). At 15 days, some of the cells began to move out of the sphere, so the size of the neural stems and the size of the corpuscles, the secret (4) cells did not show any, 'black fluorescent. Fluorescence elimination of the sphere after 5 days of sputum In addition to showing the removal of cells, the cells in the sphere have also differentiated. In the immunofluorescence staining test ±, the labeled protein of the god transfected cell cultured in the Cai Ju riding · i = the table shown in the fifth figure (four) in) and the sixth figure climbed her.). It was found that the sphere was in the sky. The strong U showed no nestm positive. The cells showing the sphere maintained the state of the squamous cells. The secret showed a slight decrease when the culture time increased. On the other hand, the performance on the entire [S] f film is low. This result shows that neural stem cells cultured on chitosan membranes can be used to maintain these cells' self-renewal ability and dryness (IV) or 13 201219572 for purification of neural stem cells from cell populations. 1.4 Deacetylation, surface potential, and water contact angle values of chitosan and tissue culture plates (TCPS) are listed in Table 1. Table 1 Comparison of characteristics of different chitosan (chitosan-1 (CS-i) and chitosan-2 (cs_2)) and tissue culture plates (TCPS) (8) de-acetylation degree (b) surface potential , and (c) surface water contact angle. Material to B-degree surface potential (mV) Surface water contact angle (°) CS-1 77.7% -3·47±0.21 87.0 Soil 2_08 CS-2 86.0% -1.97± 0.15 74.2±3.67 TCPS NA 64.9 Soil 4.57 Chitin The deacetylation degree of glycan-1 and chitosan-2 was about 777% and 860%, respectively. The higher the degree of chitosan deacetylation, the lower the surface water contact angle, the higher the hydrophilicity, ie the hydrophilicity.

Chitosan·2 is larger than several dicans (4), and both surfaces are more hydrophilic than TCPS, because cells are easier to attach in a matrix with high hydrophilicity, and neural stem cells are attached to TCPS. The sugar is coming up well. Moreover, the attachment of neural stem cells to chitosan

The ratio is better than that of the chitosan _1, and the spheroid formation time delay on the diced vinegar. The degree of deacetylation of chitosan is increased, and the hydrogen bond in the amino group and the molecule is increased, which may cause surface hardening and strong crystallinity, which is also consistent with our results. The surface potentials of chitin and chitosan-2 are both negative 'but the value is close to neutral, and the surface is more negative. In the results of this example, 'the neural stem cells are not very good. And the poly (10) situation is presented. The surface hardness of the chitosan-1 (indentation depth is about 5〇nm) is similar to the inside (indentation depth. Ah. On the other hand, the surface hardness of chitosan-2 is higher than the internal and higher than the chitin Sugar], chitin f-glucose _2 is coarser and more densely crystallized on the surface. Due to the harder and more crystalline surface, the neural stem cells may be attached to a few squares. Ding Juyiyiyi is better and the formed sphere is smaller. 14 201219572 Example 2 Culture of adult neural stem cells/nerve precursor cells, adipose stem cells 2.1 alginate film with two alginate films. Preparation of two alginates Films were prepared, including alginate A#1 (M/G ratio=0.5 'molecular weight 110,000, purchased from Hayashi, Japan) and lower than the β-D-mannuronic acid/a-galuronic acid ratio. Sorghum/G ratio alginate A#2 (M/G ratio=1.59, molecular weight 12,000 - 80,000, Sigma, USA). Alginate A#1 and A#2 were first dissolved in 9 mg/ml sodium sulphate, respectively. The solution was brought to a solution concentration of 40 mg/ml. The pH value of the solution was adjusted to 7, and after autoclaving (125 ° C and 1.5 atm) for 30 minutes, 2-[4-(2-hydroxyethyl) 0 was added. 2-[4-(2-hydroxyethyl)piperazin_ 1 -yl] ethanesulfonic acid 'purchased from HEPES, Invitrogen) maintains the same pH value as the human body. Both alginate can be made into a smooth or wrinkled film. The alginate film is added with 400 μl alginate per well to a 24-well culture plate (purchased from Coming, USA) and air-dried for 60 minutes. Then, 102 mM calcium chloride was slowly added dropwise along the edge of the well. 2.2 Extraction and culture of rat adipose stem cells (rADASs) Adipose tissue was obtained subcutaneously from rats (Spraque-Dawley rate, SD rate), using acid buffering Wash the liquid, cut into small pieces, centrifuge for the first time (1500 rpm, 5 mins). Pour the upper layer of fat into a new centrifuge tube and add a little acid buffer, then cut the small pieces as much as possible for a second centrifugation. (1500 rpm, 5 mins). Take the upper layer of fat and prepare the digestive juice: 1 mg/mi collagen type I (collagenase type I 'purchased in Sigma) / HBSS solution (HBSS buffer solution is prepared as shown in Table 4). 4HBSS buffer solution component concentration (g/1) NaHC03 0.35 [S1 15 201219572 KC1 0.4 KH2P 4 0.06 NaCl 8 Na2HP04 * 7H2〇 0.09 CaCl2 0.14 MgCl2 · 6H2〇 0.1 MgS04 * 7H2〇 0.098

