TWI300095B - - Google Patents

Description

1300095 IX. Description of the invention: • Technical field to which the invention pertains The present invention relates to a method for culturing stem cells, and more particularly to a method for culturing stem cells using a cell culture medium containing human umbilical cord serum. [Prior Art] Stem Cell is a non-differentiated primary cell in an organism, which has the ability to continuously replicate, renew, and differentiate into mature cells having a specific pattern and function for a long time. In general, the source of human stem cells can be mainly divided into embryonic stem cells (which are derived from embryos remaining after infertility treatment, embryonic primordial germ cells that are terminated by pregnancy, or obtained by cell fusion) and tissue stem cells (which are taken from From bone marrow, peripheral blood, liver, skin, etc. isolated from mature individual tissues, or blood stem cells in the placenta/umbilical cord; or by somatic cell fusion technology. Stem cells can be divided into three major categories according to their ability to differentiate. One is a totipotent stem cell, which is a fully capable embryo that can differentiate into a complete embryo or organism, and a pluripotent stem. Cells), which are cells that have multiple abilities but cannot develop into a complete embryo or organism and that form a certain tissue or organ; three are multipotent stem cells, including stem cells of a specific tissue, such as Neural stem cells, blood stem cells, liver stem cells, skin stem cells, and the like. Human stem cells can now be used as drug development (design, screening 1300095, &#mechanism, t-full test) to produce specific cells or tissues, or transplant, update of cells, tissues (including disease medical , regenerative medicine, group = work, elderly medicine, etc.). In addition, continuous stem cells can also be used for research and treatment of gene expression, cell differentiation, disease mechanisms, and cancer. 'This, in general, the willingness to provide stem cells is not high, in order to be able to apply this: a limited number of human stem cells in medical treatment and research, so • Maozhan culture technology for the proliferation of human stem cells obtained, Become an extremely important topic. It is customary to culture cells in vitro, and these cells must provide a suitable growth environment and provide the nutrients needed for cell growth with appropriate cell nutrient solution, and the most commonly used animals in the conventional nutrient solution are animals. serum. Serum refers to the blood after removing cells and platelets. Animal serum contains many substances needed for cell growth and survival. The type and quality of conventional serum can have a great impact on the growth of cells. The serum from different species has different contents and amounts. Therefore, when the serum is used incorrectly, the cells often fail. Survive.

