TWI419970B - A method to producing a spheroid population of adult stem cells - Google Patents

Description

Method for producing adult stem cells as a spheroid cell population

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.

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; stem cells are divided into totipotent stem cells according to their potential for differentiation. , multipotent stem cells, pluripotent stem cells, and unipotent stem cells, for example, every cell before morula retains pluripotency. 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, 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 glial cells, called unipotent stem cells.

Stem cells can be further divided 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, hematopoietic stem cells, mesenchymal stem cells, neural stem cells (NSCs), hepatic stem cells (oval cells), and the like. Among them, mesenchymal stem cells include but are not limited to bone marrow mesenchymal stem cells (BMSCs), placenta-derived mesenchymal stem cells (PDMSCs), adipose-derived adult stem cells (ADASs), and gingival stem cells (Human gingival). 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 (neurons), astrocytes, and oligodendrocytes under appropriate conditions, and adult stem cells are often produced during in vitro proliferation and culture. It is difficult to maintain the problem of differentiation activity and functionality.

On the other hand, it is generally known that materials suitable for culturing stem cells are polystyrene, gelatin, poly-α-hydroxy acid, polylactic acid glycolic acid, etc., which have the advantages of stable biocompatibility, but these The material is used for the proliferating culture process of adult stem cells, has the disadvantage of being difficult to differentiate or losing function, and affects the effect of adult stem cell proliferation and differentiation.

At present, it has been found that cell morphology can regulate the lineage of differentiation factors of stem cell treatment, and the cell morphology of the sphere has been confirmed to be related to the differentiation potential. For example, the colony of embryonic stem cells can self-renew from cell-cell contact; embryo body ( Embryoid bodies (EB) are three-dimensional spatial cell aggregation of embryonic stem cells, which can be initially differentiated under the simulated normal embryo growth environment. However, there is still no effective method for adult stem cells to aggregate into spheres.

In order to solve the problem that conventional adult stem cells are difficult to maintain stemness and differentiation in vitro, the object of the present invention is to provide a method for producing adult stem cells into a spheroid cell population, comprising culturing adult stem cells in vitro to be compatible with one organism. a film formed by a polymer, wherein the biocompatible polymer is chitosan, alginate, hyaluronic acid, polycaprolactone, tremella polysaccharide or any combination thereof; and collected by adult The stem cells aggregate into a population of spheroid cells, wherein the spheroid cell population expands and has the ability to self-renew and differentiate into a single cell; the adult stem cells are selected from the group consisting of neural stem cells, neural precursor cells, adipose stem cells, gingival stem cells, and mesenchymal stem cells. A group consisting of 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 It is 3/0.177 to 3/4.425, and 0.1 mg/cm ² , 0.5 mg/cm ² , 2.5 mg/cm ² is used for the present invention, because a slide of 1.77 cm ² is 3 mg (chitosan): 0.177 mg ( HA) 3 mg (chitosan): 0.885 mg (HA), 3 mg (chitosan): 4.425 mg (HA) are effective, and are double-layered materials that are negatively adsorbed on the surface of chitosan (positive) by hyaluronic acid, otherwise transparent The acid can't catch the surface and dissolve into the water. The somatic cell can be a nerve cell. In another aspect, 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 β3 (TGF-β3).

Another object of the present invention is to provide an adult stem cell having differentiation ability, which is prepared by contacting an adult stem cell with a film formed of a biocompatible polymer for an effective period of time, wherein The biocompatible polymer is chitosan, 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 into one 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 functional region of the amino acid sequence of SEQ ID NO: 1 - RGD attachment sequence (MSC binding domain-RGD, CBD-RGD) can enhance the transdifferentiation ability of the spheroid cell population; the biocompatible polymer may be 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); it can be chitosan and tremella polysaccharide Combination, and the ratio of chitosan to tremella polysaccharide is about 3:0.4 to 3:4.5 (w/w); it can also be a combination of polycaprolactone and hyaluronic acid; the somatic cell can be a nerve cell .

It is still another object of the present invention to provide a pharmaceutical composition comprising the aforementioned adult stem cells and a pharmaceutically acceptable carrier. The carrier may be, but is not limited to, an excipient such as water, a filler such as sucrose or starch, a binder such as a cellulose derivative, a diluent, a disintegrant, an absorption enhancer or a sweetener. . The pharmaceutical composition of the present invention can be produced according to a conventional method for preparing a stem cell therapy agent, and the adult stem cells of the present invention are mixed with one or more carriers to prepare a desired dosage form, which can include a tablet, a powder, and a tablet. Agents, capsules or other liquid preparations, but not limited to this. The pharmaceutical composition of the present invention can be applied to clinical stem cell therapy to promote regeneration of nerve cells, cardiomyocytes or chondrocytes, and the pharmaceutical composition comprising adult stem cells can be used to avoid the difficulty and ethical consideration of embryonic stem cells. .

An advantage of the present invention is to provide a novel mammalian pluripotent adult stem cell, a pharmaceutical composition thereof, and a method for producing the same, which are self-renewing and maintaining dryness by contact with a biocompatible polymer. (stemness) outstanding effect.

The embodiments of the present invention are further described in the following description, and the embodiments of the present invention are set forth to illustrate the present invention, and are not intended to limit the scope of the present invention. In the scope of the invention, the scope of protection of the invention is defined by the scope of the appended claims.

The natural biocompatible polymer used in the embodiment of the present invention is chitosan, alginate, hyaluronic acid or any combination thereof, wherein chitosan is composed of amino-polysaccharide. The chitosan comprises a glycosamino-glycan which is the same component as the extracellular matrix, and is therefore biocompatible and biodegradable. Alginate is a natural soft polymer that promotes axon regeneration. Alginate can be cross-linked with calcium ions under physiological conditions (at room temperature or body temperature, physiological pH and isotonic pressure); Calcium alginate is a biocompatible and non-immunogenic material, so it is a material with high stability and high biocompatibility. Hyaluronic acid (HA) is a material that promotes cell migration, proliferation, and interstitial secretion, and does not form a film when it is water-soluble. In the present invention, it is combined with chitosan to form a film. Accordingly, the present invention discloses a novel method of 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, which causes the adult stem cells to aggregate. Forming a population of spheroid cells, and treating the population of spheroid cells of the present invention with trypsin detaches the globules from the membrane. If only the stem cell aggregation phenomenon mentioned in the prior literature is treated with trypsin One cell separates.