Mix the fat with the above digestive juices and place in a cell culture incubator (100 rpm, 1 hour). The third centrifugation (15 rpm, 10 minutes). The cell fluid was filtered using a 70 m cell sieve (purchased from Falc〇n, BD Bioscience) for a fourth centrifugation (1500 rpm, 5 mins). The above oil layer was aspirated, and then the culture solution was added and placed in a 75T-flask dish (purchased from Falcori, BD Bioscience). The cells were attached to the culture material every other day. The supernatant was withdrawn and a new culture solution was added. After the cells were cultured to 8 minutes, the cells were subcultured with 0.25% trypsin/ethylenediaminetetraacetic acid (purchased from Gibco) at a cell density of 50,000 cells/T-flask. 2.3 Morphological analysis and differentiation of neural stem cells/neural precursor cells into alginate membranes The condition of mouse brain neural stem cells/neural precursor cell extraction was as described in Example 1. The obtained cells were cultured on a 2D alginate membrane. The NSPCs were implanted in a 24-well culture plate containing cyanobacterial gel (2,5χ104 cells were implanted in 2 ml of culture medium). Replace half of the culture medium every other day. After 12-24 hours, NSPCs were found to accumulate on the alginate membrane. After three days, the cell clusters on the alginate membrane were found to have no uniform size (Fig. 7). In the expression of differentiation, NSPCs can express the genes of nerve cells and glial cells when they are formed on 2D alginate membranes. However, NSPCs which are cultured on TCPS and exhibit fiber-like morphology cannot express nerve cells and nerves. Glue cell genes (see Figure 8). [S] 16 201219572 Fat dry fine 神神', dry blister culture μ day The size of the ball on A#1 and A#2 is 55 bodies formed when the table is not 14 days old, and the sphere size is 28 days. Time is similar): Number of implants: 5χ104/well Neural stem cell Adipose-derived stem cells in the ΑΜΛ 否 否 聚 是 是 是 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 138 138 138 138 27.94 A#2 upper sphere diameter (mm) 121.10 soil 36.90 141.25 ± 31.84 Example 3

Preparation of two kinds of molecular weight chitosan films by using two kinds of molecular weights, τ and _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ The system uses a molecular weight different from the above-mentioned examples of chitosan _3 (described in Kiotec), molecular weight of about 170kDa, and chitosan_1 (purchased from Fkika), molecular weight of about 510 kDa #取〇. 5 g of chitin polydomain powder was dissolved in 49 5mi of secondary water, stirred at room temperature for half an hour and then added with 0.5 m of acetic acid to continue for one day. It is a 1% chitosan solution. Table 3 Deacetylation of two kinds of chitinases and molecular weight Deacetylation degree molecular weight chitosan-3 97% 170kDa chitosan-1 77.7% 510kDa 1% chitosan solution 2〇〇μ1 Dilute into a 1.5cm round slide and dry it naturally for 1-2 days at room temperature. After confirming the film dry, cover the slide with 〇·5Ν NaOH for 5 minutes, then wash it with secondary water for 3 times, then continue. Soak in the secondary water to the next day; the next day, the second water is poured off. After the secondary water remaining naturally at room temperature, it is chitosan _3 and chitosan·i > . In addition, a growth factor-free polystyrene tissue culture plate (tjssue cuIture [y 17 201219572 polystyrene surface, TCPS) was used as one of the control groups. The other control group is a polyvinyl alcohol (PVA) film. Because the literature indicates that the tooth germ cells are on the pVA membrane, the cells can aggregate into cell spheres to help the hard bone differentiation; the preparation is to weigh 2.5 g of polyvinyl alcohol (Poly vinyl). Alcoh〇1, PVA 'purchased from sigma, H〇t water s〇lubk) powder dissolved in 50ml of 1-personal water, after dissolving it by warming and autoclaving, the temperature is reduced to 5% at room temperature. . 150 μl of a 5 〇 / 〇 polyvinyl alcohol solution was evenly dropped into a 1.5 cm circular slide, and placed in a 60 ° C oven for one day to form a polyvinyl alcohol film. 3.2 Preparation of cellulose-binding functional region--GRD-attached sequence (CBD-RGD) with a cellulose-binding functional region _RGD attachment sequence (CBD_RGD) modified by chitosan-1 (CBD-RGD) for Professor Chen Zhenhan of Zhongxing University The gene is the gene of the cellulolytic enzyme cellulobiohydrolase I (CBHI) of the fungus Tric〇derma konigii. At the N-terminus of CBHI, there is a 36 amino acid-sized cellulose binding domain (CBD). The fifth amino acid modified by the polymerization enzyme chain reaction method was changed from the original tyr〇Sin to trypt〇. Phan, in order to increase the affinity of CBD for cellulose, and add a PT linker and a rgd sequence gene at the 3th end of the CBD gene. RGD is the smallest functional site in cell adhesion factor (Fibronectin) that promotes cell attachment. The cellulose-binding functional region _RGD attachment sequence (CBD-RGD) has an amino acid sequence as SEQ ID NO: 1 due to CBD-RGD. There is a very high molecular 2: (25 KDa), using only Coulomb gravity, a bifunctional protein that promotes cell attachment to cellulose. In the embodiment of the present invention, the film formed by chitosan-1 is combined with CBD_RGD, and the film is soaked in 75% alcohol for 3 hours, and then washed twice with phosphate buffer solution, and the film of chitosan-1 is placed for half an hour. Dry; uniformly deposit CBD-RGDGpg^Oy) on the film of chitosan 丨 (4pgCBD-RGDA.77 cm2) ' After 1-2 hours of CBD-RGD drying, wash once with phosphate buffer A film of chitosan-1 modified with CBD-RGD. 201219572 3.3 Extraction and culture of rat adipose stem cells (rADASs) The extraction and culture of rat adipose stem cells (rADASs) were as in Example 2. 3.4 Amorphous analysis of adipose-derived stem cells in three chitosan films and differentiation into cardiomyocytes This example is divided into two parts. The first part is to compare tissue culture plates (T), polyvinyl alcohol (P), chitosan. Sugar 3 (CS3) and chitosan 1 (CS1). The T, P, CS3 and CS1 films were soaked in 75% alcohol for 3 hours, rinsed with the acid buffer, and placed in a 24-well culture plate; rat adipose-derived stem cells were prepared using 0.25% trypsin (purchased from Gibco). (Laboratory cell generation number: 2-6 generations) Put down 'collected in the culture solution, put 5xi〇4 cell solution in each well, shake the plate, evenly disperse the cells, and then put them into the cell culture incubator. . 5-azacytidine (5-aza) is a compound that demethylates DNA. According to research, 'demethylation by 5-aza irregularly' induces cells to become cardiomyocytes, so it is replaced with lOMSaza on the fourth day. The culture medium of (5-azacytidine, Sigma) was divided into the group with 5-aza (w 5-aza) and 5-aza (w/o 5-aza), so w/〇5 In the group of -aza, the original culture solution is still added. On the fifth day, the culture solution was aspirated, replaced with a 5-aza-free medium, and cultured for 1 week (total u days) using reverse transcription polymerase chain reaction (RT-PCR). The results of comparing the ability of myocardial differentiation are shown in Table 5 and ninth and tenth. The second part is to compare the myocardial differentiation monthly b force of T, CS1 and CS1 combined with CBD-RGD (CS1R). The first part was consistent with the experimental conditions of the sputum. The experimental group was changed to τ, CS1, and CS1R'. The ability to differentiate myocardium was analyzed by RT-PCR and immunofluorescence staining. The results are shown in Table 5 and Figure IX and Figure 10. Table 5 Cell size of adipose stem cells (rADASs) formed on different materials Day 1 Day 3 Day 11 [S] 19 201219572

