A method for isolating mesenchymal stem cells from umbilical cord amniotic membrane using cell culture medium

Abstract

The present invention relates to a method for isolating a mesenchymal stem cell population from the amniotic membrane of an umbilical cord, the method comprising culturing umbilical cord tissue in a culture medium, the culture medium comprising DMEM (Dulbecco's Modified Eagle's Medium), F12 (Ham's F12 Medium), M171 (Culture Medium 171) and FBS (Fetal Bovine Serum). The present invention also relates to a mesenchymal stem cell population isolated from the amniotic membrane of an umbilical cord, wherein at least about 90% or more of the cells of the stem cell population express each of the following markers: CD73, CD90 and CD105, and lack the expression of the following markers: CD34, CD45 and HLA-DR. The present invention also relates to a pharmaceutical composition of the mesenchymal stem cell population.

Description

[0001] Cross-references to related patent applications

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 404,582, filed on October 5, 2016, the contents of which are incorporated herein by reference in their entirety for all purposes. Technical Field

[0003] The present invention relates to a method for isolating mesenchymal stem cells (or such stem cell populations) from the amniotic membrane of an umbilical cord, and a mesenchymal stem cell population isolated from the amniotic membrane of an umbilical cord. The present invention also relates to a cell culture medium for isolating mesenchymal stem cells from the amniotic membrane of an umbilical cord. The present invention also relates to a pharmaceutical composition and use of the isolated mesenchymal stem cell population. The present invention also relates to a method for treating a disease or disorder, the method comprising administering the mesenchymal stem cell population of the present invention or a pharmaceutical composition containing such a mesenchymal stem cell population to a subject in need thereof. Background Art

[0004] Mesenchymal stem cells isolated from the amniotic membrane of the umbilical cord were first reported in U.S. Patent Application 2006 / 0078993 (granted U.S. Patents 9,085,755 and 9,737,568) and the corresponding international patent application WO2006 / 019357. Since then, umbilical cord tissue has attracted much attention as a source of pluripotent cells; due to the wide availability of the umbilical cord, the umbilical cord, especially stem cells isolated from the amniotic membrane of the umbilical cord (also known as "cord lining stem cells"), have been regarded as an excellent alternative source of cells for regenerative medicine. See Jeschke et al., Umbilical Cord Lining Membrane and Wharton's Jelly-Derived Mesenchymal Stem Cells: the Similarities and Differences; The Open Tissue Engineering and Regenerative Medicine Journal, 2011, 4, 21-27.

[0005] A subsequent study compared the phenotype, proliferation rate, migration, immunogenicity and immunomodulatory capacity of human mesenchymal stem cells (MSCs) derived from the amniotic membrane of the umbilical cord (umbilical cord lining (CL-MSC), umbilical cord blood (CB-MSC), placenta (P-MSC) and Wharton's jelly (WJ-MSC)) (Stubbendorf et al., Immunological Properties of Extraembryonic HumanMesenchymal Stromal Cells Derived from Gestational Tissue, STEM CELLS AND DEVELOPMENT Volume 22, Number 19, 2013, 2619-2629). Stubbendorf et al. concluded that MSC populations derived from extraembryonic gestational tissues showed different potentials to escape immune responses and exert immunomodulatory effects. The authors also found that CL-MSCs showed the most promising potential for cell therapy because they showed low immunogenicity, but they also showed enhanced proliferation and migration potential, so future studies should focus on the best disease models in which CL-MSCs can be administered.

[0006] Although mesenchymal stem cells of the amniotic membrane can be easily obtained using protocols such as those described in U.S. Patent Application 2006 / 0078993 and International Patent Application WO2006 / 019357, for clinical trials using these umbilical cord lining MSCs it would be advantageous to have a method that allows the isolation of a highly homogenous population of these umbilical cord lining MSCs that can therefore be used in clinical trials.

[0007] Therefore, one object of the present invention is to provide a method for isolating a mesenchymal stem cell population that meets this need from the amniotic membrane of the umbilical cord. Therefore, another object of the present invention is to provide a highly homogenous mesenchymal stem cell population isolated from the amniotic membrane of the umbilical cord. Summary of the invention

[0008] The objects of the present invention are achieved by a method, a population of mesenchymal stem cells, a corresponding pharmaceutical composition and a cell culture medium having the features stated in the independent claims.

