WO2013123607A1 - 用于成体干细胞的体外无血清培养的方法和培养基 - Google Patents

用于成体干细胞的体外无血清培养的方法和培养基 Download PDF

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WO2013123607A1
WO2013123607A1 PCT/CN2012/000192 CN2012000192W WO2013123607A1 WO 2013123607 A1 WO2013123607 A1 WO 2013123607A1 CN 2012000192 W CN2012000192 W CN 2012000192W WO 2013123607 A1 WO2013123607 A1 WO 2013123607A1
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cells
serum
stem cells
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human
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French (fr)
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陈怡如
林挺宇
何宗晏
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光丽生医股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • C12N5/0037—Serum-free medium, which may still contain naturally-sourced components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • C12N5/0056—Xeno-free medium

Definitions

  • the invention relates to a method for amplifying culture of serum-free adult stem cells in vitro and a culture solution thereof. Background technique
  • 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-renewal 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 the embryo; adult cells are derived from a wide variety of tissues. In adult tissues, stem cells and precursor cells serve as a body repair system that complements adult tissues. In the embryonic development phase, the thousand cells can differentiate into any specialized cells, but still maintain normal metastasis of new tissue (such as blood, skin or intestinal tissue).
  • stem cell research also known as regenerative medicine
  • stem cell research has the potential to alter the response to human diseases by repairing specific tissues or growing organs.
  • National Institutes of Health report of the US government pointed out that "important technical barriers 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:
  • cord blood stem cells have been used to treat many childhood diseases such as Jeddah Syndrome, Henda Syndrome, and Syndrome, Acute Lymphocytic Leukemia.
  • Umbilical 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 disease source 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 you are using other donor stem cells,
  • Peripheral blood cells are:
  • the bone marrow contains the source of the most important hematopoietic 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 thousand cells in the bone marrow to the peripheral blood, and then collect them through the blood separator. Obtained stem cells. These stem cells belong to the hematopoietic cells of CD34+, which are often used to improve the central nervous system and cardiovascular diseases, and are mainly used to treat diseases of hematopoietic system degradation or abnormal immune system.
  • G-CSF white blood cell growth hormone
  • 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
  • peripheral blood stem cells can be used to replace bone marrow transplantation.
  • Embryonic stem cells are obtained from undifferentiated internal microtubules of embryos (early embryos consisting of 50-100 cells), belonging to nearly pluripotent stem cells, having the ability to differentiate into three germ layers, exhibiting high-end granzyme activity, and can be cultured in vitro. More than a year can still maintain the ability to self-renew.
  • 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 Trial. Due to the high titer of embryonic stem cells, the methods and indications for clinical use need to be more rigorously verified.
  • stem cell lines are controversial. (Because stem cell lines must acquire human embryos, they are purified by culture, etc. Therefore, many people think that stem cell forests are considered part of humans).
  • the fat extracted by body sculpting was usually disposed of as medical waste. It has been confirmed by medical experts that fat contains a large number of mesenchymal cells. Interstitial cells have the potential for in vitro proliferation and multiple differentiation. Tissue and organ regeneration and repair.
  • the acquisition method is low invasive and harmless to the human body.
  • iPS induced pluripotent stem cell
  • Adipose tissue is rich in many stromal cells, progenitor cells and mesenchymal cells with regenerative capacity.
  • Purified adipose-derived stem cells can be obtained by in vitro isolation and culture. Therefore, compared with other adult-type cells, adipose-derived stem cells have the advantage of being easy.
  • the stromal vascular fraction (SVF) can be obtained by separating the adipose tissue in vitro, and is a combination of stromal cells, blood cells, vascular endothelial cells and fat cells.
  • CD34+ hematopoietic stem cells CD31+ endothelial cells, CD45+ immune cells, and CD105+ mesenchymal stem cells.
  • Adipose-derived stem cells have a stem cell population with mesenchymal-cell characteristics but slightly different cell tropism.
  • Scientists can determine whether it is a thousand cells by examining cell surface antigens such as CD34, CD105, CD73, CD44 and CD90. .
  • the cooperation between various cells and fat cells is an important key to the treatment of diseases.
  • adipose stem cells or adipose stromal cells.
  • Adipose stromal cells belong to adherent cells when cultured in vitro, and because adipose stromal cells are multipotent cells, they have the ability to differentiate across germ layers, so 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 the thousand cells can be judged by observation of cell type, cell antigen labeling, and expression of a thousand cell genes.
  • Fat cell growth has its own microenvironment, which is also known as Niche in science.
  • the stem cell is a patch cell, and it needs to contain adipose stem cells in the extracellular matrix of the culture environment.
  • Unique integrin ligands, these ligatids belong to some extracellular matrix, which has the function of helping cells to attach and crawl.
  • the medium, the medium supplement and the co-cultured material, structure, and space are all important factors affecting cell self-renewal.
  • the cells When the culture is enlarged, the cells are kept the most primitive and closest to the first generation.
  • the identity of the primary cell is the ability to maintain stem cell self-renewal.
  • Stem cells are the cells of origin; during the differentiation of cells, cells often lose the ability to divide again 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. Thousand 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, which can be used for transplantation and anti-aging medicine. Extend human 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 eliminate more than 80% of various diseases.
  • the difficulty in treating with adult thousand cells is that not all tissues and organs can separate stem cells, and the number is small. If thousands of cells isolated from other organs develop into specific tissue cells, their transplantation and functionality are lower. Thousands of cells isolated from the original organs.
  • the characteristics of stem cells cultured in vitro may be changed 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 by a thousand cells for treatment, the thousand 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.
  • 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, through the addition of different serum substitutes and cytokines, and the regulation of their optimal concentration formula, and the establishment of a detection system for mesenchymal stem cell function, a large number of correct in vitro can be established. Serum proliferation culture system. Summary of the invention
  • the main object of the present invention is to provide a different serum substitute and cytokine addition, and to adjust its optimum concentration formula, and to establish a detection system for mesenchymal stem cell function, and to establish a large and correct external serum-free proliferation culture system.
  • a secondary object of the present invention is to provide a method for the in vitro culture of serum-free adult fine-grained adults, which comprises the following steps:
  • Step 1 Provide human tissue
  • Step 2 hydrolyzing the tissue obtained in the step 1 with an enzyme, and separating the unhydrolyzed tissue from the human adult stem cells by centrifugation;
  • Step 3 The human adult stem cells obtained in the second step are cultured in a serum-free stem cell culture medium containing autologous growth factor (PRGF);
  • PRGF autologous growth factor
  • Step 4 The adherent cells in the primary culture medium in the third step are taken out, and subcultured in a serum-free stem cell culture medium containing autologous growth factors to complete the human adult stem cell expansion culture.
  • the human tissue is selected from the group consisting of umbilical cord, bone marrow, placenta, fat, blood, and deciduous teeth.
  • the enzyme used to hydrolyze 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, and the phage-growth factor (PRGF) isolated from the autologous blood is added to the serum-free stem cell culture solution; the ratio of the autologous growth factor added to the serum-free cell culture medium is 0.1 ⁇ 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.
  • the serum-free culture solution containing the autologous growth factor, and autologous growth factor (PRGF) isolated from autologous blood is added to the adult stem cell culture solution; the ratio of the autologous growth factor to the adult stem cell culture solution is 0.1 to 10% (volume ratio).
  • PRGF autologous growth factor
  • the autologous growth factor (PRGF) is obtained after isolation and purification from human autologous blood.
  • the human adult stem cells are 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.