In each of the embodiments of the present invention, Examples 1 and 2 are two-dimensional (2D) films made by using chitosan of different molecular weights and alginate of different M/G ratios, and comparing neural stem cells culture thereon. Comparison of 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 with 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 materials, and the different materials are de-acetylated, surface potential, And water contact angle analysis of each material produces a spherical cell population effect. Further confirming the cell type and differentiation effect of the spheroid cell population, wherein F1B-fluorescent green protein (F1B-GFP) exhibits green fluorescence in undifferentiated neural stem cells, so the analysis of neural stem cell differentiation detects F1B. - The expression of fluorescein, which in turn is combined with immunofluorescence to detect the labeled nestin of neural stem cells and the marker β3-tubulin of undeveloped neurons.

In the present invention, "a neural stem cell" or "a neural precursor cell" refers to an undifferentiated central nervous system (CNS) pluripotent stem cell.

"An adult stem cell with differentiation ability" refers to a body cell in which an adult stem cell is easily differentiated into a plurality of types. The treatment of adult stem cells in accordance with the method of the present invention enables adult stem cells to self-renew and differentiate and enhance the plasticity of adult stem cells.

By "a period of effective time" is meant a period of time necessary for the biocompatible macromolecule to form a spheroid cell population with the adult stem cells of the invention, for example, the neural stem cells are contacted with chitosan for a period of time to agglomerate to form a sphere. The effective period of contact with the biocompatible polymer may be from 4 hours to 14 days or more, preferably from 1 day to 4 days in the present specification.

"A pharmaceutically acceptable carrier" means a substance used to achieve or enhance the successful delivery of a pharmaceutical composition of the invention, which may be an excipient (such as water), a filler (such as sucrose or starch), a binder (such as cellulose derived). But not limited to this, a diluent, a disintegrant, an absorption enhancer or a sweetener.

"About", "about" or "approximately" generally means within 20%, preferably 10%, and most preferably 5%. 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. For example, "a 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 × 1.5 cm was prepared, and a 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 neural stem cell spheres on the chitosan membrane, and the cell marker (nestin, labeled with neural stem cells; β3-tubulin, labeled with neurons) Immunofluorescence staining. It was fixed in 4% paraformaldehyde at different cycle times of cell culture. Rinse three times with phosphate buffer. 0.5 μ Triton X-100 (t-octylphenoxypolyethoxyethanol, Sigma, USA) 500 μl was added for 10 minutes to destroy the cell membrane and nuclear membrane. The sample was rinsed several times with phosphate buffer, and 20 μl of the primary antibody was added and cultured in a dark room at 37 ° C for 1 hour. To stain the cells, add 0.1% of 4,6-diamidino-2-phenylindole dihydrochloride (purchased in DAPI, Sigma, D9542, USA) to 200 μl. 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-β3-tublin mouse-mono IgG (purchased from Santa Cruz, USA; :1000). The dilution ratio of the secondary antibody 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 myocardial differentiation ability of the examples of the present invention utilizes an erect fluorescent microscope (NIKON): the sample is rinsed with a phosphate buffer solution, fixed at room temperature for 20 minutes by adding 4% paraformaldehyde, and washed three times with phosphate buffer. After 10 minutes, add 3% BSA/phosphate buffer for 30 minutes at room temperature, aspirate, add 3% BSA prepared primary antibody, the dilution ratio is the following table, sealed with parafilm and placed in a refrigerator at 4 ° C overnight; The next day, the primary antibody was aspirated and rinsed three times with phosphate buffer solution (the following steps were protected from light throughout the whole process), and the secondary antibody prepared with 3% BSA was added, covered with aluminum foil paper and protected from light, and placed in a 37 ° C incubator for 1 hour; The secondary antibody was aspirated, phosphate buffer was rinsed three times, DAC dye prepared with 3% BSA was added for 3 minutes, aspirated, phosphate buffer was washed three times, phosphate buffer was added, and the material was removed from the well by needle and clip. The plate was taken out and placed on a glass slide and observed with a erect fluorescent microscope (purchased from NIKON).

The cell surface protein characteristics analyzed in the examples of the present invention were analyzed by flow cytometry using a flow cytometer (purchased from FACScan, BD, USA). First, the cells were removed from the culture plate, washed at 5 × 10 ⁵ cells/ml in phosphate buffer, centrifuged at 1000 rpm for 5 minutes, and the cells were collected three times, and 100 μl of phosphate buffer was added to reconstitute the cells. Add 10 μl of primary antibody to the cell solution, react for 30 to 60 minutes, rinse with phosphate buffer three times, add 10 μl of secondary antibody for 30 to 60 minutes in the dark, wash with phosphate buffer, centrifuge at 1000 rpm for 5 minutes, repeat Three times to remove unreacted antibody, and finally 500 μl of phosphate buffer was added to re-dissolve 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 the neural stem cells, the neural precursor cells, and the adipose stem cells in the adult stem cells are exemplified in the examples, the gingival stem cells and the placental stem cells are isolated from humans, but the present invention is not limited thereto, but is applicable. It is isolated from mammals including humans, rats, mice and monkeys.

Example 1

Culture of adult neural stem cells/neural precursor cells with chitosan films of two molecular weights

1.1 Preparation of chitosan film

Two chitosan-1 (purchased from Fluka, USA) and chitosan-2 (purchased from Sigma, USA) were used to prepare chitosan at a concentration of 1%, and 0.5 g of chitosan was weighed. The sugar powder was dissolved in 49.5 ml of secondary water, stirred at room temperature for half an hour, and then 0.5 ml of acetic acid was added, and the mixture was gently stirred at room temperature for 12 hours. The filter was used to filter impurities on the next day as a 1% chitosan solution, wherein the chitosan-1 has a molecular weight of about 510 kDa and the chitosan-2 has a molecular weight of about 400 kDa. The resulting solution was filtered through a mesh of 100 μm, and then coated on a glass coverslip (100 μl of each 15 mm diameter slide) and allowed to air dry for two days. The chitosan membrane was immersed in a 0.5 N 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 Rat brain neural stem cell extraction and culture

Neural stem cells were isolated from the brain of a gene-transforming mouse containing the promoter F1B-fluorescence green protein (F1B-GFP) gene. The two-month-old mouse was ground and shredded into smaller pieces to obtain a single cell suspension. The minced cells were implanted in a 35-mm culture dish containing no supplemental matrix and adhesion factor in DMEM/F-12 medium (Dulbecco's modified Eagle's medium and Ham's F-12, purchased from Giboco, USA) 1:1 medium. Contains 10% fetal 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 μg/ml of glycoside antibiotic geneticin (G418, available 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 implanting the neural stem cells into the culture material, the chitosan membrane on the glass coverslip was immersed in 75% ethanol, rinsed with phosphate buffered saline (PBS), and then placed in a 24-well culture plate. In a hole. Neural stem cells were implanted at a density of 5 x 10 ⁴ cells per well. 10% fetal calf serum (purchased from Gibco, USA), 400 μg/ml glycoside antibiotic (G418), and streptomycin-penicillin (100 U/ml) were added as a culture solution in DMEM/F-12 medium. Incubate in a humidified incubator at 37 ° C, 5% CO ₂ , and change the culture medium twice a week; in addition, tissue culture polystyrene surface (TCPS) and gelatin (gelatin) As a control group, the results are shown in the first figure and the second figure.