[0009] In a first aspect, the present invention provides a method for isolating a population of mesenchymal stem cells from the amniotic membrane of an umbilical cord, the method comprising culturing the umbilical cord tissue in a culture medium comprising DMEM (Dulbecco's modified Eagle's medium), F12 (Ham's F12 medium), M171 (medium 171) and FBS (fetal bovine serum).

[0010] In a second aspect, the present invention provides a population of isolated mesenchymal stem cells from the amniotic membrane of the umbilical cord, wherein at least about 90% or more of the cells of the stem cell population express each of the following markers: CD73, CD90 and CD105. Preferably, the isolated mesenchymal stem cell population lacks expression of the following markers: CD34, CD45 and HLA-DR. In an embodiment of this second aspect, at least about 91% or more, about 92% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the cells of the isolated mesenchymal stem cell population express each of CD73, CD90 and CD105. In addition, in these embodiments of the second aspect, at least about 91% or more, about 92% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the cells of the isolated mesenchymal stem cell population lack expression of markers CD34, CD45 and HLA-DR. The mesenchymal stem cell population can be obtained by the method for isolating a mesenchymal stem cell population of the first aspect.

[0011] In a third aspect, the present invention provides a pharmaceutical composition comprising the mammalian cell of the present invention (the second aspect).

[0012] In a fourth aspect, the present invention provides a method for preparing a culture medium for separation, the method comprising mixing the following to obtain a culture medium with a final volume of 500 ml:

[0013] i. 250 ml of DMEM

[0014] ii.118ml M171

[0015] iii.118ml DMEM / F12

[0016] iv. 12.5 ml fetal bovine serum (FBS) to give a final concentration of 2.5% (v / v).

[0017] In a fifth aspect, the present invention provides a cell culture medium obtainable by the method described in the fourth aspect.

[0018] In a sixth aspect, the present invention provides a method for isolating mesenchymal stem cells from amniotic membrane of an umbilical cord, the method comprising culturing amniotic membrane tissue in a culture medium prepared by the method described in the fourth aspect.

[0019] In a seventh aspect, the present invention provides a cell culture medium comprising:

[0020] - DMEM at a final concentration of about 55 to 65% (v / v),

[0021] - F12 at a final concentration of about 5 to 15% (v / v),

[0022] - M171 at a final concentration of about 15 to 30% (v / v) and

[0023] - FBS at a final concentration of about 1 to 8% (v / v).

[0024] In an eighth aspect, the present invention provides use of the cell culture medium of the seventh aspect for isolating mesenchymal stem cells from amniotic membrane of umbilical cord.

[0025] In a ninth aspect, the present invention provides use of the cell culture medium described in the seventh aspect for culturing mesenchymal stem cells from amniotic membrane of the umbilical cord. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The present invention will be better understood by referring to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

[0027] Figure 1 shows a technical information sheet of Lonza's Dulbecco's modified eagle medium, which includes the catalog number of DMEM used to prepare an exemplary example of the culture medium of the present invention (PTT-6) in the experimental section;

[0028] Figure 2 A technical information sheet of Lonza's Ham's F12 medium is shown;

[0029] Figure 3 A technical information sheet of Lonza's DMEM:F12 (1:1) medium is shown, which includes the product catalog number of the DMEM:F12 (1:1) medium used to prepare an exemplary example (PTT-6) of the medium of the present invention in the experimental section;

[0030] Figure 4 shows a technical information sheet of M171 medium of Life Technologies Corporation, which includes the product catalog number of M171 medium used to prepare an exemplary example of the medium of the present invention (PTT-6) in the experimental section;

[0031] Figure 5 An ingredient list is shown, which includes the commercial suppliers and product catalog numbers of each ingredient used to prepare PTT-6 medium in the experimental section;