(D. In the prior art, human stem cells are usually cultured in a medium containing animal fetal serum (for example, Fetal Calf Serum (FCS)). This method of culturing stem cells using fetal bovine serum has been widely used. It is used by those skilled in the art and is an operating procedure for stem cell culture as recognized by the U.S. Food and Drug Administration (A). Although fetal bovine serum has been widely used to culture stem cells, if these human stem cells cultured in fetal bovine serum are used in humans, it may be due to non-6 1300095 • λj protein shells contained in animal fetal serum. It induces an allergic reaction in the human body, and if the fetal bovine serum contains viruses and other pathogenic bacteria (for example, rabies protein), the patient implanted with the stem cells is at risk of being infected by the infection source. In order to avoid the possible problem of the use of non-human serum to cultivate stem cells (4), the financial person (4) made human serum or sputum into serum to replace the animal sputum serum, and cultured the secret human stem cells. However, the composition of serum is very complex. It contains proteins, • long-term factors, hormones, lipids, metabolites, inorganic salts, etc., and the quality of serum is also affected by the health, age, nutrition, etc. of animals; The biomedical technology 'has no complete understanding of all the components, sputum and function of animal and human serum. Therefore, adult sera may lack certain supplies due to their growth, daily harvest, purpose and function. Stem cell growth • (4) An important substance, so its effect on promoting human stem cell growth is not significant; in addition, 'the use of synthetic serum also has the same problem. The problem of Lu is such that human stem cells are greatly limited in the application of disease treatment. It has become a major issue in the development of stem cell medical related technologies, how to develop a culture method that excludes fine infections of viruses and other pathogens and can effectively promote the growth of human stem cells. SUMMARY OF THE INVENTION Therefore, the present invention has been made in an effort to solve the above disadvantages of the prior art. In order to solve the problems of the prior art, the object of the present invention is to provide a cell culture medium containing human umbilical cord serum for culturing human bone marrow 1300095, whereby the human bone mineral stem cells after culture are applied to the disease! To avoid allergies caused by the use of non-human serum - and the risk of wood-borne viruses or pathogens. Bone Shovel The object of the present invention is a method for cultivating human cercaria stem cells according to the present invention, the steps comprising: a transgenic group, wherein the sessile nutrient comprises a human (:) human The bone marrow stem cells are transferred to the cell culture medium; and, (); cultured and propagated under the preset conditions of δ and human osteophyte stem cell growth. The inventors of the present invention (4) culture method of lining cells, the cells used may further include - nutrient additives such as hormones, proteins, biological hormones or growth factors, but are not limited thereto. Among them, the growth factor can be applied to the present invention, and examples thereof include small plate growth factor (PDGF), epithelial cell growth (EGF), basic fibroblast growth factor (bFGF) and the like. Insulin growth factor (igf_ ι), etc., but not limited to this. The culture method of human bone marrow stem cells according to the present invention has a growth effect recorded by a culture method using non-human serum. Since the human (4) stem cell culture method of the present invention uses the human umbilical cord serum as a raw material in the cell culture medium, the cell culture medium is not only safe and hygienic, but also conveniently available. In addition, the cultured bone marrow stem cells have the advantages of good growth state and the like. 1300095 The method for culturing human bone marrow stem cells of the present invention allows human osteophyte stem cells to maintain intact differentiation without affecting their subsequent differentiation. In addition, the human bone marrow stem cell culture method of the present invention uses the human umbilical cord serum at the time of birth of the fetus, so that the human bone marrow stem cells cultured by the method of the present invention can be propagated and reproduced under conditions and conditions similar to the birth of the fetus. Cultured human bone marrow stem cells have strong growth and development. Further, since the human bone marrow stem cells cultured according to the present invention have good differentiation, they are highly suitable for use in medical treatment such as cell transplantation. [Embodiment] The method for culturing human bone marrow stem cells according to the present invention is to prepare a cell culture medium containing human umbilical cord serum, which can be prepared by a conventional cell culture medium formula, but contains The animal serum (for example, fetal bovine serum) is replaced with human sputum serum. The cell culture medium which can be used in the present invention, and examples thereof include MEM medium (MEM), α-MEM medium (α_ΜΕΜ), BME medium (Basal Media Eagle, BME), DMEM medium (Dulbecco's) Modified Eaglefs modium, DMEM), MEM/F12 medium, Ham, s