Ball size (μιη) 5-aza - - + TXXXXP 350 soil 50 400 士 100 XX CS3 35±15 70±60 70±60 30±20 CS1 25 士15 55 士45 55±45 55±45 CS1R X 175± 90 140±65 160±75 T: TCPS; P: PVA; CS3: Chitosanl; CSl: Chitosanl; CS1R: Chitosanl+CBD-RGD. No added 5-aza; +: Add 5-aza; X: Cell-free sphere.

It can be seen from Table 5, ninth and tenth that the rat adipose stem cells cultured in the tissue culture plate cannot be globulated. 'The rat adipose stem cells on the polyethylene glycol will aggregate the most on the third day', but then whether or not there is When 5-aza was added, the cells all ran out of the aggregated cell sphere, so on the 11th day, the morphology of the cells was similar to that of the cultured tissue culture plate. In CS3, on the first day, cells can be found to gradually aggregate into cell spheres with a size of about 35μηι. On the third day, a single cell sphere can be found, which is larger than the first day, about 70μηι; It was found that the larger cell ball gradually drifted away, and on the 11th day, the CS3 (1) cell was smaller than the CS3 (+) cell. In CS1, the cells can be found to gradually aggregate into cell spheres on the first day. The size of the sphere is about 25μπι; on the third day, a single cell sphere can be found, which is larger than the first day, about 55μηι; although CS1 is formed. The ball is smaller than the CS3, but the ball is not easy to float; CSI (-) and CS1 (+) on the 11th day are about 55μηι. In CS1R, on the first day, the cells were found to be unsphered. 'The cells spread out on the material' are similar to TCPS, but there is a tendency to gradually aggregate. On the third day, 'a large cell ball can be found' with a maximum of 250μτη. The balls formed on the CS1R are large but 'nearly drift away; on the 11th day, the cell sphere formed by CS1R is larger than CS3 and CS1. On the ηth day, the balls gathered by the cs3, CS1, and 201219572 CS1R cells will be found. Some cells will run out of the cell ball. The group without the rhyme will be out of the cell group with the 5-aza group. The trend is even stronger. This is the first. The experimental result of the sickle is the best for the CS1 film. The bigger the cell sphere, the better the differentiation effect, but the hidden ball will go out and stick back to the material and (3) can't keep the big ball' and leave the remaining cell ball. It is smaller than CS1, so the differentiation effect is worse than CS1. Comparing the results of the first part, the expression of adipose-derived stem cells into cardiomyocytes in the material PVA, CS3, CS1 was ±, and the genes of the fine transcription factors showed that CS1 (+) and CS1 (1) were better than other groups. The base of late myocardial protein has the best performance due to α-MHC and CS1 (10). Therefore, the second part of the experiment is based on CS1R and CS1 of cbd_ coffee modification CS1. In the second part of the experiment, the cells will gradually aggregate into the ball on the third day. The size of the cell sphere is much larger than that of CS3 and CS1, but it is not easy to drift away from the shape of the large ball formed by (3). It can be seen from the experiment that (3) R(1) and CS1R(1), when differentiated to the uth day, the size of the sphere is about 155 post, but the cslR(+) with 5_aza added has better differentiation ability and the cell oligo from the cell ball; RT-PCR analysis verified that (3) is in GATM, and

Tr〇_n ϊ all show a better tendency than the Cs) differentiation, so there is a plus 5. Correction increases the differentiation of silk: the size of the ball and the formation of the ball also the effect of mosquito differentiation.