[0032] FIG6 shows the results of flow cytometry experiments in which the expression of mesenchymal stem cell markers CD73, CD90 and CD105 of mesenchymal stem cells isolated from umbilical cords was analyzed. For these experiments, mesenchymal stem cells were isolated from umbilical cord tissue by culturing the umbilical cord tissue in three different culture media, and then the mesenchymal stem cells were subcultured in the corresponding culture media. In these experiments, the following three culture media were used: a) 90% (v / v) DMEM supplemented with 10% FBS (v / v), b) PTT-4 culture medium described in U.S. Patent Application 2006 / 0078993 and corresponding International Patent Application WO2006 / 019357, which consists of 90% (v / v) CMRL1066 and 10% (v / v) FBS (see paragraph

[0183] of WO2006 / 019357) and c) PPT-6 culture medium of the present invention, the composition of which is described herein. In this flow cytometry analysis, two different samples of the umbilical cord lining mesenchymal stem cell (CLMC) population were analyzed in each of the three culture media used. The results are shown in Figures 6a to 6c In more detail, Figure 6a Shown is the percentage of isolated mesenchymal umbilical cord lining stem cells expressing stem cell markers CD73, CD90 and CD105 following isolation from umbilical cord tissue and culture in DMEM / 10% FBS. Figure 6b The percentage of isolated mesenchymal umbilical cord lining stem cells expressing stem cell markers CD73, CD90 and CD105 after isolation from umbilical cord tissue and culture in PTT-4 is shown, and Figure 6c Shown are the percentages of isolated mesenchymal umbilical cord lining stem cells expressing stem cell markers CD73, CD90 and CD105 following isolation from umbilical cord tissue and culture in PTT-6.

[0033] Figure 7 shows the results of flow cytometry experiments in which the expression of stem cell markers (CD73, CD90 and CD105, CD34, CD45 and HLA-DR (human leukocyte antigen-antigen D related) of mesenchymal stem cells isolated from the umbilical cord was analyzed and compared with the expression of these markers of bone marrow mesenchymal stem cells. For this experiment, mesenchymal stem cells of the amniotic membrane of the umbilical cord were isolated from the umbilical cord tissue by culturing the umbilical cord tissue in the PPT-6 medium of the present invention, while the bone marrow mesenchymal stem cells were isolated from human bone marrow using standard procedures. Figure 7aThe percentage of isolated mesenchymal umbilical cord lining stem cells expressing stem cell markers CD73, CD90 and CD105 and lacking expression of CD34, CD45 and HLA-DR following isolation from umbilical cord tissue and culture in PPT-6 medium is shown. Figure 7b Shown are the percentages of isolated bone marrow mesenchymal stem cells expressing CD73, CD90 and CD105 and lacking expression of CD34, CD45 and HLA-DR. DETAILED DESCRIPTION OF THE INVENTION

[0035] As described above, in the first aspect, the present invention relates to a method for isolating a mesenchymal stem cell population from the amniotic membrane of the umbilical cord, the method comprising culturing the umbilical cord tissue in a culture medium comprising DMEM (Dulbecco's Modified Eagle's Medium), F12 (Ham's F12 Medium), M171 (culture medium 171) and FBS (fetal bovine serum). It was surprisingly found in the present application that the use of such a culture medium provides the separation of a mesenchymal stem cell population from the amniotic membrane of the umbilical cord, wherein greater than 90% or even 99% or more of the cells in the mesenchymal stem cell population are positive for three mesenchymal stem cell markers CD73, CD90, and at the same time these stem cells lack the expression of CD34, CD45 and HLA-DR (see the experimental part), meaning that 99% or even more of the cells in the population express the stem cell markers CD73, CD90 and CD105 but do not express the markers CD34, CD45 and HLA-DR. Such highly homogenous and well-defined cell populations are ideal candidates for clinical trials and cell-based therapies because they fully meet the generally accepted criteria for human mesenchymal stem cells for cell therapy, as defined, for example, by Dominici et al., “Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement”, Cytotherapy (2006) Vol. 8, No. 4, 315-317, Sensebe et al., “Production of mesenchymal stromal / stem cells according to good manufacturing practices: a, review”, Stem Cell Research & Therapy 2013, 4: 66, Vonk et al., Stem Cell Research & Therapy (2015) 6: 94 or Kundrotas Acta Medica Lituanica. 2012. Vol. 19. No. 2. P. 75–79. In addition, using a bioreactor such as the Quantum Cell Expansion System, large quantities of mesenchymal stem cells can be obtained, such as 300 million to 700 million mesenchymal stem cells per run (see also the Experimental Section). Thus, the present invention allows the number of stem cells required for therapeutic applications (such as use in wound healing) to be provided in a low-cost manner. In addition, all components used to prepare the culture medium of the present invention are commercially available in GMP quality.Therefore, the present invention opens the way for the GMP production of such highly homogenous populations of mesenchymal stem cells from the amniotic membrane of the umbilical cord.