F10 medium, Ham's F12 medium, RPMI 1640 medium (Roswell Park Memorial Institute, RPMI) and Medium 199 medium, etc., but are not limited thereto. In addition, in order to enable the culture method of the human bone marrow stem cells of the present invention, a better stem cell proliferation culture effect of 8 9 1300095 can be obtained, and the content of the human umbilical cord serum in the cell culture medium is preferably 5 to 3 G% (four), more preferably For 6~2()%(four). In general, the various media are similar in composition and are rich in four essential elements, including amino acids, carbohydrates, inorganic salts and vitamins. Therefore, the culture medium for human stem cells of the present invention can further comprise a nutritional supplement which can promote the growth of human bone marrow stem cells, and can be applied to the nutritional supplement of the present invention. Examples include, but are not limited to, hormones, proteins, biological hormones, antibiotics, and growth factors. The above-mentioned growth factor which can be applied to the present invention is not particularly limited as long as it is a growth factor which can be applied to cell culture, can be used to promote growth of human bone marrow stem cells, and conforms to relevant medical and health regulations. All of them can be used in the present invention, and examples thereof include platelet growth factor (PDGF), epithelial cell growth factor (EGF), basic fibroblast growth factor (bFGF), and insulin-like growth factor (IGF). -1) Special 'but not limited to this. Further, the medium used in the present invention may further contain phenol red (Phenol Red) as an acid-base indicator to determine whether or not the pH in the medium is maintained within a normal range. In order to effectively maintain the acidity and alkalinity of the medium, the medium of the present invention may further comprise an acid-base buffer, and examples thereof include sodium hydrogencarbonate (NaHC 3 ), but are not limited thereto. Thereafter, the human bone marrow stem cells to be cultured are transferred to the above-prepared cell culture medium. Next, the cell culture medium containing human bone marrow stem cells is cultured and allowed to grow under a predetermined condition suitable for the growth of the human bone marrow stem cells, whereby the human bone marrow fine 1300095 cells can be efficiently propagated. • Those skilled in the art of the present invention can also easily infer from the contents contained in the specification of the present invention that other human stem cells can be cultured by the culture method of the present invention, except that the human bone stem cells can be cultured by the culture method of the present invention. The method of culture is known as the dry secret of the culture, and the source thereof can be divided into embryonic stem cells and tissue stem cells. As an example of a secret stem cell, 'the embryo remaining after infertility treatment, the original embryonic lean cell which is terminated by pregnancy', and the embryonic stem cell obtained by cell fusion, but not limited to this. As an example of tissue stem cells, stem cells isolated from bone minerals, peripheral blood, liver and skin tissues of mature individuals, and blood stem cells isolated from the placenta or umbilical cord are included. The above-mentioned predetermined condition suitable for the growth of the human bone marrow stem cell means: the culture temperature of the human (four) stem cell growth and the upper gas composition of the medium. The above culture temperature is not particularly limited in the present invention, and the stem cells to be cultured are adjusted and closed, and generally, the culture is performed. The cultured stem cells have a good growth state, and the culture temperature is preferably between 32 and 42 C and more preferably between 35 and 39 C. The upper gas of the medium can constitute a gas composition of a conventionally cultured stem cell, which comprises oxygen and carbon dioxide, and is not limited thereto, but in order to make the cultured stem cells have a better raw state, oxygen in the upper gas of the medium The content is preferably between 9 〇 and just % (v/v), and the carbon dioxide content is preferably between. The invention will be further illustrated by reference to the following examples, which are not to be construed as limiting. </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; Example 1 Preparation of Cell Culture Medium Containing Human Umbilical Cord Serum To obtain human umbilical cord serum for testing, a desired umbilical cord blood was aspirated by inserting a needle containing no anticoagulant agent into the human umbilical cord. Next, the cord blood was centrifuged (3 rpm, 1 〇 minute), and the supernatant was boiled at 56 ° C for 30 minutes, and then filtered (0.22 / / m) as a follow-up. The DMEM medium is prepared by the method and formula, and then the prepared human umbilical cord serum is added to the basic DMEM medium as a subsequent cell culture application. Example 2 Comparison of cell growth The human bone marrow stem cells to be subjected to proliferation culture were separately transferred to the following four DMEM cell culture media to culture to compare the growth of human bone marrow stem cells. The four media are: (a) serum free (Serum Free) DMEM cell culture medium, hereinafter referred to as SF medium; (8) DMEM cell culture