Csir>csi >tcps. > J can also be found by immunostaining. 'GATM and all are only in the cell sphere. 'Single-cells have almost no fluorescence, and cells that run out of the cell sphere have no performance. The gene expression of CBD-RGD modified CS4 can be seen in the eleventh figure. The early gene (10) and the late gene a-MHC Troponin I have the best differentiation ability (CSR(+), followed by c), so add 5-aza. It is helpful for myocardial differentiation. It can also be seen that cbdrgd modification (3) does have better differentiation ability than unmodified CS1. Therefore, adipose-derived stem cells have differentiation effects on both CS1 and CS1R membranes in both gene expression and immunochemical staining. [S] 21 201219572 In addition, CBD-RGD modified biopolymers include, but are not limited to, chitosan. The biocompatible polymers mentioned in the present invention can be modified with CBD_RGD to promote adult stem cells into globular shape. Example 4: Culture of dental stem cells and foreskin fibroblasts with a chitosan membrane 4.1 Chitosan membrane preparation Using a moderate viscosity of _], a deacetylation degree of 777%, an average molecular weight of about 5 m diced poly _ fresh (four) dissolved in 1% acetic acid (purchased in sh_), filtered • coated 15 coffee cover slips (purchased in coffee pottery. Chitosan film dried at room temperature, 〇.5 The dihydric hydroxide also neutralizes the acidity of the liquid. Then the second steaming water washes the sodium hydroxide on the chitosan film. Then immerse all the diced polydamine in the Μ% alcohol and the UV In order to make the silk, the last one is washed with phosphorus leaching buffer to wash off the remaining 75% of the alcohol. For the comparison of chitosan results, another 86% of the deacetylation, molecular spoon 40〇k〇a The chitosan (purchased _) was prepared in the same manner as described above. In the present example, 4 chitosan is used to represent chitosan, and chitosan is used to represent chitosan (Sigma). _ 4.2 People's Sakisaki cell and human surface noodles and fresh-keeping and cultivation: It is obtained from healthy adult teeth. The human tooth paste is secreted in the minimum necessary alpha culture solution (α-jyjgM, purchased from (10) such as Likou secret fetal bovine serum and 2% antibiotic antifungal drugs (2 reads _ penicillin (basal), 2 μ_ streptomycin (basal) and $ n〇g / ml amphotericin) (purchased in Gibb〇 The cell culture flask (c〇ming) is cultured in a pit, "2; I. In the box. Change the basic culture solution once every two days. In human gingival stem cells, cover up to 60/〇 to 7〇〇/0 In the case of 〇25〇/〇 蛋白酶 / 乙 乙 乙 乙 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 Human foreskin fibroblasts, [s] 22 201219572 HSF). Human foreskin fibroblasts were supplemented with 10% fetal bovine serum (purchased from Gibco) in 200 mg of high-sugar source DMEM (purchased from Gibco), purchased from Gibco ), 1% MEM non-essential amino acid 100X solution (purchased in Sigma), and 1% penicillin/streptomycin (5000 units/mL penicillin (basal) and 50 (% g/mL streptomycin (basal)) cultured in cells In the culture flask, HSF was cultured in a 37 ° C '5% C02 incubator, and the basic medium was changed once every two days. When the human foreskin fibroblasts covered 80% to 90%, the value was 0. .25% trypsin/ethylenediaminetetraacetic acid solution (purchased from Gibco). This example uses the 28th generation of foreskin fibroblasts (HSF). 4.3 Morphological analysis of human gingival stem cells on chitosan film In the case of differentiation into chondrocytes, human gingival stem cells (4 >< 104 cells/ml) were implanted into each of the chitosan membranes placed in a 24-well tissue culture plate, and the culture solution was changed once every two days. Three days after cell implantation, chondrocyte-inducing medium was added to induce cartilage formation of human gingival stem cells on both chitosan membranes and TCPS. Chondrocyte-inducing medium contains DMEM (purchased from Gibco) containing high sugar source, TGF_p3 (Cyt〇Lab/pepr〇Tech, purchased from Rehovot, Israel), dex 7 Μ dexamethasome (purchased in Sigma) ), 50 pg/ml of 1-ascorbic acid-2- tartaric acid (purchased in Sigma), L-valine (purchased from Sigma) of Qingca 1 and ITS-premix of lOpg/ml 1 (purchased in Sigma), 1% fetal calf serum (purchased from Gibco), and 2/〇 antibiotic-antimycotic. Human gingival stem cells were cultured in a medium containing the inducing medium for 7 days and 14 days 'every two days. The morphology of human dental silver stem cells cultured on chitosan·1, chitosan-2 and TCPS films for three days after implantation was shown in Fig. 12. 4.4 Morphological analysis and differentiation of human foreskin fibroblasts on chitosan membranes. Human foreskin fibroblasts under the same conditions were used as a control group, human foreskin fibrils [S1 23 201219572 «Cell (〇 Cells/ml) implanted in each of the 24-well tissue culture plates and the TCPS' series with basic growth culture and cartilage sylvestris transfer medium, change the culture solution every two days - times, human The morphology of the foreskin fibroblasts cultured on the chitosan-1 film for three days after implantation is shown in Fig. 12. Referring to Fig. 12, the human foreskin fiber cells cultured on the chitosan·i film are free of spheroid formation (Fig. 12A). However, it has been cultivated in several clusters of cells to form (12th Figure 8), which clearly shows that not all of the chitosan-1 binding membranes will form spheres, because in this embodiment, they do not form themselves. A person with a spheroid breaks a stem cell but can form a sphere on a few scorpions, while a human foreskin» _ mother is not a county. The first busy graph D shows the situation where no one sphere is formed on the Tcps. In addition, when human gingival stem cells are cultured in chitosan films of different molecular weights, the ratio of 'forming spheres is different; in the twelfth map (b, ^, three days after implantation of human gingival stem cells, cultured in a few squares Sugar·! The film formed by the film is much more than that on the chitosan-2 film. This means that chitosan] may induce the formation of human gingival stem cell spheres more easily than the chitosan J. Thirteen® 'detailed human broken cell culture in chitin