  • Figure 1 shows the growth of adipose-derived cells in serum-free culture with or without the addition of PRGF.
  • P0 is the first generation culture
  • P1 is the first generation of the generation, and so on.
  • Figure 2 is a graph showing the growth magnification of adipose-derived cells in serum-free culture with or without added PRGF.
  • P1/P0 is the magnification from the first generation to the first generation
  • P2/P0 is the magnification from the first generation to the second generation.
  • Figure 3 is a phase difference magnified photograph (200X) of the B1 cell culture of BN998619 on the fourth day of serum-free culture. (A) No PRGF was added, and (B) PRGF was added.
  • Figure 4 is a phase difference magnified photograph of the B3 cell culture of BN726415 on the fourth day of serum-free (200X), (A) no PRGF added, and (B) PRGF added.
  • Figure 5 shows the expression of surface antigen markers such as CD34, CD45, HLADR, CD44, CD73, and CD90 by flow cytometry analysis of P7 cells of BN262813 in culture medium without serum and without added PRGF for four days, and using CellQuest software. do analysis.
  • Figure 6 shows the expression of surface antigen markers such as CD34, CD45, HLADR CD44, CD73, and CD90 by flow cytometry analysis of P7 cells of BN262813 in serum-free medium supplemented with PRGF for four days, and analyzed by CellQuest software. .
  • Figure 7 shows the ratio of the expression of surface antigens in which adipose-derived stem cells were added without serum in serum-free culture.
  • Figure 8 shows the gene expression of the BN456812 and P2 cells.
  • the dried 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 PRGF added, and the right picture shows the group with no PRGF added.
  • G gapdh, 347 bp
  • S sox2, 139 bp
  • O oct4, 103 bp
  • N nanog, 142 bp
  • K klf4, 182 bp
  • U utfl, 117 bp
  • T htert, 258 Bp
  • M marker
  • Figure 9 shows the thousand cell gene expression of BN262813 and P3 cells.
  • the fat-dried fine moon pack was examined by RT-PCR, and then subjected to 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.
  • G gapdh, 347 bp
  • S sox2, 139 bp
  • O oct4, 103 bp
  • N nanog, 142 bp
  • K klf4, 182 bp
  • U utfl, 117 bp
  • T htert, 258 bp
  • M marker
  • Figure 10 shows P2 cells of BN456812 undergoing (A) adipocytes and (B) osteoblast differentiation to day 14; 200-fold magnification, bright field.
  • Figure 11 shows P5 cells of BN262813 undergoing (A) adipocytes and (B) osteoblast differentiation to day 14; 200-fold magnification, bright field. '
  • Figure 12 shows P3 cells of BN726415 undergoing (A) adipocytes and (B) osteoblast differentiation to day 14; 200-fold magnification, bright field.
  • Figure 13 shows P2 cells of BN998619 undergoing (A) adipocytes and (B) osteoblast differentiation to day 14; 200-fold magnification, bright field.
  • Example 1 Isolation and in vitro culture of adipose mesenchymal stem cells
  • the stromal vascular cell population is obtained by separation of adipose tissue, and further subjected to culture to obtain adipose stem cells (fat stromal cells), and the present invention will be further described.
  • the adipose tissue obtained by surgery is 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 act on the enzyme.
  • the mixture was shaken in a 37 ° C incubator for 45 minutes, then rinsed with phosphate buffer.
  • the supernatant was carefully removed by centrifugation at 500 RCF for 5 minutes, leaving the stromal vascular cell mass agglomerated. After repeated washing three times, inoculation.
  • the cells were cultured in a T-shaped flask containing serum-free medium. After 1 to 3 days, the unattached cells were removed, and the spindle cells to which the T-shaped flasks were attached were adipose stem cells. This cell was continuously cultured in a serum-free medium for characterization.
  • T-shaped flask was pre-treated with MesenCult®-XF Attachment Substrate (Stem Cell Technology) to inoculate cells for cell attachment. After the cells were attached, the cells were continuously cultured in a 37-inch, 5% C0 2 incubator. When the cell density was as long as 80%, the cells were subcultured.
  • the PRGF was directly added to the serum-free medium during the subculture or inoculation, and the PRGF was added to the sputum-free PRGF and added to the serum-free medium of 10 ml.
  • the serum medium contains 2 ⁇ 10 5 to 1 ⁇ 10 6 platelets and released the growth factor. If the medium is replaced in the subculture, the PRGF is added in proportion.
  • CPT camouflage blue tube blood
  • CPT tube contains anticoagulant, Ficoll and a layer of solid glue, each can collect about 8 ml of blood.
  • 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, it is plasma, platelet rich plasma (PRP), solid glue layer. Containing a FicolK red blood cell; aspirate the upper plasma layer into a new 50 ml centrifuge tube and aspirate the white PRP layer into a new 15 ml centrifuge tube.
  • PRP platelet rich plasma
  • 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 centrifugation, the supernatant was added to PRP and mixed thoroughly, and placed in a reaction at 37 ° C for 40 minutes, that is, the preparation of PRGF was completed.
  • Flow cytometry was used to quantify the number of platelets in PRP. 50 ⁇ PRP and 50 ⁇ 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.
  • the stem cells obtained in this study were assayed for cell surface antigen by flow cytometry (Flow Cytometry, BD FACScalibur). After the cells were detached and washed with phosphate buffer, they were dissolved in an appropriate amount of phosphate buffer, and the corresponding antigens were stained with corresponding immunofluorescent antibodies, including IgG, CD34, CD45, CD44, CD73, CD90. , HL-ADR, etc. After 15 minutes of incubation under room temperature, the appropriate amount of phosphate buffer was added and analyzed by flow cytometry. The data was collected by flow cytometry and analyzed by CELL QuestTM (BD) software.
  • BD CELL QuestTM
  • Adipose stem cells differentiate in vitro
  • the obtained fat cells were cultured at 3000 cells/cm 2 in Dulbecco's Modified Eagle Medium (DMEM high glucose, GIBCO) to add P 10 rnM ⁇ -glycerophosphate (Sigma), 0.1 M Dexamethasone ( Sigma), 0.2 mM Ascorbic acid (Sigma), 10% (v/v) fetal bone serum (Hyclone) hard bone induction medium, medium changed every three days.
  • DMEM high glucose, GIBCO Dulbecco's Modified Eagle Medium
  • P 10 rnM ⁇ -glycerophosphate Sigma
  • 0.1 M Dexamethasone Sigma
  • 0.2 mM Ascorbic acid Sigma
  • 10% (v/v) fetal bone serum (Hyclone) hard bone induction medium medium changed every three days.
  • the obtained fat cells were cultured in DMEM at 10000 cells/cm 2 and added with 0.5 mM 3-Isobutyl-l-methylxanthine (IBMX, Sigma), 10 ⁇ g/L Insulin (Sigma), 100. ⁇ Indomethacin (Sigma), 1 ⁇ Dexamethasone (Sigma), 10% (v/v) fetal calf serum (Hyclone) fat-inducing medium, medium changed every three days.
  • Differentiated adipocytes were stained with Alkaline phosphatase (ALP).
  • ALP Alkaline phosphatase
  • the differentiation medium was aspirated, rinsed 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 placed at 37 °C.
  • the cells were aspirated for 2 minutes, then stained with hematoxylin solution (Sigma) for 1 minute; rinsed with secondary water until the secondary water turned pale blue and then observed under a microscope. .
  • the differentiated osteoblasts were stained with Oil red-0, 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. Rinse with water, 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 the second time. The water turned into a light blue color and then observed under a microscope.