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 gelatin. On the other hand, the cells cultured on the chitosan film began to aggregate in 1-2 days, 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 chitosan-1 are roughly larger than that of chitosan-2 (as shown in the second figure).

In order to confirm 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, by the scanning electron microscopic image of the third figure, the side view of the sphere can clearly show the three-dimensional structure, and the optical image The size of the sphere is shown to increase over time, up to about 100 μm.

In addition, the fourth to sixth graphs show the degree of differentiation of neural stem cells. The fluorescence intensity of neural stem cells, with green fluorescence, indicates undifferentiation. The green fluorescence began to fade after 15 days, but it remained clear (as shown in Figure 6d). At 15 days, some of the cells began to move out of the sphere, so the size of the neural stem cell spheres changed quite rapidly, and the removed cells did not show any green fluorescence. Fluorescence elimination of the sphere after 15 days In addition to showing the removal of cells, the cells in the sphere have also differentiated.

In the immunofluorescence staining assay, the marker protein of neural stem cells cultured in chitosan-1 is shown in the fifth panel (nestin) and in the sixth panel (β3-tubulin). The spheres were found to be strongly positive for nestin at 21 days, indicating that the cells of the sphere maintained the state of the neural stem cells for 21 days; the display of Nestin showed a slight decrease as the incubation time increased. On the other hand, the performance of β3-tubulin was low throughout the process. This result shows that the cultivation of neural stem cells on the chitosan film can be a method of maintaining the self-renewal ability and stemness of these cells, or for purifying neural stem cells from a cell population.

1.4 Deacetylation, surface potential, and water contact angle values for chitosan and tissue culture plates (TCPS) are listed in Table 1.

Table 1 Comparison of characteristics of different chitosan (chitosan-1 (CS-1) and chitosan-2 (CS-2)) and tissue culture plates (TCPS) (a) deacetylation degree ( b) surface potential, and (c) surface water contact angle.

The deacetylation degree of chitosan-1 and chitosan-2 was about 77.7% and 86.0%, respectively. The higher the chitosan deacetylation degree, the lower the surface water contact angle, and the higher the hydrophilicity, that is, the hydrophilic chitosan-2 is larger than the chitosan-1, and the surface of the chitosan is hydrophilic. The sex is higher than TCPS, because the cells are easier to attach in a highly hydrophilic matrix, and the neural stem cells are attached to TCPS better than chitosan. Moreover, the attachment of neural stem cells to chitosan-2 was better than that of chitosan-1, and the formation of spheres on chitosan-1 was delayed. 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 consistent with our results. The surface potentials of chitosan-1 and chitosan-2 were all negative, but the values were close to neutral, and the surface was less negative. In the results of this example, the neural stem cells were not attached. Very good and present a gathering situation. The surface hardness of chitosan-1 (indentation depth of about 50 nm) is similar to the inside (indentation depth 100-150). On the other hand, the surface hardness of chitosan-2 is higher than that of the interior and is also higher than that of chitosan-1, and the chitosan-2 is coarser than the chitosan-1 and has a more dense crystal structure on the surface. Due to the harder and more crystalline surface, neural stem cells may adhere to chitosan-2 better than chitosan-1 and form smaller spheres.

Example 2

Culture of adult neural stem cells/neural precursor cells and adipose stem cells with two alginate films

2.1 Preparation of alginate film

A film was prepared using two alginate, including alginate A#1 with a lower M/G ratio (β-D-mannuronic acid/α-L-guluronic acid ratio) (M/G ratio=0.5, molecular weight 110,000, Alginate A#2 (M/G ratio=1.59, molecular weight 12,000-80,000, Sigma, USA) purchased from Hayashi, Japan) and higher M/G ratio. Alginate A#1 and A#2 were first dissolved in 9 mg/ml sodium chloride solution to make the solution concentration 40 mg/ml. Adjust the pH of the solution to 7, and autoclave (125 ° C and 1.5 atm) for 30 minutes after adding 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (2-[4-( 2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid, purchased from HEPES, Invitrogen, maintains the same pH value as the human body. Both alginate used in the examples of the present invention can be made into a smooth or wrinkled film. The alginate film was prepared by adding 400 μl of alginate per well to a 24-well culture plate (purchased from Corning, USA), air-dried for 60 minutes, and slowly dropping 102 mM calcium chloride 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), washed with phosphate buffer, cut into small pieces, and centrifuged for the first time (1500 rpm, 5 mins). Pour the upper layer of fat into a new centrifuge tube and add a little phosphate buffer, then cut as much as possible and perform a second centrifugation (1500 rpm, 5 mins). Take the upper layer of fat and prepare the digestive juice: 1 mg/ml collagen type I (collagenase type I, purchased from Sigma) / HBSS solution (HBSS buffer solution is prepared as shown in Table 4).

Table 4 HBSS buffer solution components

The fat was mixed with the above digestive juice and placed in a cell culture incubator (100 rpm, 1 hour). The third centrifugation (1500 rpm, 10 minutes). The cell broth was filtered using a 70 m cell sieve (purchased from Falcon, 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 Falcon, BD Bioscience). The culture material was attached to the cells 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 50000 cells/T-flask.

2.3 Morphological analysis and differentiation of neural stem cells/neural precursor cells into alginate films

The rat brain neural stem cell/neural precursor cell extraction step was as described in Example 1. The obtained cells were cultured on a 2D alginate membrane, and the NSPCs were implanted in a 24-well culture plate containing cyanobacterial gel (2.5 × 10 ⁴ cells were implanted in 2 ml of the culture solution). 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 performance 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).

The size of the astrocytes and neural stem cells cultured on A#1 and A#2 for 28 days is shown in the table (the spheres are formed when cultured for 14 days, and the sphere size is similar to that at 28 days):

Example 3

Culture of adipose-derived stem cells with chitosan film modified with two molecular weight chitosan films and cellulose-binding functional region-RGD attachment sequence (CBD-RGD)

3.1 Preparation of two molecular weight chitosan films

This example uses chitosan-3 (purchased from Kiotec) having a molecular weight different from that of the foregoing examples, having a molecular weight of about 170 kDa, and chitosan-1 (purchased from Fluka) having a molecular weight of about 510 kDa. 0.5 g of chitosan powder was weighed and dissolved in 49.5 ml of secondary water, and stirred at room temperature for half an hour, and then 0.5 ml of acetic acid was added to continue stirring for one day. The filter was used to filter impurities on the next day, which was a 1% chitosan solution.