[0036] In this context, it is noted that the culture medium of the present invention allows the isolation of a population of mesenchymal stem cells (also referred to herein as "mesenchymal stem cells") from the amniotic membrane under conditions that allow the proliferation of mesenchymal stem cells / progenitor cells without differentiation of the mesenchymal stem cells / progenitor cells. Thus, after the mesenchymal stem cells are isolated from the amniotic membrane as described herein, the isolated population of mesenchymal stem cells / progenitor cells has the ability to differentiate into a variety of cell types, such as described in U.S. Patent Application No. 2006 / 0078993, U.S. Patent No. 9,085,755, International Patent Application No. WO2006 / 019357, U.S. Patent No. 8,287,854 or WO2007 / 046775. For example, as described in U.S. Patent Application No. 2006 / 0078993, the mesenchymal stem cells of the amniotic membrane of the umbilical cord have a spindle shape, express the following genes: POU5f1, Bmi-1, leukemia inhibitory factor (LIF), and secrete activin A and follistatin. Mesenchymal stem cells separated in the present invention can, for example, be differentiated into any type of mesenchymal cells, such as, but not limited to adipocytes, skin fibroblasts, chondrocytes, osteoblasts, tenocytes, ligament fibroblasts, cardiomyocytes, smooth muscle cells, skeletal muscle cells, adipocytes, cells producing mucin, cells derived from endocrine glands such as cells producing insulin (e.g., β-islet cells) or neuroectodermal cells. The stem cells separated in the present invention can be differentiated in vitro so that the differentiated cells are subsequently used for medical purposes. An illustrative example of this method is that mesenchymal stem cells are differentiated into β-islet cells producing insulin, which can then be applied to patients suffering from insulin deficiency (such as diabetes) (also referring to WO2007 / 046775 for this aspect) by implantation. Alternatively, the mesenchymal stem cells of the present invention can be used in their undifferentiated state for cell-based therapy, for example, for wound healing purposes, such as for the treatment of burns or chronic diabetic wounds. In these therapeutic applications, the mesenchymal stem cells of the present invention can both promote wound healing by interacting with surrounding diseased tissues and differentiate into corresponding skin cells (see again, for example, WO 2007 / 046775).

