TW201331366A - In-vitro serum-free culturing method for somatic stem cell proliferation - Google Patents

Abstract

This invention makes use of human fat mesenchymal stem cell culturing with the addition of different serum substituents and cytokines as well as the control of an optimal concentration formulation to establish a function detection system for mesenchymal stem cells, thereby establishing a large-scale and correct in-vitro serum-free proliferation and a culturing system. This invention relates to an in-vitro serum-free culturing method for the somatic stem cell proliferation, in which the plasma rich in growth factor (PRGF) is added to a serum-free stem cell culture medium for carrying out culturing and sub-culturing of human somatic stem cells. The human somatic stem cells cultured by this method, after multiple cycles of the sub-culturing, can still maintain a substantially undifferentiated state.

Description

In vitro serum-free adult stem cell amplification culture technique

The invention can establish a large number of correct and correct cells by culturing human adipose mesenchymal stem cells, by adding different serum substitutes and cytokines, and regulating the optimal concentration formula, and establishing a detection system for mesenchymal stem cell function. In vitro serum-free proliferation culture system.

Stem cells are primitive and unspecialized cells that are under-differentiated and have the potential to regenerate various tissues and organs. In all multi-cellular tissues, they can divide into a variety of specialized cells via mitosis and differentiation, and can use self. Updated to provide more stem cells. For mammals, stem cells fall into two broad categories: embryonic stem cells and adult stem cells. Embryonic stem cells are derived from inner cell masses in blastocysts; adult stem cells are derived from a wide variety of tissues. In adult tissues, stem cells and precursor cells serve as body repair systems that complement adult tissues. During the embryonic development phase, stem cells can differentiate into any specialized cells, but still maintain normal metastasis of new tissue (such as blood, skin or intestinal tissue).

The discovery of Ernest A. McCulloch and James E. Till at the University of Toronto in the 1960s opened the door to stem cells. Today, we can use in vitro culture techniques to grow or transform stem cells into several specialized cells, or to form cells that make up specific tissues such as muscles or nerves. Adult stem cells with high plasticity have been used in medical practice. There are many sources of stem cells, including embryos, cord blood, bone marrow, fat and amniotic fluid. The use of embryonic stem cells to grow into therapeutic tissues and even organs is a very novel and challenging subject. It develops under good CMC labor standards and may become a new medical method in the future, solving the shortage of current organ transplantation. The problem. Medical researchers believe that stem cell research (also known as regenerative medicine) has the potential to alter the response to human disease by repairing specific tissues or growing organs. However, the National Institutes of Health report of the US government pointed out that "important technical obstacles still exist and it will take several years of concentrated research to overcome."

The sources of stem cells can be divided into the following five categories:

1. Cord blood stem cells:

The blood remaining in the placenta and umbilical cord after birth is rich in hematopoietic stem cells. Since 1988, cord blood stem cells have been used to treat many childhood diseases such as Jeddah Syndrome, Henda Syndrome, Lara Syndrome, and Acute Lymphocytic Leukemia. Cord blood is collected by the umbilical cord; after the umbilical cord is cleaned and disinfected, the cord blood is taken out from the umbilical vein, and the infectious agent and the human leukocyte surface antigen are tested, and then stored in liquid nitrogen for storage. When used, first thaw and then inject into the patient's vein. If the treatment of other donor stem cells is used, it is called allogeneic transplantation; if the stem cells are from the patient, they are autografted.

2. Peripheral blood stem cells:

The bone marrow contains the source of the most important hematopoietic stem cells in the body, while the peripheral blood stem cells refer to the use of white blood cell growth hormone (G-CSF) to drive the stem cells in the bone marrow to the peripheral blood, and then collect them through a blood separator. Stem cells. These stem cells belong to CD34 ⁺ hematopoietic stem cells, which are often used clinically to improve central nervous system and cardiovascular diseases. They are also mainly used to treat diseases of hematopoietic system degradation or abnormal immune system. Patients suffering from major pain require general anesthesia, and bone marrow transplantation in the bone marrow of the hematopoietic, mesenchymal stem cells and matrix precursor cells, etc., is a wide range of indications or systemic hereditary serious disease treatment, if only for the part For systemic diseases, peripheral blood stem cells can be used to replace bone marrow transplants.

3. Embryonic stem cells:

Embryonic stem cells are obtained from undifferentiated internal cell masses of blastocysts (early embryos consisting of 50-100 cells), belonging to near-omnipotent stem cells, having the ability to differentiate into three germ layers, exhibiting high-end granzyme activity and can be cultured in vitro for two years. The above time can still maintain the ability to self-update. The current research on embryonic stem cells is still in its infancy. Many studies are still based on animal models other than humans, such as mice, cattle or sheep. As of 2011, a human trial is officially registered in the US clinical trials. Because of the high titer of embryonic stem cells, the methods and indications for clinical use need to be confirmed by more rigorous experiments. In addition, since the source of human embryonic stem cells involves moral and ethical constraints, the establishment of stem cell lines is controversial. (Because stem cell lines must acquire human embryos, they are purified by culture, etc. Therefore, many people think that stem cell lines are considered part of humans).