  • the fat-derived cells obtained in this study were analyzed by reverse transcription-chain-chainase chain reaction (RT-PCR) to analyze the expression of genes related to undifferentiated stem cells.
  • the cultured cells are washed with phosphate buffer and collected.
  • RT-PCR reverse transcription-chain-chainase chain reaction
  • 1 ml of TriZol (10296-010, Invitrogen) reagent leave it at room temperature for five minutes, and add 100 ⁇ l of hydrazine.
  • ⁇ ( ⁇ . 151, MRC) solution mixed with Vortex to a pink solution, allowed to stand at room temperature for 15 minutes, and centrifuged at 15,000 g for 15 minutes at 4 °C.
  • the eppendorf 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 during the suction process. The other two layers. 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.
  • RNA extraction was completed after reconstitution with water containing DEPC.
  • Aspirate the appropriate amount of RNA add NCode cDNA synthesis Kit (All 193-050, Invitrogen), complete the RT with PCR machine and add GoTaq Green Master Mix (M7122, Promega) to run PCR.
  • the PCR setting conditions are different due to the different Tm values of different Primers. There are adjustments.
  • Analyze the undifferentiated genes associated with stem cells such as oct4, nanog, sox2, cMyc, Lin28, hTERT, etc. This patent analyzes oct4, sox2, klf4, nanog, utfl, hTERT and control group gene GAPDH.
  • the primers used to analyze each gene are listed in the following table:
  • the isolated stromal vascular cell population is further cultured into a fat one, and the culture and subsequent recording are as described later.
  • the adipose stem cells are cultured in a serum-free medium by the above method.
  • PRGF equivalent to IxlO 7 platelet separation is added per ml of the medium to compare the growth curve of the cells with or without added PRGF.
  • cell qualitative results The cell counts of the cells in the serum-free culture of the present invention are listed in the following table:
  • the number of cells collected in this table was cultured from a stromal vascular cell population isolated from 17 ml of adipose tissue.
  • 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.
  • P0-P1 early algebra
  • 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.
  • the cells gradually became aging, because the PRGF was rich. Containing growth factors, it is speculated that it can help the initiation of thousand cell signaling and maintain the ability of stem cells to self-renew, so the growth rate can still be maintained.
  • PRGF protein kinase kinase kinase kinase
  • sternness signaling pathways Interaction with interaction is very important.
  • adipose cells When adipose 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 fat cells can be known from the fat cell type. Fat cells are adherent cells in culture and have a spindle cell type similar to fibroblasts.
  • the adipose-derived stem cell type cultured in the serum-free environment is normal, and the adipose stem cells exhibit a normal spindle cell type in the early subculture (P0 ⁇ P1), in the culture group with and without the addition of PRGF.
  • the cell type with the added PRGF is smaller than the circle which is not added, and is closer to the undifferentiated state; in addition, it is found that the cell density can be grown to a fuller, and there is an addition.
  • PRGF cells can reach about 55,000 cells/cm 2 before P3, which is also reflected in The cells grow faster and the number of cells is larger.
  • the adipose thousand cells amplified in serum-free culture were analyzed by flow cytometry.
  • the cell population was found to be CD34-, CD45-> HLADR-, CD44+, CD73+ CD90+, which is a cell population similar to mesenchymal stem cells.