Table 3 Deacetylation and molecular weight of two chitosan

Take 200 μl of 1% chitosan solution and evenly drop into a 1.5 cm round slide and dry it naturally for 1-2 days at room temperature. After confirming the film dry, cover the slide with 0.5N NaOH for 5 minutes, then use twice. After washing the water for 3 times, continue to soak in the secondary water to the next day; the next time the second water is poured off, and the secondary water remaining naturally at room temperature remains chitosan-3 and chitosan. Sugar-1 film. In addition, a tissue culture polystyrene surface (TCPS) without growth factor 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). Alcohol, PVA, purchased from Sigma, Hot water soluble) The powder was dissolved in 50 ml of secondary water and dissolved in a high-temperature autoclave. The temperature was lowered to normal temperature to be 5% polyvinyl alcohol solution. 150 μl of a 5% 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 Chitosan-1 modified by cellulose-binding functional region-RGD attachment sequence (CBD-RGD)

The cellulose-binding functional region-RGD-attached sequence (CBD-RGD) was provided by Professor Chen Zhenhan of Zhongxing University and derived from the gene of the cellulolytic enzyme cellulobiohydrolase I (CBH I) of the fungus Tricoderma konigii. At the N-terminus of CBH I, there is a 36 amino acid-sized cellulose binding domain (CBD). The password of the fifth amino acid modified by the polymerization enzyme chain reaction method was changed from the original tyrosin to tryptophan. The CBD has affinity for cellulose, and a PT linker and a RGD sequence gene are added to the 3th end of the CBD gene. RGD is the smallest functional site in cell adhesion factor (Fibronectin) to promote cell attachment, and the cellulose binding functional region-RGD attachment sequence (CBD-RGD) has the amino acid sequence as SEQ ID NO: 1, due to CBD-RGD It has a very high molecular weight (25 KDa) and uses Coulomb's gravitation to form 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, then washed twice with phosphate buffer solution, and placed for half an hour for chitosan-1. The film was dried; CBD-RGD (4 μg/20 μl) was evenly dropped on the film of chitosan-1 (4 μg CBD-RGD/1.77 cm2), and after 1-2 hours of CBD-RGD drying, buffered with phosphate. The liquid was washed once to form a film of chitosan-1 modified with CBD-RGD.

3.3 Extraction and culture of rat adipose stem cells (rADASs)

Extraction and culture of rat adipose stem cells (rADASs) was as in Example 2.

3.4 Morphological analysis and differentiation of adipose-derived stem cells into three chitosan films into myocardial cells

This example is divided into two parts. The first part is a comparative tissue culture plate (T), polyvinyl alcohol (P), chitosan 3 (CS3) and chitosan 1 (CS1). The T, P, CS3 and CS1 films were soaked in 75% alcohol for 3 hours, rinsed with phosphate buffer, and placed in a 24-well culture plate; rat adipose-derived stem cells were obtained using 0.25% trypsin (purchased from Gibco) ( The cell number of the laboratory is 2-6 generations, and the cells are collected in the culture solution. After 5× ^{10 4} cell solution is placed in each well, the plate is shaken to uniformly disperse the cells, and then cultured in a cell culture incubator.

5-azacytidine (5-aza) is a compound that demethylates DNA. According to research, it is induced that cells become cardiomyocytes by irregular demethylation of 5-aza, so it is replaced with 10M on the fourth day. The culture medium of 5-aza (5-azacytidine, Sigma) was divided into groups with 5-aza (w 5-aza) and 5-aza (w/o 5-aza), so w In the group of /o 5-aza, the original culture solution is still added. On the fifth day, the culture solution was aspirated, replaced with a culture medium containing no 5-aza, and further cultured for 1 week (11 days in total) using reverse transcription polymerase chain reaction (RT-PCR). The ability to differentiate cardiac muscles was compared and the results are shown in Tables 5 and IX and X.

The second part is to compare the myocardial differentiation ability of T, CS1 and CS1 combined with CBD-RGD (CS1R). The second part was consistent with the experimental conditions of the first part. The experimental group was changed to T, CS1, CS1R. The ability of differentiated myocardium was analyzed by RT-PCR and immunofluorescence staining. The results are shown in Table 5 and ninth and tenth.

Table 5 Cell size of adipose stem cells (rADASs) formed on different materials

T: TCPS; P: PVA; CS3: Chitosanl; CS1: Chitosanl; CS1R: Chitosanl+CBD-RGD.

-: No added 5-aza; +: Add 5-aza; X: Cell-free ball.

From Table 5, ninth and tenth, it can be found that the rat adipose stem cells cultured in the tissue culture plate can not form a ball, and the rat adipose stem cells on the polyvinyl alcohol will aggregate the most on the third day, but then whether or not there is no addition. 5-aza, 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 plate. In CS3, on the first day, cells can be found to gradually aggregate into cell spheres with a sphere size of about 35 μm. On the third day, a single cell sphere can be found, which is larger than the first day, about 70 μm. It was found that the larger cell spheres gradually drifted away, and on the 11th day, CS3(-) was found to be smaller than the CS3(+) cell sphere. In CS1, the cells were gradually aggregated into cell spheres on the first day, and the size of the spheres was about 25 μm. On the third day, a single cell sphere was found, which was larger than the first day, about 55 μm; although CS1 was formed. The ball is smaller than the CS3, but the ball is not easy to float; CS1(-) and CS1(+) are about 55μm on the 11th day. In CS1R, on the first day, the cells were found to be unspheronized, and the cells spread out on the material, similar to TCPS, but there is a tendency to gradually aggregate. On the third day, a large cell sphere can be found, and the largest one can reach 250 μm. The balls formed on CS1R are large, but they hardly drift away; on the 11th day, the cell sphere formed by CS1R is more than 100 μm larger than CS3 and CS1. On the 11th day, the balls gathered by CS3, CS1, CS1R cells will be found, some cells will run out of the cell ball, and the group without 5-aza will run out of the cell ball than the group with 5-aza. The trend is even stronger.