4. Adipose stem cells:

In the past, the fat extracted by people for body sculpting was usually disposed of as medical waste. It has been confirmed by medical experts that fat contains a large number of mesenchymal stem cells. The mesenchymal stem cells have the potential for in vitro proliferation and multiple differentiation, which can be applied to tissues and tissues. Regeneration and repair of organs.

Its main features are:

(1) The acquisition method is low invasive and harmless to the human body.

(2) It is easy to obtain a large amount.

(3), can be cultured in vitro.

(4) It can be applied to the body with a wide range of tissues. After entering the body, it can be automatically moved to the wound site for repair.

5. Skin cells:

At the end of 2007, scientists from both the United States and Japan successfully transformed skin cells into an induced pluripotent stem cell (iPS) and successfully transformed these stem cells into parts of the body's organs. By implanting specific genes into skin cells, skin cells can be transformed into cells that resemble embryonic stem cells.

Adipose tissue is rich in many stromal cells, progenitor cells and mesenchymal stem cells with regenerative capacity. We can obtain autologous adipose tissue through liposuction or liposuction. Purified adipose stem cells can be obtained by in vitro isolation and culture. Therefore, compared with other adult stem cells, adipose stem cells have the advantage of being easy. Adipose tissue can be isolated in vitro to obtain a stromal vascular fraction (SVF), which is a combination of stromal cells, blood cells, vascular endothelial cells and adipose stem cells. These include CD34 ⁺ hematopoietic stem cells, CD31 ⁺ endothelial cells, CD45 ⁺ immune cells, and CD105 ⁺ mesenchymal stem cells. These different cell populations play their part in the human body and play an important role. . Adipose-derived stem cells are a group of stem cells with mesenchymal stem cell characteristics but slightly different cell tropism. Scientists can determine whether they are stem cells by examining cell surface antigens such as CD34, CD105, CD73, CD44 and CD90. In the application and treatment of cells, the cooperation between various cells and adipose stem cells is an important key to the treatment of diseases.

After the stromal vascular cell population is purified in vitro, the cells are characterized by a characteristic analysis called adipose stem cells (or adipose stromal cells). Adipose stromal cells belong to adherent cells when cultured in vitro, and since adipose stromal cells are stem cells with multipotency and have the ability to differentiate across germ layers, they maintain their cells in a state of non-differentiation when expanded in vitro ( Undifferentiated state) is very important, in other words, the ability to maintain self-renewal of stem cells. The growth state of stem cells can be judged by observation of cell type, cell antigen labeling, and stem cell gene expression. Adipose-derived stem cells grow in their own microenvironment, which is also known as Niche in science. For example, adipose-derived stem cells belong to adherent cells, and in the extracellular matrix of culture environment, it is necessary to contain integrins specific to adipose stem cells. Ligands, these ligands belong to some extracellular matrix, which has the function of helping cells to attach and crawl. In addition to the extracellular matrix in culture, the medium, medium supplement and co-cultured materials, structure, space, etc. are all important factors affecting cell self-renewal. When the culture is enlarged, the cells are kept the most primitive and closest to the primary cells (primary The identity of cell is the ability to maintain stem cell self-renewal.

Use of stem cells

Stem cell is the origin cell; in the process of cell differentiation, cells often lose the ability to re-divide due to high differentiation, and eventually die. In order to make up for this deficiency, the organism retains a part of undifferentiated primitive cells in the process of development and adaptation. Stem cells are a kind of cells with self-renewal and differentiation potential. Scholars believe that they can be used to treat hereditary diseases, malignant tumors and other chronic diseases. Stem cells are used to grow into tissues and organs. They can be used for transplantation and anti-aging medicine and prolong humanity. Life and quality of life.

Stem cell transplantation treatment technology is known as the most leaping medical treatment in human history. It can repair and renew various organs of the human body and can eliminate more than 80% of various diseases.

The difficulty in treatment with adult stem cells is that not all tissues and organs can separate stem cells, and the number is small; if stem cells isolated from other organs develop into specific tissue cells, their transplantation and functionality are lower than the original. Stem cells isolated from organs. The characteristics of stem cells cultured in vitro may change during the culture process, and thus there are cases where the results of in vitro studies differ from the actual conditions applied to the human body in clinical practice. If the patient is to be separated from the stem cells for treatment, the stem cells may also have potential factors for the disease, and it is time consuming to perform transplant pairing to find the donor. In research, the function of adult stem cell differentiation has not been fully clarified, and there may still be risks in the treatment.