  • 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.
  • Table 3 The results of the cellular 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.
  • the FSC plots of SSC can see the concentration of the community (Fig. 6). Compared with the group without PRGF, the community is more dispersed (Fig. 5).
  • 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 Maintain a state of growth without differentiation.
  • 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.
  • RT-PCR analysis of adipose stem cell gene expression can be found that oct4, sox2, nanog, klf4, utfl 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 self-renewal of thousands of cells; klf4 is also an important protein found in both embryonic stem cells and mesenchymal stem cells. The titer of the thousand cells plays an important role.
  • telomere reverse transcriptase which is human telomerase Reverse transcriptase, which can prolong the shortening of human telomere, is directly related to the lifespan of human cells.
  • adipose stem cell differentiation The adipose-derived stem cells cultured in vitro have the ability to differentiate into mesoderm cells, such as fat and bone cells.
  • mesoderm cells such as fat and bone cells.
  • PRGF adipose stem cells cultured with PRGF
  • mesenchymal stem cells it was found that the differentiation ability of fat cells is very strong, and only a large number of oil droplets are produced after differentiation for seven days, probably because of the relationship between the source and the stem cells (Fig. 10, Fig. 11, Fig. 12, Fig. 13).
  • 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.
  • PRGF solution which contains many growth factors, including VEGF, PDGF-BB, FGF2, etc.
  • the particles of CD41 which are expressed in the sample are analyzed by flow cytometry, and the internal reference group is added, and the actual number of CD41+ particles can be calculated by using the formula.
  • the PRGF platelet content of BN456812 was 4371 particles/ ⁇ 1, ⁇ 262813, the PRGF platelet content was 94101 particles/ ⁇ 1, the GF726415 PRGF platelet content was 53995 particles/ ⁇ 1, and the BN998619 PRGF platelet content was 11028 particles/ ⁇ 1.

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Abstract

提供了一种用于成体干细胞体外无血清培养的方法。方法包括向无血清干细胞培养基中添加自体生长因子，进行人类成体干细胞的原代培养和继代培养。用所述方法培养的人类成体干细胞经过多次传代后，仍然保持实质上未分化的状态。还提供了用于人类成体干细胞体外培养的无血清培养基，其中含有自体生长因子。

Description

用于成体干细胞的体外无血清培养的方法和培养基 技术领域
本发明涉及一种体外无血清成体干细胞放大培养的方法及其培养液。 背景技术
干细胞是原始且未特化的细胞， 它是未充分分化、 具有再生各种组织器官的 潜在功能， 存在所有多细胞组织里， 能经由有丝分裂与分化来分裂成多种的特化 细胞， 而且可以利用自我更新来提供更多干细胞。 对哺乳动物来说， 干细胞分为 两大类： 胚胎千细胞与成体干细胞， 胚胎干细胞取自嚢胚里的内细胞团； 而成体 千细胞则来自各式各样的组织。 在成体组织里， 干细胞与先驱细胞担任身体的修 复系统， 补充成体组织。 在胚胎发展阶段， 千细胞能分化为任何特化细胞， 但仍 会维持新生组织 （像是血液、 皮肤或肠组织） 的正常转移。 „
1960年代 Ernest A. McCulloch与 James E. Till在多伦多大学的发现， 开启 了研究干细胞的大门。 如今， 我们可以用体外培养技术使干细胞生长或转变成数 种特化细胞， 或形成各种特定组织 (像是肌肉或神经)的组成细胞。 可塑性高的成 体干细胞已常态地运用在医疗上。 千细胞的来源有很多， 包括胚胎、 脐带血、 骨 髓、 脂肪与羊水等等。 利用胚胎干细胞成长为具治疗性的组织甚至器官， 是一个 非常新颖且具挑战性的课题， 在良好的 CMC产程规范下发展， 或许未来会跻身 成为新的医疗方式， 解决现今器官移植上来源短缺的问题。 医学研究者认为干细 胞研究 (也称为再生医学)有潜力通过用于修复特定的组织或生长器官， 改变人类 疾病的应对方法。 但是美国政府的国家卫生研究院报告指出， "重要的技术障碍 仍然存在， 还需要几年的集中研究才能克服。"
干细胞的来源可分为以下五种类：
1.脐带血干细胞：
婴儿出生后残留在胎盘和脐带中的血是富含造血千细胞。 自 1988年起， 脐 血干细胞就用于治疗根达综合症， 亨达综合症， 和拉综合症， 急性淋巴细胞性白 血病等许多儿童疾病。 脐血由脐带釆集； 脐带经过清理消毒后， 从脐静脉取出 脐血后，须针对传染病源和人类白血球表面抗原进行检验，再放入液氮冻存备用。 在使用的时候首先解冻， 再注入病人静脉。 若是使用其它捐赠人干细胞的治疗方
确认本 法， 称为异体移植； 如果干细胞来自患者本人即为自体移植。
2.周边血千细胞：
骨髓中存有人体内最主要造血千细胞的来源， 而周边血干细胞则是指通过施 打白血球生长激素 (G-CSF), 将骨髓中的千细胞驱动至周边血液中， 再经由血液 分离机收集取得的干细胞。此类干细胞属于 CD34+的造血千细胞， 临床上常可听 闻用于改善中枢神经系统与心血管方面的疾病，也主要用于治疗造血系统退化或 是免疫系统不正常的疾病等； 而因骨髓移植病人受到的疼痛较大需要全身麻醉， 且骨髓移植系移植骨髓中的造血、 间质干细胞和基质前驱细胞等， 属适应症较广 泛或是全身性的遗传性严重并症治疗， 如果仅针对部分系统性疾病作治疗， 周边 血干细胞可用于取代骨髓移植。
3.胚胎干细胞：