Therefore, the experimental results of the first part are the best for the CS1 film, and the larger the cell sphere, the better the differentiation effect, but the large ball of PVA will run out and paste back to the material, while CS3 can't keep the big ball, leaving the rest. The cell sphere 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 PVA, CS3, and CS1 films showed that CS1(+) and CS1(-) were more common than other groups. Good performance; while the late myocardial protein genes α-MHC, CS1 (+) have the best performance. Therefore, the second part of the experiment is based on CS1R and CS1 of CBD-RGD modified 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 will not be easy to drift away from the large ball formed by CS3. It can be seen from the experiment that CS1R(+) and CS1R(-) have a size of about 155μm when they are differentiated to the 11th day, but the addition of 5-aza to CS1R(+) is better and the cell sphere is The cell oligos ran out; RT-PCR analysis confirmed that CS1R(+) showed a better tendency to differentiate than CS1R(-) in GATA4, α-MHC and Troponin I, so the addition of 5-aza could actually increase Differentiation effect; the size of the ball and the formation of the ball also determine the effect of myocardial differentiation, CS1R>CS1>TCPS.

It can also be found by immunostaining that both GATA4 and α-MHC are expressed only in the cell sphere, and a single cell has almost no fluorescence, and cells that run out of the cell sphere have no fluorescence. The gene expression of CBD-RGD modified CS4 can be seen in the eleventh figure. The early gene GATA4 and the late gene α-MHC Troponin I have the best differentiation ability of CSR(+), followed by CSR(-), so add 5-aza. It is helpful for myocardial differentiation. It can also be seen from this that CBD-RGD modified CS1 does have better differentiation ability than unmodified CS1. Therefore, adipose-derived stem cells have prominent myocardial differentiation effects in both CS1 and CS1R films in gene expression and immunochemical staining.

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 the formation of adult stem cells into spheres.

Example 4

Cultivating gingival stem cells and foreskin fibroblasts with two chitosan membranes

4.1 Preparation of chitosan film

Using a medium viscosity of chitosan-1, a deacetylation degree of 77.7%, an average molecular weight of about 510 kDa of 1% chitosan (purchased from Fluka) dissolved in 1% acetic acid (purchased in Showa), filtered and coated On a 15mm coverslip (purchased from Marienfeld). The chitosan film was air-dried at room temperature, and the acidity of chitosan was neutralized with a 0.5 N sodium hydroxide solution. The excess sodium hydroxide on the chitosan film was washed with double distilled water. Then, all the chitosan membranes were immersed in 75% alcohol and irradiated with ultraviolet rays to achieve a bactericidal effect. The chitosan membrane was finally washed with phosphate buffer to remove excess 75% of the alcohol. To compare the results of chitosan, a chitosan membrane (purchased in Sigma) having a deacetylation degree of 86% and a molecular weight of about 400 kDa was prepared in the same manner as above. In the present example, chitosan-1 represents chitosan (Fluka) and chitosan-2 represents chitosan (Sigma).

4.2 Extraction and culture of human gingival stem cells and human foreskin fibroblasts

Human gingival tissue is obtained from healthy adult gums. Human gingival stem cells are cultured in a medium containing minimal essential alpha (α-MEM, purchased from Gibco) plus 10% fetal bovine serum and 2% antibiotic-antimycotic (2 units/ml penicillin (basal), 2 μg/ml streptavidin The cells (basin) and 5 ng/ml amphotericin) (purchased from Gibco) were cultured in a cell culture flask (Corning) at 37 ° C in a 5% CO ₂ incubator. Replace the basic culture solution once every two days. It was collected as a 0.25% trypsin/ethylenediaminetetraacetic acid solution (purchased from Gibco) when human gingival stem cell coverage reached 60% to 70%. This example uses third to seventh generation gingival stem cells.

By obtaining healthy human adult foreskin fibroblasts (HSF). Human foreskin fibroblasts were supplemented with 10% fetal bovine serum (purchased from Gibco), 200 mM glutamine (purchased from Gibco), 1% MEM non-essential amino acid 100X solution in DMEM containing high sugar source (purchased from Gibco). Purchased in Sigma), and 1% penicillin/streptomycin (5000 units/mL penicillin (basal) and 5000 μg/mL streptomycin (substrate)) were cultured in cell culture flasks. The HSF was cultured in a 37 ° C, 5% CO ₂ incubator, and the basic culture solution was changed once every two days. When human foreskin fibroblasts cover 80% to 90%, they are collected in 0.25% trypsin/ethylenediaminetetraacetic acid solution (purchased from Gibco). This example used the 28th generation of foreskin fibroblasts (HSF).

4.3 Morphological analysis and differentiation of human gingival stem cells on chitosan film into chondrocytes

Human gingival stem cells (4 x 10 ⁴ cells/ml) were implanted into each of the chitosan membranes placed in a 24-well tissue culture plate, and the culture medium 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. The chondrocyte-inducing medium contained DMEM containing high sugar source (purchased from Gibco), 10 ng/ml of TGF-β3 (CytoLab/PeproTech, purchased from Rehovot, Israel), 10 ^-7 M of dexamethasome (purchased from Sigma), 50 μg /ml of 1-ascorbic acid-2-phosphate (purchased from Sigma), 40 μg/ml of L-valine (purchased from Sigma), 10 μg/ml of ITS-premix 10× (purchased from Sigma), 10% Fetal bovine serum (purchased from Gibco), and 2% antibiotic-antimycotic. The human gingival stem cells were cultured in an incubator containing the inducing culture solution for 7 days and 14 days, and the inducing culture solution was changed once every two days. The morphology of human gingival stem cells cultured on chitosan-1, chitosan-2 and TCPS films for three days after implantation is shown in Fig. 12.

4.4 Morphological analysis and differentiation of human foreskin fibroblasts on chitosan membranes into chondrocytes

In the same condition, human foreskin fibroblasts were used as a control group, and human foreskin fibroblasts (4×10 ⁴ cells/ml) were implanted into each of the chitosan membranes and TCPS placed in a 24-well tissue culture plate. The culture medium was continuously cultured with the basic growth medium and chondrocytes, and the culture solution was changed once every two days. The morphology of the human foreskin fibroblast cultured on the chitosan-1 film for three days after implantation was shown in Fig. 12.

Referring to Fig. 12, human foreskin fibroblasts cultured on chitosan-1 film have no spheroid formation (Fig. 12A). However, human gingival stem cells cultured on chitosan-1 film have spheroid formation (Fig. 12B), which clearly shows that not all cells cultured on chitosan-1 film will form spheres because In this embodiment, human gingival stem cells which do not form spheres themselves can form spheres on the chitosan-1 film, and human foreskin fibroblasts are not formed. Fig. 12 shows the case where human gingival stem cells are cultured on TCPS without spheroid formation. In addition, when human gingival stem cells are cultured in chitosan films of different molecular weights, the proportion of formed spheres is also different; in the twelfth image (B, C), three days after the implantation of human gingival stem cells, the cells are cultured in several The glycan-1 film formed a much larger sphere than on the chitosan-2 film. This indicates that chitosan-1 may easily induce the formation of human gingival stem cell spheres compared to chitosan-2.