A technique conventionally used for large-scale cultivation of stem cells in vitro is to use serum as an additive for a medium which provides various components required for cell growth in cell culture, such as hormones, growth factors, and binding proteins. However, the use of serum also has the potential to be contaminated by pathogens such as bacteria, mycoplasma or viruses, plus the high cost of the serum, large batch variability and interference with subsequent recovery and purification of the product. Therefore, for the culture of human adipose mesenchymal stem cells, by adding different serum substitutes and cytokines, and regulating their optimal concentration formula, and establishing a detection system for mesenchymal stem cell function, a large number of correct in vitro can be established. Serum proliferation culture system.

Therefore, in view of the above, as well as the possibility, the inventor of the present invention has improved and innovated, and after years of painstaking research, finally succeeded in research and development of this in vitro serum-free adult stem cell amplification culture technology.

The main object of the present invention is to provide different serum substitutes and cytokines, and to regulate the optimal concentration formula, and to establish a detection system for mesenchymal stem cell function, and to establish a large number of correct in vitro serum-free proliferation culture systems.

A secondary object of the present invention is to provide a method for amplifying culture of serum-free adult stem cells in vitro, which comprises the following steps: Step 1: providing a human tissue; Step 2: hydrolyzing the tissue obtained in step one by centrifugation The unhydrolyzed tissue is separated from the human adult stem cells; Step 3: The human adult stem cells obtained in the second step are subjected to primary culture in a serum-free stem cell culture medium containing autologous growth factor (PRGF); Step 4: Take out step 3 The adherent cells in the primary culture medium are subcultured in a serum-free stem cell culture medium containing autologous growth factors to complete the expansion of human adult stem cells.

The human tissue is selected from the group consisting of umbilical cord, bone marrow, placenta, fat, blood, and deciduous teeth.

The enzyme for hydrolyzing human tissue is trypsin or collagen decomposing enzyme.

The primary culture step is carried out for 3 to 15 days.

The serum-free stem cell culture solution containing the autologous growth factor is added to autologous growth factor (PRGF) isolated from autologous blood in a serum-free stem cell culture solution; the autologous growth factor is added to the serum-free stem cell culture solution. The ratio is 0.1 to 10% (volume ratio).

The step further comprises: confirming the surface antigen labeling characteristics of the primary cultured and subcultured human adult stem cells by flow cytometry.

Among them, human adult stem cells after subculture are still in a state of being substantially undifferentiated.

It is an object of the present invention to provide a serum-free adult stem cell culture solution for culturing human adult stem cells, which comprises an autologous growth factor and a serum-free stem cell culture solution.

Wherein the serum-free medium containing the autologous growth factor is added with autologous growth factor (PRGF) isolated from autologous blood in an adult stem cell culture solution; the proportion of the autologous growth factor added to the adult stem cell culture solution It is 0.1 to 10% by volume.

The autologous growth factor (PRGF) is obtained after separation and purification from human autologous blood.

It is used for primary culture and subculture of human adult stem cells.

The human adult stem cell line is selected from the group consisting of cord blood stem cells, bone marrow stem cells, placental stem cells, adipose mesenchymal stem cells, human blood cells, and deciduous stem cells.

Among them, human adult stem cells after subculture are still in a state of being substantially undifferentiated.

The present invention is exemplified by the following examples, but the present invention is not limited by the following examples.

Example 1: Isolation and in vitro culture of interstitial stem cells

In the following embodiments, the stromal vascular cell population is isolated by adipose tissue, and further subjected to culture to obtain adipose stem cells (fatty stromal cells), and the present invention will be further described.

1. Vascular cell population separation

The adipose tissue obtained by surgery was centrifuged to 1000 RCF, centrifuged for 10 minutes to remove blood water, large connective tissue and other impurities to leave a layer of pure adipose tissue, followed by adding an appropriate amount of phosphate buffer solution and collagenase to activate at 37 ° C. The incubator was shaken for 45 minutes in the incubator, then rinsed with phosphate buffer, carefully removed at 500 RCF, centrifuged for 5 minutes, leaving the stromal vascular cell mass underneath, repeatedly washed three times, inoculated to contain no The serum medium is cultured in a T-shaped flask, and after 1 to 3 days, the unattached cells are removed, and the spindle cells to which the T-shaped bottle has been attached are the adipose stem cells. This cell was continuously cultured in serum-free medium for characterization.