胚胎干细胞是从嚢胚 (由 50-100个细胞组成的早期胚胎)未分化的内部细 ^团 中取得， 属于近全能干细胞， 有三胚层的分化能力， 表现高端粒酶活性， 可以在 体外培养两年以上的时间仍可维持自我更新的能力。胚胎干细胞现在的研究现在 仍处于刚起步的阶段。 许多研究仍建立在人类以外的动物模式， 例如老鼠、 牛或 是羊等， 截至 2011 年， 在美国临床试验官方注册有一项人体试验正在进行中。 胚胎干细胞由于效价高， 所以临床使用上的方法与适应症需要更严谨的实验证 实， 另外， 由于人类的胚胎千细胞取得来源涉及道德伦理上的约束， 在干细胞株 的建立上有所争议。（因为干细胞株必须取得人类胚胎，在经过培养纯化等。 因此 许多人认为干细胞林视同为人的一部份)。
4.脂肪千细胞：
以往人们因塑身而抽出的脂肪， 通常是作为医疗废弃物处置， 现经由医学专 家研究证实， 脂肪中含有大量的间质千细胞， 间质千细胞具有体外增生及多重分 化的潜力， 可运用于组织与器官的再生与修复。
其主要特性为：
(1)、 取得方式侵入性低， 对人体无害。
(2)、 容易大量的取得。
(3)、 可进行体外增生培养。
(4)、 可运用于身体的组织类型广泛， 输入体内后能自动移至创伤部位， 进行 修补。 5.皮肤细胞：
在 2007年末， 美国和日本两组科学家同时成功把皮肤细胞转化成一种可诱 导万能干细胞 (induced pluripotent stem cells， iPS)， 并成功使这些干细胞转化成为 身体器官的一部份。 通过向皮肤细胞植入特定的基因， 可诱导皮肤细胞改造， 变 成类似胚胎千细胞的一种细胞。
脂肪组织中富含许多具有再生能力的基质细胞、 前趋细胞与间质千细胞。 吾 人可经由抽脂或是切脂取得自体的脂肪组织， 经体外分离与培养可得纯化的脂肪 干细胞， 故相较于其它的成体千细胞， 脂肪干细胞具有取得容易的优点。 脂肪组 织经体外分离可得基质血管细胞群 (stromal vascular fraction， SVF)， 为基质细胞、 血液细胞、血管内皮细胞与脂肪千细胞等共同组合而成。其中含有 CD34+的造血 干细胞、 CD31+的内皮细胞、 CD45+的免疫细胞、 还有 CD105+的间质干细胞等， 这些不同的细胞群， 在人体中均各司其职， 扮演着重要的角色。 脂肪干细胞 ^其- 中为具有间质千细胞特性， 但细胞趋性略有不同的干细胞群， 科学家早期可通过 检验 CD34、 CD105、 CD73、 CD44和 CD90等细胞表面抗原来判定其是否为千 细胞。 在细胞的应用及治疗方面， 各种细胞与脂肪千细胞之间的合作， 对于疾病 的治疗是重要的关键。
基质血管细胞群经体外培养纯化后，其细胞经特性鉴定分析称为脂肪干细胞 (或脂肪基质细胞)。 脂肪基质细胞在体外培养时属于贴附型细胞， 且因脂肪基质 细胞为具有多潜能 (multipotency)的千细胞， 具有跨胚层的分化能力， 故在体外放 大培养时， 维持其细胞不分化的状态 (undifferentiated state)十分重要， 换句话说， 就是维持干细胞自我更新 (selfrenew)的能力。 利用细胞型态的观察、 细胞抗原标 记染色、 千细胞基因表现等， 可以判断千细胞的生长状态。 脂肪千细胞生长有属 于自己适合生长的微环境 (microenvironment), 科学上也常称作 Niche, 譬如说月旨 肪干细胞属于贴附型细胞，在培养环境的胞外基质中就需要含有属于脂肪干细胞 特有 integrin的 ligands, 这些 ligatids属于一些月包夕卜基质 (extracellular matrix), 具 有帮助细胞附着、 爬行的功能。 在体外培养时除了胞外基盾， 培养基、 培养基添 加物与共同培养的材料、 结构、 空间等都是影响细胞自我更新的重要因素， 在放 大培养时，维持细胞最原始与最贴近初代细胞 (primary cell)的相同性 (identity)就是 维持干细胞自我更新的能力。
千细胞的用途 干细胞 (Stem cell)即起源细胞； 在细胞的分化过程中， 细胞往往由于高度分 化而完全失去了再分裂的能力， 最终衰老死亡。 生物体在发展适应过程中为了弥 补此不足， 保留了一部分未分化的原始细胞。 千细胞是一类具有自我更新和分化 潜能的细胞， 学者相信有应用于治疗遗传性疾病、 恶性肿瘤和其它种慢性病等； 以干细胞为种子培育成组织和器官，可用于移植及抗衰老医学及延长人类的寿命 和生活质量。
干细胞移植治疗技术， 被誉为人类有史以来的最飞跃式医疗手段， 实现人体 各个器官修复和更新， 能消除目前 80%以上的各类疾病。
利用成体千细胞进行治疗的困难，在于并非所有的组织器官皆能分离出干细 胞， 且数量艮少； 若以其它器官分离出来的千细胞发育成特定的组织细胞， 其移 植和功能性皆低于原器官分离出的千细胞。 经由体外培养的干细胞， 其特性可能- 会在培养过程中改变， 因而有体外研究结果与临床应用于人体的实际状况有所差 异的情形发生。 若是要由患者本身分离千细胞来进行治疗， 其千细胞也可能有发 病的潜在因子， 若进行移植配对来寻找捐赠者又很耗时。 研究上， 成体干细胞分 化的功能尚未完全厘清， 应用于治疗上仍可能有风险存在。
传统用于体外大量培养干细胞的技术是使用血清来做为培养基的添加物，血 清在细胞培养中提供细胞生长所需的各种成分， 例如荷尔蒙、 生长因子及结合蛋 白等。 然而使用血清也同时潜在着细菌、 霉浆菌或病毒等病源污染的危险， 加上 血清的成本昂贵、 批次变异性大且会干扰产物后续的回收纯化。 因此， 针对人类 脂肪间叶干细胞的培养， 通过不同血清取代物与细胞激素的添加， 及调控其最适 的浓度配方， 并建立间叶干细胞功能性的检测系统， 可以建立大量且正确的体外 无血清增殖培养系统。 发明内容
本发明的主要目的在于提供不同血清取代物与细胞激素的添加，及调控其最 适的浓度配方， 并建立间叶干细胞功能性的检测系统， 可以建立大量且正确的体 外无血清增殖培养系统。
本发明的次要目的为提供一种体外无血清成体干细月 文大培养的方法，该方 法包含下列步骤：
步骤一： 提供人体組织； 步骤二： 以酵素水解步骤一所得的组织， 并以离心方式将未水解的组织与人 类成体干细胞予以分离；
步骤三：将步骤二所得人类成体干细胞于含有自体生长因子 (PRGF)的无血清 干细胞培养液中进行初代培养；
步骤四： 取出步骤三中初代培养液中的黏贴型细胞， 于含有自体生长因子的 无血清干细胞培养液进行继代培养， 以完成人类成体干细胞放大培养。
所述人体组织选自于脐带、 骨髓、 胎盘、 脂肪、 血液及乳牙所组成的群组。 其中用以水解人体组织的酵素为胰蛋白分解酵素或胶原蛋白分解酵素。
所述初代培养的步骤进行 3至 15日。
所述含有自体生长因子的无血清干细胞培养液， 添加分离自自体血液的 ί体- 生长因子 (PRGF)于无血清干细胞培养液中；该自体生长因子添加于无血清千细胞 培养液的比例为 0.1~10% (体积比)。
所述步骤还包含： 以流式细胞仪确认初代培养及继代培养的人类成体干纟田胞 的表面抗原标记特征。
其中经过继代培养后的人类成体干细胞仍保持在实质未分化的状态。
本发明的目的为提供一种培养人类成体干细胞的无血清成体千细胞培养液， 其包含自体生长因子及无血清干细胞培养液所制得者。
所述含有自体生长因子的无血清培养液， 添加分离自自体血液的自体生长因 子 (PRGF)于成体干细胞培养液中；该自体生长因子添加于成体干细胞培养液的比 例为 0.1~10% (体积比）。
所述自体生长因子 (PRGF)是由人类自体血液分离纯化后所获得的。
用以进行人类成体干细胞的初代培养及继代培养。
所述人类成体干细胞选自于脐带血干细胞、 骨髓干细胞、 胎盘干细胞、 脂肪 间叶干细胞、 人类血液细胞及乳牙干细胞所组成的群组。
其中经过继代培养后的人类成体千细胞仍保持在实质未分化的状态。
附图说明
图 1为脂肪千细胞细胞于无血清培养下有无添加 PRGF的生长情形。 P0为初 代培养， P1为继代第一代，依此类推。实施例于培养角瓶中以无血清培养四天后， 继代算细胞， 白色的组别为没有加 PRGF的组别， 灰色的组别为有加 PRGF的组 *: -/+ PRGF组别有显著差异（p < 0.05)， n=4， t-test单尾统计。 图 2为脂肪千细胞细胞于无血清培养下有无添加 PRGF的生长放大倍率图。 P1/P0为初代到第一代的放大倍数， P2/P0为初代到第二代的放大倍率。 实施例于 培养角瓶中以无血清培养四天后， 继代算细胞， 白色的组别为没有加 PRGF的组 别， 灰色的组别为有加 PRGF的组别。
*: -/+ PRGF组别有显著差异（p < 0.05)， n=4, t-test单尾统计。
图 3 为 BN998619 的 P1 细胞培养在无血清下第四天的相位差放大照片 (200X), (A)没有添加 PRGF, (B)有添 PRGF。