Further reference to Fig. 13 shows the live angiography of human gingival stem cells cultured on chitosan-1 in the same range at 12, 14, 16, 18, 20, 22 and 24 hours after implantation. Human gingival stem cells cultured on chitosan-1 began to form spheres 12 hours after implantation, and initially formed spheres after 24 hours. Human gingival stem cells adhered to chitosan-1 to exhibit a fiber-like morphology just after implantation. However, as the culture time increases, more and more human gingival stem cells gather into the ball, and human gingival stem cells attached to chitosan-1 are less and less.

In addition, referring to Fig. 14 shows human gingiva cultured in chitosan and TCPS, with or without cartilage-inducing culture solution at 0 to 3 days after implantation, and 0, 1, 2, 7 and 14 days after the addition of the cartilage-inducing culture solution. Stem cell morphology. Human gingival stem cells are fiber-like throughout the culture process. As cell culture time increases, human gingival stem cells form relatively larger cell spheres. 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-1 film added with the basic growth medium increases with time, it seems to be self-spherical about 14 days after cartilage induction (or 17 days after implantation). Secretion. 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 gingival stem cells cultured in TCPS. It is particularly important that the chondrocyte differentiation potential of human gingival stem cells cultured in a cartilage-inducible chitosan film is higher than that of human gingival stem cells cultured on a cartilage-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, and therefore, NANOG and Oct 4 are often used as differences. Markers of differentiated cells, Oct 4 is especially used to prevent cell differentiation. In addition, neural ridge cells are a group of transient pluripotent and migratory cells. Specific two-segment neural ridges SLUG and Sox10 determine the specification and differentiation of the neural ridge. 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 indicates that the spheres of human gingival stem cells formed only on chitosan have higher neurological gene expression and dry gene expression than general fiber 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 was confirmed by the spheroids of human gingival stem cells on the chitosan film on the SLUG, Sox 10, NANOG and Oct 4 gene expressions 0 to 3 days after implantation. Thus, the formation of spheres helps maintain the pluripotency, migration and dryness of human gingival stem cells. In addition, it was observed from live cell imaging that the above human gingival stem cells were spherical on chitosan, and the spherical shape was formed by continuous cell-matrix interaction and cell-cell interaction of human gingival stem cells resulting in dynamic movement. The dynamic state of the sphere also confirms the ability of the human gingival stem cells to migrate from the neural ridge gene. In this study, it was observed that in all forms of human gingival stem cell formation, human gingival stem cells forming spheroids continued 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 globular morphology of human gingival stem cells still maintains pluripotency and dryness, which is not found in humanoid gingival stem cells in the fibrillar form.

In addition, bone marrow-derived MSCs are cultured on the chitosan-1 membrane and also form a sphere (see Figure 15).

Example 5

Placental and adipose-derived stem cells cultured with chitosan and chitosan-hyaluronic acid membrane

5.1 Preparation of chitosan and chitosan-hyaluronic acid (HA) film

The chitosan-1 powder has a molecular weight of 510 kDa (purchased from Fluka) dissolved in 1% acetic acid to obtain a 1% chitosan solution. The solution (300 μl) was applied to each well of a 24-well culture plate. When the sample is placed in the laminar flow chamber 24, the solvent evaporates, which is a chitosan film. Sodium hydroxide (0.5 N) was added to each well for 30 minutes, and then washed several times with phosphate buffer.

Preparation of chitosan-hyaluronic acid film, hyaluronic acid solution (purchased in scientific research or Shiseido, the effect is the same) 300μl added to the chitosan film in different amounts of 0.1, 0.5 or 2.5mg/cm ² . Hyaluronic acid has a strong negative charge and will immediately bind to the positively charged chitosan membrane. Chitosan and different hyaluronic acid doses of chitosan-hyaluronic acid membrane are abbreviated as C, CH0.1, CH0.5 and CH2.5. The chitosan-hyaluronic acid membranes of different amounts of hyaluronic acid were washed 5 times with phosphate buffer to remove unbound hyaluronic acid and then lyophilized. The effective absorption rates of CH0.1, CH0.5 and CH2.5 were 94%, 95% and 89%, respectively. The total amount of hyaluronic acid absorbed increases as the amount of hyaluronic acid initially added increases. In addition, a tissue culture polystyrene surface (TCPS) without growth factor 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 placenta was washed several times with phosphate buffer and then disrupted and treated with 0.25% trypsin at 37 °C for about 10 minutes. After shredding and enzyme treatment, the homogenate was cultured in DMEM-LG (Dulbecco's Modified Eagle Medium-low glucose) (purchased in Gibco) complete medium, 10% fetal bovine serum (purchased in Gibco), 10 mg/l penicillin - Streptomycin and 10 mg/l L-glutamine (purchased from Tedia). The cells were cultured at 37 ° C with water-saturated air and 5% CO 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 in adipose tissue. The cells are extracted by enzymatic extraction from adipose tissue. The adipose tissue was first broken into several pieces and then gently mixed in a phosphate buffer of type I collagenase (purchased in Sigma-Aldrich) at 200 U/ml for 30 minutes at 37 °C. The homogenate was cultured in DMEM-LG (Dulbecco's Modified Eagle Medium-low glucose) (purchased in Gibco) complete medium, and 10% (v/v) fetal bovine serum (purchased from Gibco), 10 mg/l penicillin-chain was added. Mycin. The incubator maintained 37 ° C / 5% CO ₂ . The culture medium was changed twice a week.

5.4 Morphological analysis and differentiation of placental stem cells and adipose stem cells on chitosan and chitosan-hyaluronic acid films into chondrocytes

Adipose stem cells and placental stem cells (3 × 10 ⁴ cells) were cultured for three days after each of the chitosan membranes containing the basic culture medium, and the culture solution was changed to a chondrocyte-inducing culture solution. The induction medium was DMEM-LG containing 10 ng/ml TGF-β3 (purchased from CytoLab/Peprotech, Rehovot, Israel), 0.1 μM dexamethasome, 50 μg/ml L-ascobate-2-phosphate, 40 μg/ml L- Proline (Sigma), 1% insulin-transferrin-selenium supplement (ITS+premix) 100× (purchased in Sigma) and 1% penicillin-streptomycin. The induction medium was changed twice a week. The other group (3 days after implantation) was still cultured in the basic culture medium as a control group.