On the d, live cells were imaged at 12, Μ, 16, 18, 2G '22 and 24 hours in the same range. The human tooth-transplanted cells cultured on several T-glycans 1 began to form spheres at η hours after implantation, and were initially taken after 24 hours. When the human is implanted, the human tooth-stained cells are attached to the form of fibrils on the poly-_-1. However, as the culture time increases, more and more human gingival stem cells become globular, and there are fewer and fewer human gingival stem cells. In addition, refer to Figure 14 for humans cultured in chitosan and Tcps, with or without cartilage-directed culture solution for G to 3 days after implantation, plus human chondrocyte culture solution after Q, Bu 2, 7 and 14 days. Gingival stem cell morphology. Human dental stem cells are in the form of fibrils throughout the culture process. As cell culture time increases, human gingival stem cells form relatively large cell spheres of m 24 201219572. In addition, many human gingival stem cells appear to migrate out of the cell sphere, forming cell clusters and cell populations. When the number of human gingival stem cell spheres on the chitosan film added to the basic growth medium increases with time, it appears that there is a self-spherical secretion about 14 days after cartilage induction (or 17 days after implantation). . On the other hand, human gingival stem cells cultured in tcps did not form a sphere at all. From the foregoing results, it is known that not all cells cultured in chitosan form spheres, such as human foreskin fibroblasts, nor all types of chitosan (eg, chitosan-1 and chitosan-2). ) have the same effect of inducing cells into a ball. Human gingival stem cells cultured on chitosan membranes have higher cartilage differentiation potential than human dental stem cells cultured in TCPS. It is particularly important that the human chondrocyte differentiation potential of the human gingival stem cells cultured without the cartilage-induced chitosan thin film is higher than that of the human gingival stem cells cultured on the chondrocyte-induced TCPS. In other words, the chondrocyte differentiation potential of human gingival stem cells can be enhanced by simply culturing on the chitosan film of the present invention. In determining the pluripotency and dryness of the human gingival stem cell sphere of the present invention, the self-renewal process of undifferentiated stem cells is determined by two dry markers, NANOG and Oct 4, so 'NAN0G and Oct 4 are often used as differences. Markers of differentiated cells, 〇ct 4 are specifically used to prevent cell differentiation. In addition, neural ridge cells are a group of cells that are transiently pluripotent and migratory. Specific two-segment neural ridges SLUG and SoxlO determine the specification and differentiation of neural ridges. In this example, we found that the SLUG, Sox 10, NANOG, and Oct 4 genes exhibited higher levels of human gingival stem cells cultured in chitosan films than human gingival stem cells cultured in TCPS. This means that the spheres of human gingival stem cells formed only on chitosan have higher neurological gene expression and dry gene expression than the general fiber-like morphology. In other words, human gingival stem cells that form spheres are associated with transient pluripotent, migratory, and mostly undifferentiated cells. In addition, there appears to be an increase in pluripotency, migration capacity and dryness in human gingival stem cell spheres. This can be confirmed by the SLUG, Sox 10, NANOG and Oct 4 gene expression on the chitosan film by human gingival stem cells 3 days after implantation. Thus, the formation of spheres helps maintain the pluripotency, migration and dryness of human gingival stem cells. In addition, it has been observed from living cell imaging that the above human dental silver stem cells are spherical on chitosan, and the spherical shape is formed by continuous cell-matrix interaction of human gingival stem cells and cell-cell interaction resulting in dynamic movement. The dynamic state of the sphere also confirms the ability of the neural crest gene to indicate the migration of human gingival stem cells in the sphere. In this study, it was observed that in all forms of human gingival stem cell formation, human gingival stem cells that form spheroids continue to enter and exit the sphere. When human gingival stem cells migrate out of the spheroid, human gingival stem cells will emanate and attach to chitosan in a fibrillar form. Therefore, the human-shaped stem cells in the form of spheres still maintain pluripotency and dryness, which are not found in the fiber-like human gingival Φ stem cells. In addition, bone marrow mesenchymal stem cells (b〇ne marr〇w_derived MSCs) are cultured on the chitosan-1 membrane and also form a sphere (see Figure 15). Example 5 The sample of the stomach 1 powder has a molecular weight of 51 GkD in the form of a small amount of polystyrene and a few butaric acid, a placenta, a stem cell, a chitosan, a chitosan, and a hyaluronic acid (HA) film.黯a) mixed with 1% acetic acid to obtain

Second, after the solvent is evaporated, it is a chitosan film. Sodium hydroxide was added to each well (the horse was shaken for four minutes, and then washed several times with the salt buffer. The sodium of the city was agglomerated, and the hyaluronic acid was positively charged. CH0.5 and CH2.5 ▲ of 4 Ding Shi through the day _» by C, CH (U, acid buffer and transparent f acid amount of a few miscellaneous · through the acid membrane to phosphorus first 5 - people to lack of unbonded „ 后 post-wei. (4) ΚΗ有效5 [S] 26 The effective absorption rates of 201219572 and CH2.5 were 94%, 95% and 89%, respectively. The total amount of hyaluronic acid absorbed increased with the increase of the amount of hyaluronic acid added initially. Growth factor polystyrene surface (TCPS) was used as a control group. 5.2 Extraction and culture of human placental stem cells (hPDMSC) The placenta donated by the healthy mother during the late pregnancy (38-40 weeks). The phosphate buffer was washed several times and then broken up and treated with 25% trypsin at 37t for about 1 minute. After shredding and enzyme treatment, the homogenate was cultured in DMEM_LG (Dulbecc〇, s Modified Eagle Medium-low glUC0se) (purchased from Gibe〇) complete medium, add 1〇0/fetal bovine serum (purchased from Gibco), 1〇 Mg/l penicillin·streptomycin and 1〇mg/〖L_Amidoxime (constructed in Tedia^ cells cultured in 37t water-saturated air, and state c〇2. The culture medium was changed twice a week. 5.3 Extraction and culture of adipose stem cells (hADAS) Adipose stem cells can be obtained from adipose tissue. The cells are extracted by enzymatic extraction from adipose tissue. The adipose tissue is first broken into several pieces and then 200 U/ml type I collagenase (purchased) In Sigma-Aldrich) phosphate buffer at 37. (: Mix for 30 minutes. Shame culture in DMEM-LG (Dulbecco's Modified Eagle Medium-low glucose) (purchased from Gibco) in the whole medium 'Add 10% (v/v) fetal bovine serum (purchased from Gibco), 10 mg/1 penicillin-streptomycin. Incubator maintained at 37 ° C / 5 ° / 〇 C02. Change medium twice a week. 5.4 placental stem cells and fat Morphological analysis of stem cells on chitosan and chitosan-hyaluronic acid film and differentiation into chondrocytes. Adipose stem cells and placental stem cells (3χ104 cells) were implanted into each chitosan containing basic culture solution. After three days of membrane culture, the culture medium was replaced with chondrocyte-inducing culture medium. The nutrient solution is DMEM-LG, containing 10 ng/ml TGF-p3 (purchased from CytoLab/Peprotech, Rehovot, Israel), 0.1 μΜ dexamethasome, 50 pg/ml L-ascobate-2-acid, 40 pg/ml L- Proline (Sigma), 1% insulin-transferrin-selenium supplement (iTS+premix) 27 201219572 ιοοχ (purchased from sigma) and m penicillin streptomycin. The induction medium was changed twice a week. The other-group (planted for 3 days) is still in the domain of the Wei riding control group. The adipose stem cells were formed on a chitin polyhedral-hyaluronic acid film for one day, and a three-dimensional sphere formed by aggregation of the fat stem cells was formed. As the osmotic acid on the chitosan increases, the resulting sphere becomes more pronounced' and the number and size are larger. The morphology of adipose stem cells and placental stem cells cultured on tcps, chitosan, chitosan, and transparent membranes on day 3 is as shown in Fig. 16. After 3 days of culture, the adipose stem cells and placental stem cells cultured in a few butts and a few hyaluronic acid membranes were mixed. The average cell size of the different cells on these membranes is listed in Table 6.