medium containing 10% Fetai Calf Serum, hereinafter referred to as FCS medium. (4) DMEM cell culture medium containing 10% human Umbilical Cord Serum, hereinafter referred to as hUCS medium; and (d) 10% human umbilical cord serum and 1 Ong/ml epithelial growth factor (EGF) DMEM cell culture medium with 8 l3〇0〇95 factor (bFGF) was grown with basic fibroblasts, hereinafter referred to as hucs^ nutrient. At the initial stage of the culture, the concentration of the cells transferred to each medium was 50 cells/cm 2 , respectively. After that, the medium was placed at π. ^, 95% air and 5% carbon dioxide, let the cells grow. The cell growth was observed under a microscope after three days and seven days, and the results are shown in the first figure. Referring to the first figure, human bone marrow _ stem cells grown in these four different media did not differ much in shape and appearance. In addition, the cell densities after three days and seven days of incorporation were calculated, and the results are shown in the second figure. Referring to the second figure, the growth density of the stem cells of the human bone marrow stem cells under the same culture conditions, the hucs medium group, and the hUCS+ medium group was significantly higher than that of the SF medium group and the FCS medium group (about 2 times higher), among which hUCS+ The medium group has the highest growth density. From this result, it was found that human umbilical cord serum can indeed obtain a better growth-promoting effect of human bone marrow stem cells than fetal calfin, and the addition of growth factor can further promote the effect. Example 3 Comparison of Nature of Growth Cells The human bone marrow stem cells obtained by FCS medium and hUCS+ medium in Example 2 were respectively treated with 2 mM ethyenediamine tetraacetate (EDTA). The buffered chyme water (PBS) was used to separate the stem cells from the surface of the dish. The cells were washed with 2% bovine serum albumin (BSA) and 〇·1% sodium trinitrate (NaNO phosphate buffered water 1300095 • (PBS). The washed stem cells were separately labeled with antibodies CD13, CD29, CD44 , CD73, CD90, CD 105, HLA-ABC or HLA-DR are reacted, and the above antibodies are pre-bound with fluorescein isothiocyanate (FITC) or phycoerythrin (PE). Finally, flow cytometry is used to detect and analyze the surface antigen of the above stem cells. The analysis results are shown in Fig. 3, Fig. 4 and Fig. 5. See Fig. 2 for the two different media respectively. The size and granularity of the stem cells obtained after the culture are compared. It can be seen from the figure that the stem cells obtained after the culture of the two different media are analyzed, and the forward scattered light (FSC, representing the cell size) and Side Scattering (SSC, representing cell granularity) has roughly the same properties: See Figures 4 and 5, respectively, for humans cultured in FCS medium and hUCS+ medium. The surface antigen detection map of myeloid stem cells. From the comparison between the fourth and fifth graphs, human bone marrow stem cells obtained from hUCS+ culture medium can be obtained and cultured in FCS medium with similar surface antigen phenotypes. Therefore, based on the analysis results of the three figures, it can be seen that human bone marrow stem cells to be proliferated and maintained in hUCS+ medium can be obtained by culture in FCS medium, which has similar cell size and granularity. The expression pattern of surface antigens, which shows that human umbilical cord serum can indeed be used to replace fetal bovine serum and is applied to the culture of stem cells. Example 4 8 14 1300095 Cell differentiation ability comparison • Preparation of osteoblast production medium [containing 0.1// Mol/l adrenal cortexone (dexamethasone), 10mmol/l /3 glycerol phosphate (cold_glyCerol phosphate) and 50//mol/l ascorbate (asc〇rbate) DMEM medium] and divided into two groups, One group added 1% fetal bovine serum (FCS), while the other group added 10% human umbilical cord serum and growth factor (hUCS+). The human bone marrow stem cells of Vi. 3 cells/cm2 were transferred into the two culture media, and cultured. After three weeks of culture, the deposition of the dance was detected by staining with Aliarin Red S stain, and it was confirmed that the human bone marrow stem cells differentiated into bone cells. The results are shown in Figure 6. See Figure 6 'The cell staining range in the hUCS+ medium group is larger and darker than in the FCS medium group; the staining results represent the differentiation of cells cultured in hUCS+ medium Preferably. That is, human bone marrow stem cells have a better stem cell differentiation result after being cultured in a medium containing human umbilical cord serum than those cultured in a medium containing fetal bovine serum. From this, it can be seen that human bone marrow stem cells cultured in human umbilical cord serum have the ability to promote differentiation of human bone marrow stem cells into osteoblasts in vitro, which is better than those used in fetal bovine serum, under the same conditions for promoting differentiation. Prepare an adipogenic medium [containing 1//m〇l/l adrenal cortex/_(dexamethasone), 5 // g/ml insulin (insulin), 0.5mmol/l isobutylmethylxanthine) and 60 / / mol/1 ° DMEM medium of indomethacin and divided into two groups, one group added 10% fetal bovine serum (FCS), and the other group added 1% human