图 4 为 BN726415 的 P3 细胞培养在无血清下第四天的相位差放大照片- (200X), (A)没有添加 PRGF, (B)有添加 PRGF。
图 5为 BN262813的 P7细胞于无血清且没有添加 PRGF的培养基下培养四 天后， 以流式细胞仪分析 CD34、 CD45、 HLADR、 CD44、 CD73、 CD90等表面 抗原标记的表现， 并利用 CellQuest软件作分析。
图 6为 BN262813的 P7细胞于无血清， 添加 PRGF的培养基下培养四天后， 以流式细胞仪分析 CD34、 CD45、 HLADR CD44、 CD73、 CD90等表面抗原标 记的表现， 并利用 CellQuest软件作分析。
图 7为脂肪干细胞于无血清培养下有无添加 PRGF的表面抗原的表现比率。 在无血清培养下四天后以流式细胞仪分析 CD34、 CD45、 HLADR、 CD44、 CD73、 CD90等表面抗原标记的表现， 白色的组别为没有加 PRGF的组别， 灰色的组别 为有加 PRGF的组别。
*: -/+ PRGF组别有显著差异（p < 0.05)， n=4， t-test单尾统计。
图 8为 BN456812、 P2细胞的千细胞基因表现。 利用 RT-PCR检验脂肪干细 胞， 之后以洋菜胶电泳分析 (agarose gel electrophoresis)。 M代表 Marker, 左边图 为有添加 PRGF的组别， 右边图为没有添加 PRGF的组别。 缩写对照： G: gapdh, 347 bp; S: sox2, 139 bp; O: oct4, 103 bp; N: nanog, 142 bp; K: klf4， 182 bp; U: utfl, 117 bp; T: htert, 258 bp; M: marker
图 9为 BN262813、 P3细胞的千细胞基因表现。 利用 RT-PCR检验脂肪干细 月包， 之后以洋菜胶电 '泳分析 (agarose gel electrophoresis)。 M代表 Marker， Marker 左边七行为没有添加 PRGF的组别，右边七行为有添加 PRGF的组别。缩写对照： G: gapdh, 347 bp; S: sox2, 139 bp; O: oct4, 103 bp; N: nanog, 142 bp; K: klf4, 182 bp; U: utfl, 117 bp; T: htert, 258 bp; M: marker
图 10为 BN456812的 P2细胞进行 (A)脂肪细胞与 (B)骨母细胞分化至第 14天； 200倍放大， 明视野。
图 11为 BN262813的 P5细胞进行 (A)脂肪细胞与 (B)骨母细胞分化至第 14天； 200倍放大， 明视野。 '
图 12为 BN726415的 P3细胞进行 (A)脂肪细胞与 (B)骨母细胞分化至第 14天； 200倍放大， 明视野。
图 13为 BN998619的 P2细胞进行 (A)脂肪细胞与 (B)骨母细胞分化至第 14天； 200倍放大， 明视野。 具体实施方式
本发明通过下面的实施例予以示范阐明， 但本发明不受下迷实施例所限制。 实施例 1 脂肪间叶干细胞的分离及体外培养
以下实施方式是利用脂肪组织分离而得基质血管细胞群，再进一步经培养而 得脂肪干细胞 (脂肪基质细胞)， 来对本发明作进一步说明。
1.基质血管细胞群分离
经手术取得的脂肪组织， 以离心方式 1000 RCF、 10分钟离心去除血水、 大 块的结締组织等杂质留下纯脂肪组织层 , 随后加入适量的磷酸緩冲液与胶原蛋白 酶酵素作用，酵素于 37°C恒温箱中震荡作用 45分钟，其后加入磷酸緩冲液冲洗， 以 500 RCF、 离心 5分钟小心去除上清液， 留下底下的基质血管细胞群团块， 重 复清洗三次后， 接种至含无血清培养基的 T型角瓶中培养， 待 1〜3天后， 将未贴 附的细胞移去， T型角瓶已贴附的梭状细胞即为脂肪干细胞。 此细胞持续以无血 清培养基培养放大， 作为特性分析之用。
2. 脂肪干细胞培养与继代
Technology), 添力口 20%的 MesenCu t⑧ -XF Supplement (Stem Cell Technology), 及 2 mM L-glutamine (GIBCO)及抗生素 50 g ml gentamicin (GIBCO)作为长期培 养基。 T型角瓶接种细胞前预先以 MesenCult®-XF Attachment Substrate (Stem Cell Technology)处理，以利细胞进行贴附。待细胞贴附后持续培养于 37Ό， 5% C02 的 培养箱， 当细胞密度长至八成满时， 将细胞进行继代。 吸取出培养基， 以磷酸緩 冲溶液清洗细胞后， 加入 MesenCult® Enzymatic Dissociation Solution (Stem Cell Technology), 于 37°C中作用 5 分钟使细胞去贴附， 之后加入等量 MesenCult® Enzymatic Inhibition Solution (Stem Cell Technology)终止酵素作用， 收集细胞， 利 用 hemacytometer计算细胞数目后， 将细胞重新接种于新的 T型角瓶， 持续培养 放大， 至细胞长至 7分满以上时以同样方式再次继代， 大约每四天继代一次， 在 有添加 P GF的培养组别上，将 PRGF在继代或是接种时直接加入无血清培养基 中， PRGF的添加量为 ΙΟΟμΙ的 PRGF加到 10ml的无血清培养基中， 每 ml的无 血清培养基中内含 2xl05到 lxlO6个血小板所释放出的生长因子， 如遇有继代中 更换培养基， 则依比例再加入 PRGF。
3. 自体生长因子 PRGF制备
准备 CPT(BD REF 362761)迷彩蓝头管釆血， CPT管内含有抗凝血剂、 Ficoll 与一层固体胶， 每支约可收集 8 ml血液。 分离时， 以 1700 RCF 于室温下离心 20分钟， 离心完后， 血液在 CPT管内分四层， 从上到下分别为血浆层 (plasma)、 血小板层 (platelet rich plasma, PRP),固体胶层含 FicolK红血球层 (Red blood cell); 将上层的血浆层吸出放入新的 50ml离心管中，将白色的 PRP层吸出放入新的 15 ml 离心管中。 依照比例 (血浆层对血小板层体积之比为 1: 2.5)吸取适量血浆加 入另外一个新的 15毫升离心管中， 之后加入依比例计算出的葡萄糖酸钙 (葡萄糖 酸钙对血浆的体积比为 0.3: 1)， 置于 37°C中反应 15分钟， 以 700 RCF室温离心 10分钟。取其离心后上清液加入 PRP中充分混合， 置于 37°C中反应 40分钟， 即- 完成 PRGF制备。
4. 血小板层 PRP与血浆中血小板定量
以 Flow cytometry定量 PRP中血小板的数量，将 50 μΐ PRP与 50 μΐ血浆加入 磷酸緩冲液中，再加入 anti-CD41-PE抗体染色，免疫染色结束，加入 Count Bright Beads (Invitrogen)当作计数的参考数值，再加入磷酸緩冲溶液将总体积补到约 0.5 ml后上机分析 (FACS Calibur 3 color, BD)。 分析结果套入下列公式即可算出 PRP 与血浆中的血小板含量。
- X £ =样品中血小板的浓度 /μΐ )
其中八=血小板的数量； B=beads的数量； C=beads使用批号中每 50μ1含有 的数量； D 羊品的体积 (μΐ)
5. 细胞表面抗原分析 本研究所得的干细胞， 以流式细胞仪 (Flow Cytometry, BD FACScalibur)进行 细胞表面抗原的测定。 将细胞去附着并以磷酸緩冲液清洗后， 回溶于适量的磷酸 緩沖液中， 分别对不同的抗原以相对应的免疫荧光抗体进行染色， 包括 IgG、 CD34、 CD45、 CD44、 CD73、 CD90、 HL-ADR等。 避光于室下温染色 15分钟 后，加入适量磷酸緩冲液后上机分析，经流式细胞仪收集数据后，以 CELL Quest™ (BD)软件进行分析。
6. 脂肪干细胞体外分化
骨母细胞 (Osteoblast)分化中， 将所得的脂肪千细胞以 3000 cells/cm2培养于 Dulbecco's Modified Eagle Medium (DMEM high glucose, GIBCO)添力 P 10 rnM β-glycerophosphate (Sigma)、 0.1 M Dexamethasone (Sigma )、 0.2 mM Ascorbic acid (Sigma), 10 % ( v/v )胎牛血清 （Hyclone) 的硬骨诱导培养基， 每三天更换一次 培养基。 脂肪细胞 (Adipocyte)分化中， 将所得的脂肪千细胞以 10000 cells/cm2培 养于 DMEM添加 0.5 mM 3-Isobutyl-l -methylxanthine (IBMX, Sigma)、 10 ^g/ L Insulin (Sigma)、 100 μΜ Indomethacin (Sigma)、 1 μΜ Dexamethasone (Sigma)、 10 % (v/v)胎牛血清（Hyclone) 的脂肪诱导培养基， 每三天更换一次培养基。