[0037] In this context, it is noted that the mesenchymal stem cell population described herein can be isolated and cultured from any umbilical cord tissue (i.e., can be derived from any umbilical cord tissue) as long as the umbilical cord tissue contains amniotic membrane (also known as "cord lining"). Therefore, as described in the experimental section of this application, the mesenchymal stem cell population can be isolated from (a portion of) the entire umbilical cord. Therefore, in addition to the amniotic membrane, the umbilical cord tissue can contain any other tissue / component of the umbilical cord. For example, as shown in Figure 16 of U.S. Patent Application 2006 / 0078993 or International Patent Application WO2006 / 019357, the amniotic membrane of the umbilical cord is the outermost part of the umbilical cord, covering the umbilical cord. In addition, the umbilical cord contains a vein (which transports oxygenated, nutrient-rich blood to the fetus) and two arteries (which carry deoxygenated, nutrient-depleted blood away from the fetus). For protection and mechanical support, these three blood vessels are embedded in Wharton's Jelly, a jelly-like substance made primarily of mucopolysaccharides. Therefore, the umbilical cord tissue used in the present invention may also include this one vein, two arteries and Wharton's jelly. Using this whole (complete) part of the umbilical cord has the following advantages: the amniotic membrane does not need to be separated from the other components of the umbilical cord. This reduces the separation step, and therefore makes the method of the present invention simpler, faster, less prone to error and more economical-these are important aspects of the GMP production required for the therapeutic application of mesenchymal stem cells. Therefore, the separation of mesenchymal stem cells can be started by the tissue separation block method (tissue explant), and if a larger amount of mesenchymal stem cells are needed, for example, for clinical trials, then the following passage culture (cultivation) of the separated mesenchymal stem cells can be carried out. Alternatively, the amniotic membrane can also be separated from the other components of the umbilical cord at first, and then the mesenchymal umbilical cord lining stem cells are separated from the amniotic membrane by culturing the amniotic membrane in the culture medium of the present invention. The culture can also be carried out by the tissue separation block method, optionally followed by passage culture of the separated mesenchymal stem cells. In the present context, the term "tissue separation block (method)" or "tissue separation block method" is used with its conventional meaning in the art, and refers to a method in which once the tissue is harvested, the tissue or a portion of the tissue is placed in a cell culture dish containing a culture (growth) medium, and over time, stem cells migrate out of the tissue to the surface of the culture dish. These primary stem cells can then be further amplified and transferred to a fresh culture dish by micropropagation (subculture) as described herein. In the present context, it is noted that in terms of producing cells for therapeutic purposes, in the first step of isolating amniotic mesenchymal stem cells from the umbilical cord, a master cell bank of isolated mesenchymal stem cells is obtained, and subsequent subculture can obtain a working cell bank.If the mesenchymal stem cell population of the present invention (particularly such a mesenchymal stem cell population in which at least about 98% or 99% express each of the markers CD73, CD90 and CD105 and lack the expression of each of the following markers: CD34, CD45 and HLA-DR) is used for clinical trials or as an approved therapeutic agent, the cell population of the working cell bank is generally used for this purpose. Both the stem cell population of the separation step (which can constitute the master cell bank) and the stem cell population of the subculture step (which can constitute the working cell bank) can be stored, for example, in a cryopreserved form.

[0038] As described above, the method of isolating mesenchymal stem cells from the amniotic membrane of the umbilical cord of the present invention has the following advantages: all components used in the culture medium of the present invention are available in GMP quality, thus providing the possibility of isolating mesenchymal stem cells under GMP conditions for subsequent therapeutic administration.

[0039] "DMEM" means Dulbecco's Modified Eagle's Medium, which was developed in 1969 and is a modification of Basal Eagle's Medium (BME) (see Figure 1 , which shows a data sheet of DMEM available from Lonza). The original DMEM formulation contained 1000 mg / L of glucose and was first reported for culturing embryonic mouse cells. Since then, DMEM has become a standard culture medium for cell culture, which can be commercially obtained from a variety of sources such as ThermoFisher Scientific (Catalog No. 11965-084), Sigma Aldrich (Catalog No. D5546), or Lonza, to name just a few suppliers. Therefore, any commercially available DMEM can be used in the present invention. In a preferred embodiment, the DMEM used herein is a DMEM culture medium available from Lonza with Catalog No. 12-604F. This culture medium is a DMEM supplemented with 4.5 g / L glucose and L-glutamine. In another preferred embodiment, the DMEM used herein is a DMEM culture medium with Catalog No. D5546 from Sigma Aldrich, which contains 1000 mg / L glucose and sodium bicarbonate, but does not contain L-glutamine.

[0040] "F12" medium refers to Ham's F12 medium. This medium is also a standard cell culture medium, a nutrient mixture originally designed for culturing a variety of mammalian and hybridoma cells when used with serum and in combination with hormones and transferrin (see Figure 2, which shows a data sheet for Ham's F12 medium from Lonza). Any commercially available Ham's F12 medium (e.g., from ThermoFisher Scientific (Cat. No. 11765-054), Sigma Aldrich (Cat. No. N4888), or Lonza, to name just a few suppliers) can be used in the present invention. In a preferred embodiment, Ham's F12 medium from Lonza is used.