15 1300095 Nourishing blood stasis β and growth factor (hucs+). Then, 5X1G3 cells/cm 2 of human bone lining cells were transferred into the above two groups of medium, and 4 liters of V., and oysters were cultured for 3 weeks, and the oil droplets were stained with Oil Red staining agent. The ability of the above human bone marrow stem cells to differentiate into adipocytes was confirmed, and the results are shown in the seventh panel. Referring to the seventh figure, the number of cells stained in the hUCS+ medium group was higher than that in the FCS medium group; the staining results indicated that the hcSM^ nutrient-based culture and the t-segmentation were better. That is, human bone marrow stem cells have better stem cell differentiation results after being cultured in a medium containing human umbilical cord A and compared with those cultured in a medium containing fetal bovine serum. From this, it can be seen that human (4) stem cells cultured in human umbilical cord serum have the ability to promote human: bone marrow stem cells to differentiate into adipocytes in vitro under the same conditions for promoting differentiation. Prepare a neural cell production medium [DME]V^^Narm containing lmm〇1/ly3_6-based thiol (mercaptoethanol) and 5 ng/ml basic fibroblast growth factor φ (bFGF) and divide them into two groups. One group added 10% fetal bovine serum (FCS), while the other group added 1% human umbilical cord serum and growth factor (hUCS+). Thereafter, 5 x 10 cells/cm 2 of human bone marrow stem cells were transferred to the above two groups of culture medium, respectively, and cultured. After seven days of culture, the nerve cell markers were detected by immunostaining, and the ability of the above human bone marrow stem cells to differentiate into nerve cells was confirmed, and the results are shown in Fig. 8. - Referring to Figure 8, the staining results represent that the differentiation of the cells cultured in the two media is almost the same. That is, human bone marrow stem cells can be cultured in a medium containing human umbilical cord serum, and can be cultured in a 16 l3〇0〇95-based culture containing fetal bovine clear culture, and it is known that the same promotion is known. Under the condition of differentiation, human bone marrow stem cells cultured in human meridian serum have the same ability to promote differentiation of human bone marrow stem cells into nerve cells in vitro as those used in fetal bovine serum. In summary, the differentiation results of bone cells, adipocytes and nerve cells have shown that human umbilical cord serum can be used to replace fetal bovine serum and is applied to the differentiation and culture of stem cells.