7. 细胞化学染色
分化的脂肪细胞以碱性磷酸酶 (Alkaline phosphatase, ALP)进行染色， 先将 Fast Blue RR Salt solution与 AS-MX phosphate Alkaline solution 以 24:1比例〉 '昆合- 置于 25°C并避光； 将分化培养基吸除， 以磷酸緩冲液冲洗后； 以 citrate buffered acetone 固定 30秒， 再以二次水浸润 45秒； 然后将先前的混合液加入培养瓶中， 置于 37 °C下避光 60分钟后吸除，以二次水浸润 2分钟后，再以 hematoxylin solution (Sigma)染细胞核 1分钟； 以二次水持续冲洗，直到二次水转变为淡蓝色后再以显 微镜观察。 分化的骨母细胞以 Oil red-0进行染色， 将分化培养基吸除， 以磷酸 緩冲液冲洗， 以 3.7 % paraformaldehyde in PBS/pH7.4 将细胞于室温下固定 15分 钟后， 以二次水冲洗， 以 Oil Red O (Sigma) 染色 10分钟后， 以 50% ethanol浸 润， 并以二次水沖洗； 再以 hematoxylin solution (Sigma) 染细胞核 1分钟； 以二 次水持续冲洗， 直到二次水转变为淡蓝色后再以显微镜观察。
8. 反转录-链锁酶连锁聚合反应
本研究所得的脂肪千细胞， 以反转录-链锁酶连锁聚合反应 （RT-PCR) 来分 析未分化干细胞的相关基因的表现。 将培养出的细胞以磷酸緩冲液清洗后， 收集 在 1.5 ml eppendorf 中， 加入 1 ml TriZol (10296-010, Invitrogen)试剂， 于室温放 置五分钟， 加入 100 μ1 Β。Ρ (ΒΡ. 151, MRC)溶液， 以 Vortex混匀至成粉红色溶液 后， 室温放置 15 分钟， 再以 15,000 g 离心 15分钟在 4°C。 离心完后， eppendorf 内会分三层， 下层是红色层， 中间一层薄薄的白色层， 上层是透明层， 将上层吸 出放入新的 1.5 ml eppendorf 中， 吸取过程中要小心不要吸到另外两层。 于新的 eppendorf 中加入 0.5 ml isopropanol,摇匀后，室温放置 30分钟，之后再以 15,000 g离心 10 分钟在 4°C，将上清液抽出，不要吸到 pellet,加入 1 ml 75% ethanol 清 洗， 再以 15,000 g离心 10 分钟在 4°C， 抽掉 ethanol后， 空气干燥 10分钟， 以 含有 DEPC的水回溶后即完成 RNA萃取。 吸取适量 RNA， 加入 NCode cDNA synthesis Kit (All 193-050, Invitrogen), 以 PCR machine 完成 RT后加入 GoTaq Green Master Mix (M7122, Promega)跑 PCR, PCR设定条件因不同 Primer 的不同 Tm值而略有调整。分析跟干细胞相关未分化的相关基因，像是 oct4, nanog, sox2, cMyc, Lin28, hTERT等，本专利分析 oct4, sox2, klf4, nanog, utfl, hTERT和控制组 基因 GAPDH。 分析各基因所使用的 primer如下表所列：
Primer name Sequence (5 'to 3') Amplicon Tm
GAPDH— Forward GCATCCTGCACCACCACCTG 57.9
GAPDH— Reverse GCCTGGTTCACGACGTTCTT 347 53.8
S0X2— Forward CCATCCACACTCACGCAAAA 51.8
SOX2— Reverse TATACAAGGTCCATTCCCCCG 139 54.4
OCT4_Forward TCCCATGCATTCAAACTGAGG 52.4
OCT4_Reverse CCAAAAACCCTGGCACAAACT 103 52.4
NANOG— Forward TGGACACTGGCTGAATCCTTC 54.4
NANOG— Reverse CGTTGATTAGGCTCCAACCAT 142 52.4
KLF4— Forward CTGCGGCAAAACCTACACAA 51.8
KLF4— Reverse GGTCGCATTTTTGGCACTG 182 51.1
UTF1— Forward CGACATCGCGAACATCCTG 53.2
UTFl_Reverse AGAATGAAGCCCACGGCCA 117 53.2 hTERT— Forward GAACAGTGCCTTCACCCTCGA 56.3 hTERT_Reverse CGGAAGAGTGTCTGGAGCAAGT 258 56.7 实施例 2添加 PRGF于无血清培养基用以放大培养成体干细胞
1. 脂肪基质血管细胞群分离
本发明用以研究的临床人类检体共四例，分别以编号 BN456812、 BN262813 , BN726415, BN998619称之。
所分离的基质血管细胞群进一步培养成脂肪千细胞，培养与继代记录如后所 述。
2. 脂肪千细胞培养
脂肪干细胞以无血清培养基培养， 方法如上述； 在培养过程中， 依照 PRP 的定量结果， 于每毫升培养基中添加相当于 IxlO7血小板分离所得的 PRGF, 比 较有无添加 PRGF的细胞生长曲线及细胞定性结果。本发明的细胞无血清培养收 得细胞数记录如下表所列：
表 1、 脂肪千细胞培养记录表， 表中记录有无添加 PRGF的情况下细胞培养结果
Figure imgf000012_0001
注： 本表所收得的细胞数， 均培养自 17 ml脂肪组织所分离的基质血管细胞群。
将上表的实验数据画成细胞生长图（如图 1)， 可以明显看出有添加 PRGF的 细胞培养组， 相较于没有添加的组别， 细胞生长较快。 细胞在早期代数生长时 (P0-P1), 因细胞较年轻， 容易维持自我更新， 两組间在细胞数上没有显著差异； 然而， 随着继代， 细胞渐趋老化后， 因为 PRGF中富含生长因子， 推测可以帮助 千细胞讯息传递启动， 维持干细胞自我更新的能力， 所以生长速率仍可维持。
若将细胞数换算成细胞放大倍率 (表 2)， 更可以清楚的看到， 到笫二代时， 细胞数已经差到两倍以上； 到第 3代， 细胞数在有添加跟没添加 PRGF得组别有 显著的差异 (如图 2); 干细胞在维持不分化生长时， 需要自分泌 (autocrine)和旁分 泌 (paracrine)彼此作用， 调控讯息传递的进行； 细胞在无血清的培养环境下， 容 易改变原有的 ½性， 因为无血清培养环境不像血清可以提供很多生长必须的生长 因子与细胞激素，甚至还有些^：量矿物元素等，若长期培养在这样恶劣的环境下， 细胞会失去原有在生理环境下的一致性 (identity); PRGF 的功用即在于供给许多 种类的自体生长因子与细胞激素， 或是一些微量化学物质等， 这些东西对于帮助 脂肪干细胞旁分泌讯息传递是不可或缺的，对于长时间体外维持一致性 (long-term maintenance) ,干细胞讯息传递 (sternness signaling pathway)的进行与交互影响十分 重要。
表 2、 脂肪干细胞生长放大倍率的数据表列 （平均 ±标准偏差， n=4)
Figure imgf000013_0001
脂肪千细胞在体外培养时， 可以通过型态观察判断细胞的状态， 从脂肪千细 胞型态了解其生长状况和分化状态。 脂肪千细胞在培养时属于贴附型细胞， 具有 类似纤维母细胞的梭状细胞型态。 本发明中， 利用无血清环境培养的脂肪干细胞 型态正常， 在早期继代数 (P0~P1)，有添加和没有添加 PRGF的培养组别中，脂肪 干细胞都呈现正常的梭状细胞型态， 不过已经可以看出有些微的差异； 没有添加 PRGF的组别中， 有些游走型态的细胞， 型态上不规则， 偶为细长， 偶为大而扁 平， 所能达到的细胞密度也较低 (如图 3A); 然而， 在有添加 PRGF的组别当中， 细胞型态圓而小较为一致， 核质比大且细胞密度较高 (如图 3B)。 细胞培养到后期 代数 (如 P3)的时后， 细胞在型态上的差异也越趋明显， 没有添加 PRGF的细胞会 变得比较细长， 越趋不规则， 也会发现有些细胞的细胞质有皱折且细胞摊平， 这 些是典型细胞老化或是基因沉默 (gene senescence)的现象 (如图 4A); 反之， 有添 加 PRGF的培养组别， 细胞排列规则， 侧向排列（lateral association)的行为良好， 细胞型态圆而小， 很贴近初代细胞的细胞型态 (如图 4B)。