2. Adipose stem cell culture and passage

Serum-free MesenCult for adipose stem cell culture -XF Basal Medium (Stem Cell Technology), adding 20% MesenCult -XF Supplement (Stem Cell Technology), and 2 mM L-glutamine (GIBCO) and antibiotic 50 μg/ml gentamicin (GIBCO) as long-term medium. T-shaped flasks are pre-exposed with MesenCult before inoculation of cells -XF Artachment Substrate (Stem Cell Technology) treatment for cell attachment. After the cells were attached, the cells were cultured at 37 ° C in a 5% CO ₂ incubator. When the cell density was as long as 80%, the cells were subcultured. Aspirate the medium, wash the cells with phosphate buffer solution, and add MesenCult Enzymatic Dissociation Solution (Stem Cell Technology), which is applied at 37 ° C for 5 minutes to attach the cells, then add the same amount of MesenCult Enzymatic Inhibition Solution (Stem Cell Technology) terminates the enzyme action, collects the cells, uses the hemacytometer to calculate the number of cells, re-inoculates the cells into new T-shaped flasks, and continues to culture until the cells grow to 7 minutes or more in the same way. Subculture again, once every four days, in the culture group with added PRGF, PRGF was directly added to the serum-free medium during the subculture or inoculation, and the amount of PRGF added was 100 μl of PRGF added to 10 ml. In the serum-free medium, the growth factor released by 2 × 10 ⁵ to 1 × 10 ⁶ platelets per ml of the serum-free medium is added to the PRGF in proportion to the replacement medium in the passage.

3. Preparation of autologous growth factor PRGF

Prepare CPT (BD REF 362761) camouflage blue tube for blood collection. The CPT tube contains anticoagulant, Ficoll and a layer of solid glue. Each tube can collect about 8 ml of blood. When separating, centrifuge at 1700 RCF for 20 minutes at room temperature. After centrifugation, the blood is divided into four layers in the CPT tube. From top to bottom, the plasma, platelet rich plasma (PRP), solid The gel layer contains Ficoll and red blood cells; the upper plasma layer is aspirated into a new 50 ml centrifuge tube, and the white PRP layer is aspirated into a new 15 ml centrifuge tube. According to the ratio (the ratio of plasma layer to platelet layer volume is 1:2.5), add appropriate amount of plasma to another new 15 ml centrifuge tube, and then add the proportionally calculated calcium gluconate (calcium gluconate to plasma volume ratio is 0.3:1), placed at 37 ° C for 15 minutes, centrifuged at 700 RCF for 10 minutes at room temperature. After the centrifugation, the supernatant was added to the PRP and thoroughly mixed, and the reaction was carried out at 37 ° C for 40 minutes to complete the preparation of PRGF.

4. Platelet layer PRP and plasma platelet quantification

Flow cytometry was used to quantify the number of platelets in PRP. 50 μl of PRP and 50 μl of plasma were added to phosphate buffer, and anti-CD41-PE antibody was added for staining. The immunostaining was completed and Count Bright Beads (Invitrogen) was added for counting. The reference value was added to the phosphate buffer solution to make up the total volume to about 0.5 ml and then analyzed by FACS Calibur 3 color (BD). The results of the analysis can be calculated by inserting the following formula to calculate the platelet content of PRP and plasma.

= concentration of platelets in the sample / μl)

Where A = number of platelets; B = number of beads; C = beads used for each 50 μl of the batch number; D = volume of the sample (μl)

5. Cell surface antigen analysis

The stem cells obtained in the present study were subjected to measurement of cell surface antigen by flow cytometry (Flow Cytometry, BD FACScalibur). After detaching the cells and washing them with phosphate buffer, they are dissolved in a suitable amount of phosphate buffer, and the different antigens are stained with corresponding immunofluorescent antibodies, including IgG, CD34, CD45, CD44, CD73, CD90. , HL-ADR, etc. After temperature in the dark for 15 min at room after the addition of an appropriate amount of phosphate buffer mechanical analysis, data collected by flow cytometry, to CELL Quest ^TM (BD) analysis software.

6. Adipose stem cells differentiate in vitro

In the osteoblast (Osteoblast) differentiation, the obtained adipose stem cells were cultured at 3000 cells/cm ² in Dulbecco's Modified Eagle Medium (DMEM high glucose, GIBCO), 10 mM β-glycerophosphate (Sigma), 0.1 M Dexamethasone (Sigma), 0.2 mM Ascorbic acid (Sigma), 10% (v/v) fetal bovine serum (Hyclone) hard bone induction medium, medium changed every three days. In the differentiation of adipocyte, the obtained adipose stem cells were cultured in DMEM at 10000 cells/cm 2 and added with 0.5 mM 3-Isobutyl-1-methylxanthine (IBMX, Sigma), 10 μg/mL Insulin (Sigma), 100 μM Indomethacin ( Sigma), 1 μM Dexamethasone (Sigma), 10% (v/v) fetal bovine serum (Hyclone) fat-inducing medium, medium changed every three days.