*Each average is the average of at least 2 sphere diameters

Table 6 Average size of spheres formed by different types of placental stem cells and adipose stem cells on different membranes TCPS C CH0.1 CH0.5 CH2.5 No spheres produced hADAS into 22±5μιη 30±15μηι 35±15μιη 40±18μιη No spheres produced hPDMC into 20 ±7μηι 33±12μηι 45±19μηι 54±25μηη The expression of the dry marker genes (Oct4, Sox2 and Nanog) of the adipose stem cells and placental stem cells of the present invention during the culture was determined. mRNA expression of Oct4, Sox2 and Nanog in adipose stem cells and placental stem cells in basic culture was analyzed by real-time RT-PCR. In Figure 17, it is shown that adipose stem cells are cultured in different materials and TCPS controls of 〇ct4, 8〇χ2 and

Nanog mRNA showed quantitative results. In chitosan and chitosan-hyaluronic acid

The Oct4, Sox2, and Nanog gene expression increased on day 3 and then decreased on day 7 and day 1. On the first and third days, the performance of these genes was highest on the CH2 5 film (ρ < 0.05). In general, these genes in placental stem cells can be converted between chitosan and chitosan-transparent, [S] 28 201219572 _ on the upward touch. In the monthly fat stem cells, these genes are called the surface tree, CH2·5 is better than other materials. In the placenta stem cell order, (10) 5 with

The performance of the feature is just as good. , a soil U fat stem cells and placental stem cells were cultured in a few days after the diced vinegar and chitosan _ _ hyaluronic acid materials, with the light torn the boot _ and the placenta dry fine, chondrocyte gene performance, the results such as Eighteenth picture. After forming a sphere, the cartilage gene