One day after the adipose stem cells were cultured on the chitosan-hyaluronic acid film, a three-dimensional sphere formed by aggregation of the adipose stem cells was formed. As the hyaluronic acid on the chitosan film increases, the spheres formed are more pronounced and larger in number and size. The morphology of adipose stem cells and placental stem cells cultured on TCPS, chitosan, chitosan-hyaluronic acid membrane on day 3 is as shown in Fig. 16. After 3 days of culture on the film, the adipose stem cells and placental stem cells cultured in the chitosan and chitosan-hyaluronic acid films were spherical. The average cell size of the different cells on these membranes is listed in Table 6.

Table 6 Average size of spheres formed by different types of placental stem cells and adipose stem cells on different membranes

*Each average is the average of at least 20 sphere diameters

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. Figure 17 shows the quantitative results of mRNA expression of Oct4, Sox2 and Nanog in adipose stem cells cultured in different materials and TCPS controls. The Oct4, Sox2, and Nanog genes in chitosan and chitosan-hyaluronic acid increased on day 3 and then decreased on days 7 and 10. On the first and third days, the performance of these genes was highest on the CH2.5 film (p < 0.05).

In general, these genes for placental stem cells can be up-regulated on chitosan and chitosan-hyaluronic acid membranes for up to seven days. In adipose stem cells, upregulation of these genes can be maintained for three days. In adipose stem cells, CH2.5 maintains these genes better than other materials. In placental stem cells, CH 0.5 and CH 2.5 are equally effective at maintaining the characteristics of these genes.

Adipose-derived stem cells and placental stem cells were cultured in chitosan and chitosan-hyaluronic acid (with TCPS). After 7 days of cartilage induction, the chondrocyte gene expression of adipose stem cells and placental stem cells was quantified by RT-PCR. Eighteen pictures. After the formation of spheres, cartilage gene expression (Sox-9, aggrecan and collagen type II) was significantly increased under cartilage induction (Fig. 18, B, D, F), compared with cultured medium (Fig. 18) , C, E). When adipose stem cells are cultured in chitosan and chitosan-hyaluronic acid with a basic culture medium, these genes are highly expressed (compared to TCPS/basic medium). On the other hand, when placental stem cells were cultured on chitosan and chitosan-hyaluronic acid in a basic culture medium, these genes showed low performance (compared with TCPS/basic medium). The adipose stem cells cultured in chitosan and chitosan-hyaluronic acid having a basic culture solution have better cartilage differentiation ability than placental stem cells, particularly chitosan-hyaluronic acid. From the above results, the placental stem cells and adipose stem cells have the highest cartilage gene cultured in chitosan-hyaluronic acid in the presence of cartilage-inducing liquid, followed by chitosan, and the lowest is TCPS. Therefore, a chitosan membrane that absorbs hyaluronic acid can greatly enhance the aggregation of placental stem cells and adipose stem cells to form a spheroid cell population, and maintain the self-renewal and dryness of placental stem cells and adipose stem cells, and enhance the differentiation of cartilage in the future. .

In the embodiment of the present invention, the placental stem cells and the adipose stem cells are spontaneously formed on the film to form a stereoscopic sphere, and a plurality 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 the sphere.

Example 6

Culture of adipose stem cells and neural stem cells with chitosan-white fungus polysaccharide

6.1 Preparation of chitosan-twinkle polysaccharide membrane

Using 1% of chitosan-1 (purchased from Fluka) of Example 5 as a substrate, and preparing the chitosan film preparation method of Example 5, first adding 3 mg of chitosan to culture in 24 wells. In each well of the plate, 3 mg of Tremella polysaccharide was added to each well in a ratio of 1:1.

6.2 Extraction and culture of adipose stem cells, neural stem cells

The extraction and culture of adipose stem cells were the same as in Example 5.

The extraction and culture of neural stem cells were the same as in Example 2.

6.3 Morphology of adipose-derived stem cells and neural stem cells cultured on chitosan-twinkle polysaccharide membrane

The morphology of the adipose stem cells cultured on the chitosan-twinkle polysaccharide membrane for 24 hours and 48 hours is as shown in Fig. 19, and the neural stem cells are also spherical.

The ratio of chitosan to tremella polysaccharides includes, but is not limited to, 1:1, and is about 3:0.4 to 3:4.5.

Example 7

Lung stem cell (LSC) cultured with chitosan, chitosan-hyaluronic acid, polycaprolactone, polycaprolactone-hyaluronic acid membrane

7.1 Preparation of chitosan, chitosan-hyaluronic acid, polycaprolactone (PCL), polycaprolactone-hyaluronic acid membrane

The preparation of chitosan and chitosan-hyaluronic acid ester was the same as in Example 5.

Polycaprolactone (purchased in Sigma) having a molecular weight of 80 kDa was dissolved in 1,4-dioxane, which was 11% of polycaprolactone. The above polycaprolactone is cast on a glass slide, which is a polycaprolactone film. Then, after scanning at a distance of 10 mm 6 m/min with atmospheric plasma (energy 1 kW, purchased from Plasmatreat, Germany), 300 ul of 2.95 mg/ml aqueous hyaluronic acid solution was dripped on it, and washed away overnight, hyaluronic acid. The plasma is grafted onto the surface at a density of 0.5 mg/cm ² .

7.2 Extraction and culture of mouse lung stem cells (LSC)

The mouse lung stem cell line is based on 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 vitro. Proc Natl Acad Sci US A. 2006 Jun 20; 103(25): 9530-5. Epub 2006 Jun 13, obtained from mouse lung.

7.3 Results of culture of lung stem cells in chitosan, chitosan-hyaluronic acid, polycaprolactone and polycaprolactone-hyaluronic acid membrane

Lung stem cells form a globular shape on chitosan and chitosan-hyaluronic acid, and the nanog/oct4 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.

<110> National Taiwan University

<120> Method for producing adult stem cells as a spheroid cell population

<130> 990544-I1

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 48

<212> PRT

<213> Synthetic

<220>

<223> Cellulose Binding Functional Region - RGD Attachment Sequence (CBD-RGD)

<400> 1

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 100 μm.

The second figure is the morphology of neural stem cells cultured on different chitosan (a. chitosan-1 b. chitosan-2) on days 1, 2, and 3; the scale bar is 100 μm.

The third panel is 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 panel is the a. 3 b. 5 c. 7 d. 15 e. 20 f. 25-day cell morphology (left) and F1B-fluorescein expression (right); scale bar is 200 μm.

The fifth panel shows the performance of neural stem cells cultured in chitosan-1 on days 1, 4, and 21 of a. F1B-fluorescent green protein and b. nestin (immunofection); the scale bar is 100 μm.