7. Cytochemical staining

The differentiated adipocytes were stained with Alkaline phosphatase (ALP), and the Fast Blue RR Salt solution was first mixed with AS-MX phosphate Alkaline solution at a ratio of 24:1 at 25 ° C and protected from light; After rinsing with phosphate buffer; fixed with citrate buffered acetone for 30 seconds, and then infiltrated with secondary water for 45 seconds; then the previous mixture was added to the flask, and stored at 37 ° C for 60 minutes, then removed. After infiltration for 2 minutes with secondary water, the nuclei were stained with hematoxylin solution (Sigma) for 1 minute; the washing was continued with secondary water until the secondary water turned pale blue and then observed under a microscope. The differentiated osteoblasts were stained with Oil red-O, the differentiation medium was aspirated, washed with phosphate buffer, and the cells were fixed at room temperature for 15 minutes with 3.7% paraformaldehyde in PBS/pH 7.4, followed by secondary water. Rinse, stain with Oil Red O (Sigma) for 10 minutes, infiltrate with 50% ethanol, and rinse with secondary water; then stain the nucleus with hematoxylin solution (Sigma) for 1 minute; continue to rinse with secondary water until secondary water After changing to light blue, observe it with a microscope.

8. Reverse transcription-chainase enzymatic polymerization

The adipose stem cells obtained in this study were analyzed by reverse transcription-chainase-chain polymerization (RT-PCR) for the expression of genes related to undifferentiated stem cells. The cultured cells were washed with phosphate buffer, collected in 1.5 ml eppendorf, and added with 1 ml of TriZol (10296-010, Invitrogen) reagent for 5 minutes at room temperature, and 100 μl of BCP (BP. 151, MRC) was added. The solution was mixed with Vortex until it became a pink solution, allowed to stand at room temperature for 15 minutes, and then centrifuged at 15,000 g for 15 minutes at 4 °C. After centrifugation, the eppendorf will be divided into three layers, the lower layer is a red layer, the middle layer is a thin white layer, and the upper layer is a transparent layer. The upper layer is sucked out into a new 1.5 ml eppendorf. Be careful not to suck in the suction process. Two floors. Add 0.5 ml of isopropanol to the new eppendorf, shake well, leave it at room temperature for 30 minutes, then centrifuge at 15,000 g for 10 minutes at 4 °C, remove the supernatant, do not suck the pellet, add 1 ml of 75% ethanol. After centrifugation at 15,000 g for 10 minutes at 4 ° C, the ethanol was withdrawn, air dried for 10 minutes, and RNA extraction was completed after reconstitution with water containing DEPC. Aspirate the appropriate amount of RNA, add NCode cDNA synthesis Kit (A11193-050, Invitrogen), add RT to the PCR machine and add GoTaq Green Master Mix (M7122, Promega) to run PCR. The PCR setting conditions are slightly adjusted due to the different Tm values of different Primers. . Analysis of undifferentiated genes associated with stem cells, such as oct4, nanog, sox2, cMyc, Lin28, hTERT, etc. This patent analyzes oct4, sox2, klf4, nanog, utf1, hTERT and the control group gene GAPDH. The primers used to analyze each gene are listed in the following table:

Example 2: Adding PRGF to serum-free medium for amplifying cultured adult stem cells 1. Adipose stromal vascular cell population separation

The clinical human samples used in the present invention are a total of four cases, which are referred to as numbers BN456812, BN262813, BN726415, and BN998619, respectively.

The isolated stromal vascular cell population is further cultured into adipose stem cells, and the culture and subculture are described later.

2. Adipose stem cell culture

The adipose-derived stem cells are cultured in a serum-free medium by the method described above. In the culture process, according to the quantitative result of PRP, PRGF equivalent to 1×10 ⁷ platelet separation is added per ml of the culture medium, and the growth curve and cell characterization of the cells with or without added PRGF are compared. result. . The cell count of the cells of the present invention collected in serum-free culture is listed in the following table:

The experimental data of the above table is plotted as a cell growth map (Fig. 1). It can be clearly seen that the cell culture group with the addition of PRGF has a faster cell growth than the group without the addition. When the cells grew in early algebra (P0~P1), because the cells were younger, they were easy to maintain self-renewal. There was no significant difference in the number of cells between the two groups; however, with the passage, the cells gradually became aging, because the PRGF was rich. Containing growth factors, it is speculated that it can help stem cell signaling to initiate and maintain the ability of stem cells to self-renew, so the growth rate can still be maintained.