, · οχ_9 ' __ and eQUagen coffee increased in the age of soft sputum (the eighteenth figure B, D, F), compared with the culture of the basic culture (the eighteenth a 'c, cut. When the adipose stem cell culture These genes have a high performance (compared to TCPS/basic medium) when there are several y-glycans and chitosan hyaluronic acid in the basic culture medium. On the other hand, when the placental stem cells are cultured in the basic culture medium These genes showed low performance (compared with TCPS/basic culture solution) when riding and a few n days (four) acid. Cultured in chitosan with basic culture solution and chitin-carrying transparent hyaluronic acid The soft tip of the softer than the placenta is fine = better ~ make money T · · lyric acid. From the top, the suture stem cells and adipose stem cells in the cartilage (four) under the air, cultivated in a few H transparent f acid soft moon The gene is expressed as the highest 'the second is chitosan, the lowest is TCPS. Therefore, there is a chitosan membrane that absorbs hyaluronic acid, which can greatly enhance the aggregation of placental stem cells and adipose stem cells - a population of spheroid cells, and maintain placental stem cells And self-renewal and dryness of adipose stem cells, and subsequent enhancement The ability of the bone to differentiate. Inventive embodiments of the invention induce placental stem cells and adipose stem cells to spontaneously form a stereosphere on the membrane. A large number of large spheres are formed on the chitosan coated with hyaluronic acid, and it is also confirmed that hyaluronic acid can enhance the formation of spheres. Example 6 Culture of adipose-derived stem cells and neural stem cells with chitosan-white fungus polysaccharide m 29 201219572 - 6.1 Preparation of chitosan-twinkle polysaccharide membrane 1% of chitosan-1 of Example 5 (purchased from Fluka) As a substrate, prepared by the method of preparing chitosan film of Example 5, first adding 3 mg of chitosan to each well of a 24-well culture plate, and adding 3 mg of Tremella polysaccharide to each well, the ratio is 1 : 6.2 Extraction and culture of adipose stem cells, neural stem cells Adipose-derived stem cells were extracted and cultured as in Example 5. The extraction and culture of neural stem cells were the same as in Example 2. _ 6.3 Adipose-derived stem cells and neural stem cells were cultured in chitosan-white fungus polysaccharide Morphological adipose stem cells on the membrane were cultured on the chitosan-twinkle polysaccharide membrane for 24 hours and 48 hours, as shown in Fig. 19, and the neural stem cells were also spherical. Chitosan and silver The ratio of the polysaccharide includes, but is not limited to, 1: 1, about 3: 0.4 to 3: 4.5. Example 7 Chitosan, chitosan-hyaluronic acid, polycaprolactone, polycaprolactone_ Hyaluronic acid membrane cultured lung stem cells (LSC) 7.1 years old butyl 5^ sugar, chitin t-glycolic acid, hyaluronic acid, polyglycoside (p〇iyCapr〇iact〇ne, pcL), poly-self Preparation of chitosan and chitosan-hyaluronic acid ester by ester-hyaluronic acid membrane was prepared in the same manner as in Example 5. Polycaprolactone (purchased in Sigma) with a molecular weight of 80 kDa was dissolved in hydrazine, 4 dioxane. Ring (l,4-di〇Xane), which is 11% polycaprolactone. The above polycaprolactone is cast on a glass slide, which is a poly-inner film. Then, after scanning at a distance of 10 mm 6 m/min with atmospheric plasma (energy 1 kw, purchased from plasmatreat, Germany), 3 ul of 2% mg/mi aqueous hyaluronic acid solution was dripped on it, and washed away overnight. Hyaluronic acid is grafted onto the surface with a density of 〇5 mg/cm2 〇[S] 30 201219572 7.2 Extraction of mouse lung stem cells (LSC) and culture of mouse lung stem cell lines according to Ling TY, Kuo MD, Li CL, Yu AL, Huang YH, Wu TJ, Lin YC, Chen SH, Yu J. Identification of pulmonary Oct-4+ stem/progenitor cells and demonstration of their susceptibility to SARS coronavirus (SARS-CoV) infection in Vic. Proc Natl Acad Sci USA. 2006 Jun 20; 103(25): 9530-5. Epub 2006 Jun 13, obtained from mouse lungs. 7.3 results of lung stem cells cultured in chitosan, chitosan-hyaluronic acid, polycaprolactone and polycaprolactone-hyaluronic acid membranes. Lung stem cells in chitosan and chitosan-hyaluronan The acid forms a globular shape and the nanog/〇ct4 gene is improved. On the polycaprolactone and polycaprolactone-hyaluronic acid film, it will also form a sphere (see Figure 20). The surface of polycaprolactone is arranged in a crystal form, so that round crystals (like surface cracks) can be seen. And the performance of the dry gene nanog/oct4 is improved (21st). Therefore, the results show that the present invention provides a pluripotent adult stem cell, a pharmaceutical composition thereof, and a method for producing the same, which are capable of achieving cell self-renewal by contact with a biocompatible polymer. Maintain the outstanding effect of stemness. [Simple description of the schema] The first panel is a neural stem cell cultured on a. gelatin and b. tissue culture plate (TCps) for three days; no spheroid formation. The scale bar is ΙΟΟμτη. The second figure is the morphology of neural stem cells cultured on different chitosan (a• chitosan 丨b• chitosan-2) on days 1, 2, and 3; the scale is 1〇〇μηι . The second panel shows the results of scanning electron microscopy (SEM) after five days of culture of chitosan-1 neural stem cells: a. Top view b. Side view. The fourth picture is the a-3 b.5 c. 7 d l5 e 2〇f 25-day cell morphology (4) and ί -S] 31 201219572 FIB-fluorescent green protein performance (right); scale bar is 2 beer . The fifth figure shows the expression of neural stem cells cultured in chitosan-1 on days 4 and 21 of a F1B_fluorescent green protein and b. nestin (immunofection); the scale bar is 1〇〇μιη. The eighth figure is the sphere morphology of a. DAPI (nucleus) and b. p3-tubulin (immuno staining) on days 4, 14, and 21 of chitosan-1; the scale bar is 100|jm. The seventh figure is the type of neural stem cells/neural precursor cells cultured in four 2D alginate membranes (a. A#1 b. A#2) three days later; the scale bar is 250 μτη. The eighth figure is the comparison of the differentiation ability of NSPCs cultured on alginate film and TCPS; TuJ1 is a nerve cell and GFAP is a glial cell. The ninth image shows the cell morphology of rat adipose stem cells cultured on TCPS (T) and PVA (P) materials on days 1, 3 and 11; -: no addition of 5-aza; +: addition of 5- The tenth figure of aza is the cells of rat adipose stem cells (rADASs) cultured on Chit〇san3 (CS3), Chit〇san 1 (CS1), Chitosanl+CBD_RGD (CS1R) materials on day 1, day and day. Ball morphology; no addition of 5-aza; +: addition of 5-aza. Figure 11 shows rat adipose-derived stem cells (rADASs) in TCPS (T), Chit〇san 1 (CSl), Chitosanl+CBD-RGD (CS1R) The capillary electrophoresis pattern after RT-PCR experiment was used on the material; Cardiomyocyte positive (CP): no addition of 5-aza; +: addition of 5-aza. The twelfth picture shows human foreskin fibroblasts cultured in A. chitosan-1, human gingival stem cells cultured in B· chitosan-1 C. chitosan-2 D. TCPS implantation for three days After the cell morphology; the scale is ΙΟΟμιη; the magnification is 1〇. The twelfth figure is a human gingival stem cell cultured on a chitosan membrane after implantation. 12 Β. Η C· 16 D. 18 Ε. 20 F. 22 G 24-hour same-range form; ΙΟΟμιη, magnification 10 times" The fourteenth figure is the human gingival stem cells cultured in the form of chitin in the first A 〇B1 C. 2 D. 3 days after implantation; and cultured in TCPS after implantation E 〇R1 G2 H.3 days morphology; human gingival stem cells cultured in 〖. containing induction culture. [] 32 201219572 _ Liquid chitosan j. Chitosan containing basic culture solution and κ containing induction culture solution TCPS L. Form of TCPS containing basic culture solution after 1 day;

Human gingival stem cells are cultured in M. chitin containing R-containing medium and chitosan containing basal medium and sputum. TCPS containing induction medium. PPS containing basic medium is 2 days after cartilage Human tooth tube stem cells cultured in Q. Chitosan containing induction medium R. Chitosan containing basic culture medium and S. TCPS containing induction medium, TCPS containing basic culture solution after cartilage induction 7-day morphology; human gingival stem cells cultured in U. containing chitosan in the induction medium. V.