从细胞培养型态观察总结可以发现，有添加 PRGF的细胞型态较没添加的圓 而小， 较贴近未分化的状态 (undifferentiated state); 另外， 发现能长到比较满的细 胞密度， 有添加 PRGF时， 细胞在 P3前可以达到约 55,000 cells/cm2, 也反映在 细胞生长变快， 细胞数较多。
3.脂肪干细胞定性
3.1 细胞表面抗原分析
于无血清培养下放大的脂肪千细胞， 以流式细胞仪分析表面抗原， 可以发现 细胞族群为 CD34-、 CD45-> HLADR -、 CD44+、 CD73+ CD90+, 为类似间质干 细胞的细胞族群。 在培养的过程中， 细胞的族群会越来越纯， 可以从流式细胞仪 FSC对 SSC的点状图中观察到 (如图 5、 图 6)， 点越密集， 代表族群越集中。 统 计实施例的细胞分析结果， 结果记录于表 3:
表 3、脂肪千细胞表面抗原分析统计，表中的 PI (passage 1)代表代数 1， P2 (passage 2)代 表代数 2, 依此类推。
Figure imgf000014_0001
脂肪干细胞在培养过程中， 如果型态不健康、 不一致、 培养状况不良如有死 细胞等， 都会影响到流式细胞仪的分析结果。 有添加 PRGF的培养组别中， FSC 对 SSC的点状图可以看到群落集中 (如图 6)， 相较下， 没有添加 PRGF的组别， 则群落较为分散 (如图 5)。 另外，有添加 PRGF的培养组别在 CD73和 CD90的表 现量上较高， 且表现的强度较一致， 分析图中的峰比较尖 (如图 6)， 也呼应到细 胞的形态观察上比较一致。 在后期代数的培养上 (如 P7), 细胞型态容易变形， 这- 型态变化会反应在 CD73和 CD90的染色结果上， 如 CD73和 CD90的染色结果 比较低， 则可能暗指说细胞没有维持在不分化的生长状态。
将细胞表面抗原的表现量用统计分析后，发现添加 PRGF培养脂肪干细胞的 组别较容易维持细胞的一致性，代表脂肪干细胞的细胞标记表现比例都比未添加 PRGF的组别高 (如图 7); 其中 CD44高 3.2%(n=7， p-0.033); CD73高 3.3%(n=7, p=0.0425); CD90高 2.9%(n=7, p=0.079);三个负向的细胞标记表现量均低。 CD44 扮演着细胞间互相沟通、 影响的功能； CD90是多种干细胞会表现的细胞标记； CD73是一种干细胞会表现的酵素。 在细胞状态维持一致时， 这些表面标记的表 现量会一致， 而且脂肪干细胞都会表现； 反之， 如果细胞特性改变， 则细胞标记 表现也会随之改变。 细胞体外大量无血清培养时， 需维持良好的一致性， 经过添 加 PRGF的培养方法较未添加的方法，在维持千细胞表面抗原的表现上有更好的 效果。
3.2 千细胞基因表现分析
利用 RT-PCR 分析脂肪干细胞的基因表现， 特别针对六种干细胞基因 (sternness gene)作分析， 可以发现 oct4、 sox2、 nanog、 klf4、 utfl和 htert都会在 脂肪干细胞中表现， oct4、 sox2、 nanog是干细胞中重要的转录因子 (transcription factor), 会调控、 启动很多下游基因的表现， 对于维持千细胞自我更新是必须的； klf4也是一个在胚胎干细胞和间质干细胞中都有的重要蛋白质， 对于维持千细胞 的效价扮演着重要的功能。 2007年日本的 yamanaka和美国的 Thomson等人也利 用这些基因的组合成功的将体细胞引导成近全能型干细胞 (induced pluripotent stem cells, IPS cells),说明这些基因对于千细胞的重要性。此外， 实验结果也并且 可以发现如果有添加 PRGF，较容易维持 utfl和 htert的基因表现（如图 8和图 9)， 意指脂肪干细胞在无血清培养下添加 PRGF时，可以维持细胞生长和不分化^状- 态； utfl全名是 undifferentiated transcription factor 1， 意即千细胞未分化时会表现 的转录因子，会调控其它干细胞基因的表现； htert全名是 human telomerase reverse transcriptase, 是人类端粒酶反转录酵素， 可以延长人类端粒缩短的时间， 跟人类 细胞的寿命有直接的关系。
3.3 脂肪干细胞分化 体外培养的脂肪干细胞具有分化成中胚层细胞的能力， 像是脂肪和骨头细- 胞， 实施例中证实利用 PRGF所培养出来的脂肪干细胞可以分化成脂肪和骨头， 具有间质干细胞的分化能力， 且实验发现脂肪细胞的分化能力很强， 只需要分化 七天即有大量油滴产生， 可能是因为来源^ J旨肪干细胞的关系 (如图 10、 图 11、 图 12、 图 13)。
4.自体生长因子 PRGF 定量
利用定量检体中血小板的数目间接定量 PRGF的浓度。 CD41为血小板会表 现的表面抗原， 在 PRGF的制作过程中， 血小板会破裂， 使得血小板中的物质释 放到 PRGF溶液中，其中含有许许多多的生长因子，包括 VEGF、 PDGF-BB、 FGF2 等， 本实施例用流式细胞仪分析样品中会表现 CD41的颗粒， 另外再加上内参考 组， 利用公式即可算出 CD41+颗粒的实际数量。 BN456812的 PRGF血小板含量 为 4371颗 /μ1、ΒΝ262813的 PRGF血小板含量为 94101颗 /μ1、ΒΝ726415的 PRGF 血小板含量为 53995颗 /μ1、 BN998619的 PRGF血小板含量为 11028颗 /μ1。

Claims

权利要求
1.一种体外无血清成体干细胞放大培养的方法， 其特征在于， 该方法包含下 列步骤：
步骤一： 提供人体组织；
步骤二： 以酵素水解步骤一所得的组织， 并以离心方式将未水解的组织与人 类成体干细胞予以分离；
步骤三： 将步骤二所得人类成体干细胞于含有自体生长因子的无血清干细胞 培养液中进行初代培养；
步骤四： 取出步骤三中初代培养液中的黏贴型细胞， 于含有自体生长因子的 无血清干细胞培养液进行继代培养， 以完成人类成体干细胞放大培养。
2.如权利要求 1所述的方法，其特征在于，所述人体组织选自于脐带、骨髓、 胎盘、 脂肪、 血液或乳牙所组成的群组。
3.如权利要求 1所述的方法， 其特征在于， 用以水解人体组织的酵素为胰蛋 白分解酵素或胶原蛋白分解酵素。
4.如权利要求 1所述的方法，其特征在于，所述初代培养的步骤进行 3至 15 日。
5.如权利要求 1项所述的方法， 其特征在于， 所述含有自体生长因子的无血 清千细胞培养液，是添加分离自自体血液的自体生长因子于无血清干细胞培养液 中。
6.如权利要求 1-5任一项所述的方法， 其特征在于， 所述自体生长因子添加 于无血清千细胞培养液的体积比例为 0.1~10%。
7.如权利要求 1所述的方法， 其特征在于， 所述步骤还包含： 以流式细胞仪 确认初代培养及继代培养的人类成体千细胞的表面抗原标记特征。
8.如权利要求 1所述的方法， 其特征在于， 经过继代培养后的人类成体千细 胞仍保持在实质未分化的状态。
9.一种培养人类成体千细胞的无血清成体千细胞培养液， 其特征在于， 包含 自体生长因子及无血清千细胞培养液所制得者。
10. 如权利要求 9所述的培养液， 其特征在于， 所述含有自体生长因子的 无血清干细胞培养液，是添加分离自自体血液的自体生长因子于成体干细胞培养 液中。
11. 如权利要求 9所述的培养液， 其特征在于， 所述自体生长因子是由人 类自体血液分离纯化后所获得的。
12. 如权利要求 9所述的培养液， 其特征在于， 用以进行人类成体干细胞 的初代培养及继代培养。
13. 如权利要求 9所述的培养液， 其特征在于， 所述自体生长因子添加于 成体干细胞培养液的体积比例为 0.1~10%。
14. 如权利要求 9所述的培养液， 其特征在于， 所述人类成体干细胞选自 于脐带血千细胞、 骨髓干细胞、 胎盘千细胞、 脂肪间叶千细胞、 人类血液细胞或 乳牙干细胞所组成的群组。 。
15. 如权利要求 9所述的培养液， 其特征在于， 经过继代培养后的人类成' 体干细胞仍保持在实质未分化的状态。
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