If the number of cells is converted to cell magnification (Table 2), it can be clearly seen that by the second generation, the number of cells has been more than doubled; to the third generation, the number of cells is added with or without added PRGF. There are significant differences in the group (Figure 2); stem cells need autocrine and paracrine to regulate the transmission of signals while maintaining undifferentiated growth; cells in a serum-free culture environment It is easy to change the original characteristics, because serum-free culture environment does not provide many growth factors and cytokines necessary for growth, and even trace mineral elements. If long-term culture is in such a harsh environment, cells will lose their originality. There is identity in a physiological environment; the function of PRGF is to supply many kinds of autologous growth factors and cytokines, or some trace chemicals, which are not suitable for helping the secretion of paracrine secretions of adipose stem cells. Lack of long-term maintenance, stemness signaling pathway A very important influence.

When adipose stem cells are cultured in vitro, the state of the cells can be judged by type observation, and the growth state and differentiation state of the adipose stem cells can be understood from the type of adipose stem cells. Adipose-derived stem cells belong to adherent cells when cultured and have a spindle cell type similar to fibroblasts. In the present invention, the type of adipose stem cells cultured in a serum-free environment is normal, and the adipose stem cells exhibit a normal spindle cell type in an early subculture number (P0~P1), a culture group with and without addition of PRGF. However, some slight differences can be seen; in the group without PRGF, some migratory cells are irregular in shape, even slender, even large and flat, and the cell density that can be achieved is also low. (Figure 3A); however, among the groups with added PRGF, the cell type is round and small, the nucleoplasmic ratio is large and the cell density is high (Fig. 3B). After the cells are cultured to the late algebra (such as P3), the difference in cell type is also more obvious. Cells without added PRGF will become slender and more irregular, and some cells will be creased. Folded and cells flattened, these are typical cell aging or gene senescence (Figure 4A); conversely, there are culture groups with added PRGF, cell alignment, lateral association The behavior is good, the cell type is round and small, and it is close to the cell type of the primary cells (Fig. 4B).

From the observation of the cell culture pattern, it can be found that the cell type with the addition of PRGF is smaller than the circle which is not added, and is closer to the undifferentiated state; in addition, it is found that it can grow to a relatively full cell density and has been added. In the case of PRGF, the cells can reach about 55,000 cells/cm ² before P3, which is also reflected in the rapid growth of cells and the large number of cells.

3. Adipose stem cell characterization 3.1 Cell surface antigen analysis

The adipose-derived stem cells expanded in serum-free culture were analyzed by flow cytometry. The cell population was found to be CD34 ^- , CD45 ^- , HLADR ^- , CD44 ⁺ , CD73 ⁺ , CD90 ⁺ , which are cell populations similar to mesenchymal stem cells. . In the process of culture, the population of cells will become more and more pure, which can be observed from the flow cytometry FSC on the dot plot of SSC (Fig. 5, Fig. 6). The denser the dots, the more concentrated the population is. The results of the cell analysis of the statistical examples are shown in Table 3:

Adipose-derived stem cells in the culture process, if the type is unhealthy, inconsistent, poor culture, such as dead cells, etc., will affect the analysis results of flow cytometry. In the culture group with added PRGF, the dot pattern of SSC can be seen in the FSC (see Figure 6). Compared with the group without PRGF, the community is more dispersed (Figure 5). In addition, the culture group with added PRGF had higher expression in CD73 and CD90, and the intensity of the expression was more consistent. The peaks in the analysis chart were sharper (Fig. 6), which also echoed the morphological observation of the cells. . In the culture of late algebra (such as P7), the cell type is easily deformed. This type of change will reflect the staining results of CD73 and CD90. If the staining results of CD73 and CD90 are relatively low, it may imply that the cells are not maintained. In a state of growth without differentiation.

After statistical analysis of the expression of cell surface antigen, it was found that the group of PRGF-cultured adipose-derived stem cells was easier to maintain cell consistency, and the cell marker expression ratio of adipose-derived stem cells was higher than that of the group without PRGF (Fig. 7). CD44 is 3.2% higher (n=7, p=0.033); CD73 is 3.3% higher (n=7, p=0.0425); CD90 is 2.9% higher (n=7, p=0.079); three negative Cell marker expression was low. CD44 plays the role of communication and influence between cells; CD90 is a cell marker that many stem cells will express; CD73 is an enzyme that stem cells will express. When the cell state is consistent, the expression of these surface markers will be the same, and the adipose stem cells will behave. Conversely, if the cell characteristics change, the cell marker performance will also change. When a large amount of serum-free cells are cultured in vitro, it is necessary to maintain good consistency. The culture method by adding PRGF has a better effect in maintaining the performance of stem cell surface antigen than the method of not adding.