[0041] "DMEM / F12" or "DMEM:F12" means a 1:1 mixture of DMEM and Ham's F12 medium (see Figure 3 , which shows a data sheet for DMEM:F12 (1:1) from Lonza. DMEM / F12 (1:1) medium is also a widely used basal medium for supporting the growth of many different mammalian cells and can be commercially obtained from multiple suppliers such as ThermoFisher Scientific (Catalog No. 11330057), Sigma Aldrich (Catalog No. D6421), or Lonza. Any commercially available DMEM:F12 can be used in the present invention. In a preferred embodiment, the DMEM:F12 medium used herein is a DMEM / F12 (1:1) medium available from Lonza with Catalog No. 12-719F (which is DMEM:F12 with L-glutamine, 15mM HEPES, and 3.151g / L glucose).

[0042] "M171" refers to Medium 171, which has been developed as a basal medium for growing normal human mammary epithelial cells (see Figure 4 , which shows a data sheet for M171 medium from Life Technologies Corporation). This basal medium is also widely used and can be commercially available from, for example, ThermoFisher Scientific or, for example, Life Technologies Corporation (catalog number M171500). Any commercially available M171 medium can be used in the present invention. In a preferred embodiment, the M171 medium used herein is the M171 medium available from Life Technologies Corporation with catalog number M171500.

[0043] "FBS" means fetal bovine serum (also referred to as "fetal bovine (calf) serum"), i.e., the blood portion retained after natural coagulation of blood and then centrifugation to remove any remaining red blood cells. Fetal bovine serum is the most widely used serum supplement in eukaryotic cell in vitro cell culture, because it has very low levels of antibodies and contains more growth factors, making it versatile in many different cell culture applications. Preferably, FBS is obtained from members of the International Serum Industry Association (ISIA), which focuses on ensuring the safety and safe use of serum and animal-derived products through appropriate traceability of origin, authenticity of labels, and appropriate standardization and supervision. Suppliers of FBS as ISIA members include AbattoirBasics Company, Animal Technologies Inc., Biomin Biotechnologia LTDA, GEHealthcare, Gibco by Thermo Fisher Scientific and Life Science Production, just to name a few examples. In a currently preferred embodiment, FBS is obtained from GE Healthcare with catalog number A15-151.

[0044] Turning now to the culture medium of the present invention, the culture medium may comprise DMEM at a final concentration of about 55 to 65% (v / v), F12 at a final concentration of about 5 to 15% (v / v), M171 at a final concentration of about 15 to 30% (v / v), and PBS at a final concentration of about 1 to 8% (v / v) for the isolation or culture of mesenchymal umbilical cord lining stem cells. The numerical values ​​of "% (v / v)" used herein refer to the volume of a single component relative to the final volume of the culture medium. This means, for example, that if DMEM is present in the culture medium at a final concentration of about 55 to 65% (v / v), 1 liter of culture medium contains about 550 to 650 ml of DMEM.

[0045] In other embodiments, the culture medium may comprise DMEM at a final concentration of about 57.5 to 62.5% (v / v), F12 at a final concentration of about 7.5 to 12.5% ​​(v / v), M171 at a final concentration of about 17.5 to 25.0% (v / v), and FBS at a final concentration of about 1.75 to 3.5% (v / v). In further embodiments, the culture medium may comprise DMEM at a final concentration of about 61.8% (v / v), F12 at a final concentration of about 11.8% (v / v), M171 at a final concentration of about 23.6% (v / v), and FBS at a final concentration of about 2.5% (v / v).

[0046] In addition to the above components, the culture medium may include supplements that are beneficial to culturing the mesenchymal umbilical cord lining stem cells. For example, the culture medium of the present invention may include epidermal growth factor (EGF). If present, EGF can be present in the culture medium at a final concentration of about 1 ng / ml to about 20 ng / ml. In some such embodiments, the culture medium may include EGF at a final concentration of about 10 ng / ml.

[0047] The culture medium of the present invention may also contain insulin. If present, the insulin may be present at a final concentration of about 1 μg / ml to 10 μg / ml. In some such embodiments, the culture medium may contain insulin at a final concentration of about 5 μg / ml.

[0048] The culture medium may further comprise at least one of the following supplements: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). In such embodiments, the culture medium may comprise all three of adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). In these embodiments, the culture medium may comprise adenine at a final concentration of about 0.05 to about 0.1 μg / ml adenine, hydrocortisone at a final concentration of 1 to about 10 μg / ml hydrocortisone, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of about 0.5 to about 5 ng / ml.