The spleen and w·TCPS containing the inducing culture solution X. The form of the TCPS containing the basic culture solution after the cartilage induction; the scale bar is · μηι, and the magnification is 10 times. The fifteenth figure is the cell morphology of the mesenchymal stem cells cultured on the chitosan membrane for 4 days. The sixteenth figure is the cultivation of different stem cells on different materials. (8) Cells cultured in TCPS U (B) was cultured in the form of cells containing the basic culture solution of chitosan and chitosan-hyaluronic acid for three days. The seventeenth figure is the dry gene (培养ct4, s〇x2 and Nan〇g) of the adipose-derived stem cells cultured on the 10th day containing the basic culture solution of chitosan and chitosan and TCPS. The mRNA shows quantitative results. The eighteenth figure is secreted in chitosan and chitosan _ hyaluronic acid membrane (compared with Tcps). The chondrocyte gene expression of adipose stem cells and placental stem cells was quantified by RT_pCR 7 days after I human induction. The nineteenth figure is the form in which the hepatic cells are cultured for a few hours (4)% of the time, and 48 hours after the (Β). The twenty-fifth figure is the form of culturing lung stem cells on the second day of polyhexanol, hexamidine vinegar and chitosan. The 20th-graph is the results of mRNA expression of lung stem cells cultured in poly-caprol vinegar, poly-caprolactone-polybutyrone membrane (5) 33 201219572 1, 3 days dry genes (actin, Oct4, Sox2 and Nanog) . [Main component symbol description] None

[S] 34 201219572 Sequence Listing <110> National Taiwan University <120> Method for producing adult stem cells as a spheroid cell population <130> 990544-11 <160> 1 <170> Patentln version 3.3

<210><211><212><213> 1 48

PRT Synthetic <220> <223>Cellulose Binding Functional Region-RGD Attachment Sequence (CBD-RGD)-<400> 1

Pro Thr Gin His Trp Gly Gin Cys Gly Gly He Gly Tyr Ser Gly Pro 15 10 15

Thr Val Cys Ala Ser Gly Thr Thr Cys Gin Val Leu Asn Pro Tyr Tyr 20 25 30

Ser Gin Cys Leu Pro Thr Thr Pro Thr Gly Arg Gly Asp Ser Ala Ser 35 40 45

Claims (1)

201219572 VII. Patent Application Range: 1. A method for producing adult stem cells as a spheroid cell population, comprising: culturing adult stem cells in vitro on a film formed by a biocompatible polymer, wherein the biocompatibility The polymer system is chitosan, alginate, hyaluronic acid, tremella polysaccharide, polycaprolactone or any combination thereof; and collecting a population of spheroid cells assembled by adult stem cells, wherein the spheroid cell population Will expand and have the ability to self-renew and differentiate as a unit of cells. Φ 2· The method of claim 1, wherein the adult stem cell is selected from the group consisting of a neural stem cell, a neural precursor cell, a fat stem cell, a dental silver stem cell, a bone marrow mesenchymal stem cell, a lung stem cell, and a placental stem cell. group. 3. The method of claim 1, wherein the biocompatible polymeric film is coated onto any substrate by coating or grafting. 4. The method of claim 1, wherein the biocompatible polymer is a combination of chitin and hyaluronic acid, and the ratio of chitin and hyaluronic acid is about 3. 0.177 to 3: 4.425 (w/w). • 5. (4) Please refer to the method described in the first paragraph of the patent. The biological hybrid polymer is a combination of chitosan and tremella polysaccharide, and the ratio of chitosan to tremella polysaccharide is about 3: to 3 The method of claim 1, wherein the biocompatible polymer is a combination of polycaprolactone and hyaluronic acid. 7. The method of claim i, wherein the somatic cell is a nerve cell. 8. The method of claim 2, wherein the spheroid cell population further has the ability to differentiate into a cardiomyocyte or a chondrocyte. The method of claim 8, wherein when the spheroid cell population is the adipose stem cell, the cardiomyocyte is differentiated. 10. The method of claim 9, wherein the method further comprises contacting the adipose stem cells with 5-azacytidine to differentiate the adipose stem cells into the cardiomyocytes. 11. The method of claim 8, wherein the spheroid cell population is differentiated into the chondrocyte when the gingival stem cell, the adipose stem cell, or the placental stem cell. 12. The method of claim U, further comprising contacting the gingival stem cell, the adipose stem cell, or the placental stem cell with a transforming growth factor P3 (TGF_P3), and Φ is allowed to differentiate into the chondrocyte. 13. An adult stem cell having differentiation ability, which is produced by: contacting an adult stem cell with a film formed of a biocompatible polymer for an effective period of time, wherein the biocompatibility The polymer system is chitosan, alginate, hyaluronic acid, tremella polysaccharide, polycaprolactone or any combination thereof; wherein the adult stem cells form a spheroid cell population, and the spheroid cell population expands and has The ability to differentiate into a neuronal cell, a glial cell, a chondrocyte, or a cardiomyocyte. 14. The adult stem cell according to claim 13, wherein the adult stem cell is selected from the group consisting of a neural stem cell, a neural precursor cell, a fat stem cell, a gingival stem cell, a bone marrow mesenchymal stem cell, a lung stem cell, and a placental stem cell. Group of. 15. The adult stem cell of claim 13, wherein the biocompatible polymer further comprises a cellulose binding functional region-RGD attachment sequence having the amino acid sequence of SEQ ID NO: 1 ( CBD-RGD). 16. The adult stem cell of claim π, wherein the biocompatible polymer is a combination of chitosan and hyaluronic acid, and the ratio of chitosan to hyaluronic acid is about 3: 0.177 to 3: 4.425 (w/w) » 17. A pharmaceutical composition comprising the adult stem cells of claim 13 and [2 201219572 - a pharmaceutically acceptable carrier. - A method for screening adult stem cells having self-renewal ability, comprising contacting a candidate adult stem cell with a film formed by a biocompatible polymer for an effective period of time - and then determining that the adult stem cells having the population of spherical cells are self-determined Renewable ability of adult stem cells 'where the biocompatible polymer is chitosan, browning acid, hyaluronic acid, tremella, polycaprolactone or any combination thereof. 19. The method of claim 18, wherein the effective time is preferably from the next day to four weeks. The method of claim 18, wherein the biocompatible polymer film can be coated on any substrate by coating or grafting.