3.2 Stem cell gene expression analysis

RT-PCR analysis of adipose stem cell gene expression, especially for the analysis of six stem cell genes, can be found that oct4, sox2, nanog, klf4, utf1 and htert will be expressed in adipose stem cells, oct4, sox2, nanog is An important transcription factor in stem cells regulates and initiates the expression of many downstream genes, which is essential for maintaining stem cell self-renewal; klf4 is also an important protein found in both embryonic stem cells and mesenchymal stem cells, for maintaining stem cells. The potency plays an important role. In 2007, yamanaka in Japan and Thomson in the United States also used these combinations of genes to successfully direct somatic cells into induced pluripotent stem cells (IPS cells), indicating the importance of these genes for stem cells. In addition, the experimental results also show that if PRGF is added, it is easier to maintain the gene expression of utf1 and htert (Figure 8 and Figure 9), meaning that adipose-derived stem cells can maintain cell growth and not when PRGF is added in serum-free culture. The state of differentiation; the full name of utf1 is undifferentiated transcription factor 1, meaning that the transcription factor expressed when stem cells are not differentiated will regulate the expression of other stem cell genes; the full name of htert is human telomerase reverse transcriptase, which is human telomerase reverse transcriptase It can prolong the shortening of human telomere and has a direct relationship with the life of human cells.

3.3 Adipose stem cell differentiation

The adipose stem cells cultured in vitro have the ability to differentiate into mesoderm cells, such as fat and bone cells. In the examples, it was confirmed that the adipose stem cells cultured by PRGF can differentiate into fat and bone, have the differentiation ability of mesenchymal stem cells, and the experiment It was found that the differentiation ability of fat cells was very strong, and only a large number of oil droplets were produced after differentiation for seven days, probably because the source was a relationship of adipose stem cells (Fig. 10, Fig. 11, Fig. 12, Fig. 13).

4. Autologous growth factor PRGF quantification

The concentration of PRGF was quantified indirectly using the number of platelets in the quantitative samples. CD41 is a surface antigen expressed by platelets. During the production of PRGF, platelets will rupture, causing the release of substances in platelets into PRGF solution, which contains many growth factors, including VEGF, PDGF-BB, FGF2, etc. In this example, the particles expressing CD41 in the sample were analyzed by flow cytometry, and the internal reference group was added, and the actual number of CD41+ particles was calculated by the formula. The PRGF platelet content of BN456812 was 4371/μl, the PRGF platelet content of BN262813 was 94101/μl, the PRGF platelet content of BN726415 was 53095/μl, and the PRGF platelet content of BN998619 was 11028/μl.

The detailed description of the preferred embodiments of the present invention is intended to be limited to the scope of the invention, and is not intended to limit the scope of the invention. The patent scope of this case.

In summary, the serum-free adult stem cell amplification culture technology provided in this case has the above-mentioned multiple functions, and should fully comply with the statutory invention patent requirements of novelty and progressiveness, and apply for it according to law, and invite you to approve the invention patent. Apply for the case, in order to invent the invention, to the sense of virtue.

Figure 1. Growth of adipose stem cell cells in serum-free culture with or without added PRGF. P0 is the first generation culture, P1 is the first generation of the next generation, and so on. EXAMPLES After four days of serum-free culture in culture flasks, the cells were subcultured, the white group was the group without PRGF, and the gray group was the group with PRGF. *:-/+PRGF group had significant differences (p<0.05), n=4, t-test one-tailed statistics.

Figure 2. Growth magnification of adipose-derived stem cells in serum-free culture with or without added PRGF. P1/P0 is the magnification from the first generation to the first generation, and P2/P0 is the magnification from the first generation to the second generation. EXAMPLES After four days of serum-free culture in culture flasks, the cells were subcultured, the white group was the group without PRGF, and the gray group was the group with PRGF. *:-/+PRGF group had significant differences (p<0.05), n=4, t-test one-tailed statistics.

Fig. 3, PN cell culture of BN998619 was subjected to phase difference magnification photograph (200X) on the fourth day without serum, (A) no PRGF was added, and (B) PRGF was added.

Figure 4. P3 cell culture of BN726415 was amplified on the fourth day of serum-free day (200X), (A) no PRGF was added, and (B) PRGF was added.

Figure 5. BN262813 P7 cells were cultured in serum-free medium without added PRGF for four days. Flow cytometry was used to analyze the expression of surface antigen markers such as CD34, CD45, HLADR, CD44, CD73, and CD90, and the CellQuest software was used. do analysis.

Figure 6. P7 cells of BN262813 were cultured in serum-free medium supplemented with PRGF for four days. Flow cytometry was used to analyze the expression of surface antigen markers such as CD34, CD45, HLADR, CD44, CD73, CD90, etc., and using CellQuest software. analysis.