Example 5 Comparison of In Vivo Differentiation and Regeneration of Animals First, vascular ligation of adult rats was performed to cause stroke symptoms in brain ischemia. Then human bone marrow stem cells and stem cells were cultured in FCS medium or hUCS+ medium. The vector was transplanted into the rat brain, and then the neurobehavioral measurement of the rats was performed on days 7, 14, 21, and 28 after the cell transplantation, and the results are shown in Fig. 9. milk

Referring to the first domain, the mice mixed with the above-mentioned _ culture scale-proliferating human bone-derived stem cells, in the mouse-only rat, after the second, 2i, 28 days, the reduced body shape asymmetry (such as the ninth &amp;The number of honey movements (as shown in Figure lb), the vertical motion time (as shown in Figure IX), and the vertical motion (as shown in Figure IX) have significant behavioral characteristics. And the human bone marrow stem cells cultured in the hUCS+ medium have the same effect as the human bone marrow stem cells obtained by the FCS culture medium. Therefore, it can be seen from the above results that the growth factor and the human body are contained.

17 1300095

Human bone-sensing stem cells cultured in serum-containing hUCS+ medium can be regenerated in the same manner as human bone marrow stem cells cultured in FCS medium containing fetal bovine serum, and have the same in vivo. Medical utility.

(D 18

1300095 [Simple illustration of the diagram] The first picture shows the growth state of human osteophyte stem cells cultured in different media after three days and seven days; (A) growth state after three days; (B) growth after seven days State FCS Medium containing fetal bovine serum hUCS Medium containing human umbilical cord serum hUCS+ medium containing human umbilical cord serum and growth factor SF serum-free medium The second picture shows human bone marrow stem cells cultured in different media after three days and seven days later Comparison of cell density; a: three days later; b: seven days later, the third figure is a comparison of cell size and granules detected by flow cytometry of human bone marrow stem cells cultured in different media; (A) containing fetal bovine serum Medium (B) medium containing human umbilical cord serum and growth factor

The fourth panel is the result of detecting cell surface antigen by flow cytometry of human bone marrow stem cells cultured in medium containing fetal bovine serum; (A) CD13; (B) CD29; (C) CD44; (D) CD73; E) CD90; (F) CD105; (G) HLA-ABC; (H) HLA-DR The fifth figure shows the detection of cells by flow cytometry using human bone stem cells cultured in medium containing human sputum serum and growth factors. a result map of surface antigens;

19 1300095

(A) CD13; (B) CD29; (C) CD44; (D) CD73; (E) CD90; (F) CD105; (G) HLA-ABC; (H) HLA-DR Figure 6 shows the medium Cultured human bone marrow stem cells in vitro to promote osteogenic differentiation results; (A) medium containing fetal bovine serum (B) medium containing human umbilical cord serum and growth factors

The seventh picture shows the staining of the results of human bone marrow stem cells cultured in different media to promote adipogenic differentiation in vitro; (A) medium containing fetal bovine jk clear (B) medium containing human umbilical cord serum and growth factor Staining map of human bone marrow stem cells cultured in different media to promote neuronal differentiation in vitro; and GFAP: glial fibrillary acidic protein

NF-200: neurofilament MAP_2: microtubule-associated protein 2 τιυ-ι:β tubulin

Nestin: Intermediate Silk Protein The ninth is a comparison of the medical improvement effects of human bone marrow stem cells cultured in different media in animals. (A) The proportion of exercise recovery; (B) The number of vertical movements 5 20 1300095 (C) Vertical movement time; (D) Vertical activity ▲: Medium containing human umbilical cord serum and growth factors: Medium containing fetal bovine serum ♦: Control Group [main component symbol description]

Claims (1)

.1300095 Office. June / M (: 3⁄4 正丨;, patent application scope: 1. A cell culture medium, containing: DMEM medium, containing: 0.1 / mol / l adrenal corticosterone, 10mmol / l /5 - Glycerol phosphate, and 50//mol/1 ascorbate; and further comprising: 10% human umbilical cord serum, 10 ng/ml epithelial cell growth factor (EGF), and 10 ng/ml basic fibroblast growth factor (bFGF), The cell culture medium differentiates human bone marrow stem cells into osteoblasts. 2. Cell culture medium, including: • DMEM medium containing: 1 #mol/l adrenal corticosterone, 5//g/ml insulin, 〇·5mmol /l isobutylmethylanthin, and 6〇# m〇1/卜引哚美辛; and further comprising: 10% human Teng V serum, l〇ng/ml of epithelial cell growth factor (egf), and 10 ng/ml of fibroblast growth factor (10) GF), which allows the cell culture medium to differentiate human bone marrow stem cells into adipocytes. 3. A method for using human umbilical cord serum, which uses a cell culture medium as described in claim 1, to differentiate human bone age cells into bone cells, and to use umbilical cord serum. Applying the cell culture medium described in item 2 of the patent scope to make the human face dry and aging into a fat fine 22