The sixth figure is the sphere morphology of a. DAPI (nucleus) and b. β3-tubulin (immuno staining) cultured on chitosan-1 on days 4, 14, and 21; the scale bar is 100 μm.

The seventh panel is a 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 μm.

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 graph shows the cell pellet 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- Aza

The tenth figure shows the cell morphology of rat adipose stem cells (rADASs) cultured on Chitosan3 (CS3), Chitosan 1 (CS1), Chitosan1+CBD-RGD (CS1R) materials on days 1, 3 and 11; -: No added 5-aza; +: Add 5-aza

The eleventh figure is a capillary electropherogram of rat adipose-derived stem cells (rADASs) after RT-PCR on TCPS(T), Chitosan 1 (CS1), Chitosan1+CBD-RGD (CS1R) materials; Cardiomyocyte positive (CP) ): No added 5-aza; +: Add 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 100 μm; the magnification is 10 times.

The thirteenth figure is the morphology of the human gingival stem cells cultured on the chitosan membrane after implantation. A. 12 B. 14 C. 16 D. 18 E. 20 F. 22 G. 24 hours in the same range; 100 μm, the magnification is 10 times.

The fourteenth figure is a human gingival stem cell cultured in the form of chitosan after implantation of A. 0 B.1 C. 2 D. 3 days; and cultured in TCPS after implantation E. 0 F. 1 G. 2 H. 3 days of morphology; human gingival stem cells cultured in I. Chitosan containing induced culture solution J. Chitosan containing basic culture solution and K. TCPS containing induction culture solution The morphology of the basic culture solution after 1 day of TCPS; human gingival stem cells cultured in M. chitosan containing induction culture solution N. chitosan containing basic culture solution and O. TCPS containing induction culture solution The morphology of TCPS in the basic culture medium is 2 days after cartilage induction; human gingival stem cells are cultured in Q. Chitosan containing induction culture solution R. Chitosan containing basic culture solution and S. TCPS containing induction culture solution T. The morphology of the TCPS containing the basic culture medium 7 days after the cartilage induction; the human gingival stem cells were cultured in U. The chitosan containing the induced culture solution V. The chitosan containing the basic culture solution and the W. containing induction culture TCPS of liquid X. The form of TCPS containing basic culture solution was 14 days after cartilage induction; the scale was 100 μm, and the magnification was 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. (A) Cell morphology cultured in TCPS (B) Cell morphology after incubation with chitosan and chitosan-hyaluronic acid containing the basic culture solution for three days.

The seventeenth figure is the quantitative result of mRNA expression of adipose stem cells cultured in the basic culture medium chitosan and chitosan-hyaluronic acid and TCPS dry genes (Oct4, Sox2 and Nanog) on the 10th day.

Figure 18 shows the chondrocyte gene expression of adipose stem cells and placental stem cells quantified by RT-PCR 7 days after cartilage induction in chitosan and chitosan-hyaluronic acid membranes (compared to TCPS).

The nineteenth figure is the morphology of the cultured adipose stem cells in the chitosan-twinkle polysaccharide film at (A) 24 hours, and (B) 48 hours later.

The twentieth figure shows the morphology of the cultured lung stem cells on the polycaprolactone and polycaprolactone-chitosan membranes on the first and second days.

The twenty-first figure shows the mRNA expression results of the stem genes (actin, Oct4, Sox2, and Nanog) on the first and third days of the culture of the lung stem cells in the polycaprolactone and polycaprolactone-chitosan membranes.

Claims (20)

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 biocompatible polymer is deacetylated 60 to 100% chitosan, alginate having a molecular weight of 9,600 to 132,000, polycaprolactone having a molecular weight of 64 to 96 kDa, or any combination thereof; and collecting a population of spheroid cells aggregated by adult stem cells, The spheroid cell population expands and has the ability to self-renew and differentiate into cells. The method of claim 1, wherein the adult stem cells are selected from the group consisting of neural stem cells, neural precursor cells, adipose stem cells, gingival stem cells, bone marrow mesenchymal stem cells, lung stem cells, and placental stem cells. The method of claim 1, wherein the biocompatible polymeric film is coated onto any substrate by coating or grafting. The method of claim 1, wherein the chitosan having a deacetylation degree of 60 to 100% is further combined with hyaluronic acid, and the ratio of chitosan to hyaluronic acid is about 3: 0.177 to 3: 4.425 (w/w). The method according to claim 1, wherein the chitosan having a deacetylation degree of 60 to 100% is further combined with the tremella polysaccharide, and the ratio of the chitosan to the tremella polysaccharide is about 3:0.4 to 3:4.5 (w/w). The method of claim 1, wherein the polycaprolactone having a molecular weight of 64 to 96 kDa is further combined with hyaluronic acid. The method of claim 1, wherein the somatic cell is a nerve cell. 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. The method of claim 9, further comprising contacting the adipose stem cells with 5-azacytidine to differentiate the adipose stem cells into the cardiomyocytes. The method of claim 8, wherein the spheroid cell population is the gingival stem cell, the adipose stem cell, or the placental stem cell, and differentiates into the chondrocyte. The method of claim 11, further comprising contacting the gingival stem cell, the adipose stem cell, or the placental stem cell with a transforming growth factor β3 (TGF-β3) to differentiate into the chondrocyte. An adult stem cell having differentiation ability is prepared by contacting an adult stem cell with a film formed of a biocompatible polymer for an effective period of time, wherein the biocompatible polymer system It is a chitosan with a degree of acetylation of 60-100%, alginate with a molecular weight of 9,600-132,000, polycaprolactone having a molecular weight of 64-96 kDa or any combination thereof; wherein the adult stem cells form a A population of spheroid cells that expand and have the ability to differentiate into a neuronal cell, a glial cell, a chondrocyte, or a cardiomyocyte. 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. The adult stem cell according to 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). The adult stem cell according to claim 13, wherein the chitosan having a deacetylation degree of 60 to 100% is further combined with hyaluronic acid, and the chitosan and hyaluronic acid are used. The ratio is approximately 3:0.177 to 3:4.425 (w/w). A pharmaceutical composition comprising the adult stem cells of claim 13 and a pharmaceutically acceptable carrier. A method for screening adult stem cells with 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 spheroid cell population are self-renewing The ability of the adult stem cells, wherein the biocompatible polymer is a chitosan having a deacetylation degree of 60 to 100%, a brown alginate having a molecular weight of 9,600 to 132,000, and a polycondensation having a molecular weight of 64 to 96 kDa. Ester or any combination thereof. The method of claim 18, wherein the effective time is preferably from 1 day to 4 days. The method of claim 18, wherein the biocompatible polymeric film is coated onto any substrate by coating or grafting.