Figure 7. The performance ratio of adipose-derived stem cells to serum-free surface antigens in serum-free culture. Four days after serum-free culture, flow cytometry was used to analyze the expression of surface antigen markers such as CD34, CD45, HLADR, CD44, CD73, and CD90. The white group was the group without PRGF, and the gray group was Add PRGF group. *:-/+PRGF group had significant differences (p<0.05), n=4, t-test one-tailed statistics.

Figure 8. Stem cell gene expression of BN456812 and P2 cells. Adipose stem cells were examined by RT-PCR and then analyzed by agarose gel electrophoresis. M stands for Marker, the left picture shows the group with the added PRGF, and the right picture shows the group with no PRGF added. Abbreviations: G: gapdh, 347 bp; S: sox2, 139 bp; O: oct4, 103 bp; N: nanog, 142 bp; K: klf4, 182 bp; U: utf1, 117 bp; T: htert, 258 Bp;M: marker

Figure 9. Stem cell gene expression of BN262813 and P3 cells. Adipose stem cells were examined by RT-PCR and then analyzed by agarose gel electrophoresis. M stands for Marker, Marker's left side acts as a group with no PRGF added, and the right side has a group with PRGF added. Abbreviations: G: gapdh, 347 bp; S: sox2, 139 bp; O: oct4, 103 bp; N: nanog, 142 bp; K: klf4, 182 bp; U: utf1, 117 bp; T: htert, 258 Bp;M: marker

Figure 10. P2 cells of BN456812 were subjected to (A) adipocytes and (B) osteoblasts to day 14; 200-fold magnification, bright field.

Figure XI, P5 cells of BN262813 were subjected to (A) adipocytes and (B) osteoblasts to day 14; 200-fold magnification, bright field.

Figure 12, P3 cells of BN726415 were subjected to (A) adipocytes and (B) osteoblasts to day 14; 200-fold magnification, bright field.

Figure 13. P2 cells of BN998619 were differentiated between (A) adipocytes and (B) osteoblasts to day 14; 200-fold magnification, bright field.

Claims (15)

A method for amplifying culture of in vitro serum-free adult stem cells, the method comprising the following steps: Step 1: providing a human tissue; Step 2: hydrolyzing the tissue obtained in step one by centrifugation, and centrifuging the unhydrolyzed tissue into human The body stem cells are separated; Step 3: The human adult stem cells obtained in the second step are subjected to primary culture in a serum-free stem cell culture medium containing autologous growth factor (PRGF); Step 4: The adhesive type in the primary culture medium in the third step is taken out. The cells are subcultured in a serum-free stem cell culture medium containing autologous growth factors to complete human adult stem cell expansion culture. The method of claim 1, wherein the human tissue is selected from the group consisting of umbilical cord, bone marrow, placenta, fat, blood, and deciduous teeth. The method of claim 1, wherein the enzyme for hydrolyzing human tissue is trypsin or collagen decomposing enzyme. The method of claim 1, wherein the primary culture step is performed for 3 to 15 days. The method according to claim 1, wherein the serum-free stem cell culture solution containing autologous growth factor is added to autologous growth factor (PRGF) isolated from autologous blood in a serum-free stem cell culture solution. The method according to any one of claims 1 to 5, wherein the autologous growth factor is added to the serum-free stem cell culture solution in a ratio of 0.1 to 10% by volume. The method of claim 1, wherein the step further comprises: confirming the surface antigen labeling characteristics of the primary cultured and subcultured human adult stem cells by flow cytometry. The method of claim 1, wherein the human adult stem cells after subculture are maintained in a substantially undifferentiated state. A serum-free adult stem cell culture medium for culturing human adult stem cells, which comprises an autologous growth factor and a serum-free stem cell culture solution. The culture solution according to claim 9, wherein the serum-free culture solution containing autologous growth factor is added to autologous growth factor (PRGF) isolated from autologous blood in an adult stem cell culture solution. The culture solution according to claim 9, wherein the autologous growth factor (PRGF) is obtained by separating and purifying human autologous blood. The culture solution according to claim 9 is used for primary culture and subculture of human adult stem cells. The culture solution according to claim 9, wherein the autologous growth factor is added to the adult stem cell culture solution in a ratio of 0.1 to 10% by volume. The culture solution according to claim 9, wherein the human adult stem cell line is selected from the group consisting of cord blood stem cells, bone marrow stem cells, placental stem cells, adipose mesenchymal stem cells, human blood cells, and deciduous stem cells. The culture solution according to claim 9, wherein the human adult stem cells after subculture are still maintained in a substantially undifferentiated state.