[0049] In the method of the present invention, umbilical cord tissue can be cultured until a suitable number of (primary) mesenchymal umbilical cord lining stem cells grow out of the tissue. In a typical embodiment, umbilical cord tissue is cultured until the cell outgrowth of the mesenchymal stem cells of the amniotic membrane reaches about 70% to about 80% confluence. It should be noted here that the term "confluency / confluence" is used in its conventional sense in the field of cell culture and is intended as an estimate / indicator of the number of adherent cells in a culture dish or culture bottle, referring to the proportion of the surface covered by cells. For example, 50% confluence means that about half of the surface is covered and there is still room for cell growth. 100% confluence means that the surface is completely covered with cells and there is no longer room for cell monolayer growth.

[0050] Once the primary cells (mesenchymal umbilical cord lining stem cells) of a suitable number are obtained from the umbilical cord lining tissue by the tissue separation block method, the mesenchymal stem cells are removed from the culture container for cultivation. By doing so, a master cell bank of mesenchymal stem cells (primary) separated containing amniotic membrane can be obtained. Generally, since mesenchymal stem cells are adherent cells, standard enzyme treatment is used to remove them. For example, the enzyme treatment may include trypsin digestion as described in International U.S. Patent Application 2006 / 0078993, International Patent Application WO2006 / 019357 or International Patent Application WO2007 / 046775, meaning that cells growing outward can be harvested for further expansion by trypsin digestion (0.125% trypsin / 0.05% EDTA). If the harvested mesenchymal stem cells are, for example, used to produce a master cell bank, the cells may also be cryopreserved and stored for further use, as described below.

[0051] Once harvested, the mesenchymal stem cells can be transferred to a culture vessel for subculture. The subculture can also be started from frozen primary cells, i.e., from the master cell bank. For subculture, any suitable number of cells can be seeded in a culture vessel such as a cell culture plate. For this purpose, the mesenchymal stem cells can be seeded at, for example, about 0.5 x 10 6 cells / ml to about 5.0 x 10 6 The cells are suspended in a medium suitable for subculture (most conveniently, the medium of the present invention) at a concentration of about 1.0 x 10 cells / ml. 6 The cells are suspended at a concentration of cells / ml for subculture. The subculture can be carried out either in a simple culture bottle or in a multilayer system that can be stacked in an incubator, such as CellStacks (Corning, Corning, NY, USA) or Cellfactory (part of Nunc, Thermo Fisher Scientific Inc., Waltham, MA, USA). Alternatively, the subculture can also be carried out in a closed, self-contained system (such as a bioreactor). Bioreactors of different designs are known to those skilled in the art, such as parallel plates, hollow fibers or microfluidic bioreactors. See, for example, Sensebe et al., "Production of mesenchymal stromal / stem cells according to good manufacturing practices: a review", supra. Illustrative examples of commercially available hollow fiber bioreactors are Cell Expansion System (Terumo BCT, Inc), which has been used, for example, to expand bone marrow mesenchymal stem cells for clinical trials (see Hanley et al., Efficient Manufacturing of Therapeutic Mesenchymal Stromal Cells Using the Quantum Cell Expansion System, Cytotherapy. 2014 August; 16(8): 1048–1058). Another example of a commercially available bioreactor that can be used to subculture the mesenchymal stem cell population of the present invention is the Xuri Cell Expansion System available from GE Heathcare. If a working cell bank for therapeutic applications is to be produced under GMP conditions and a large number of cells are required, then, for example, Culturing mesenchymal stem cell populations in the automated system of the Cell Expansion System is particularly beneficial.

[0052] The subculture of the mesenchymal umbilical cord lining stem cells of the present invention occurs in the culture medium of the present invention. Therefore, the culture medium of the present invention can be used to separate mesenchymal stem cells from amniotic membrane and for subsequent culture of isolated primary cells by subculture. Also for subculture, mesenchymal stem cells can be cultured until a suitable amount of cells are grown. In an illustrative embodiment, the mesenchymal stem cells are subcultured until the mesenchymal stem cells reach about 70% to about 80% fusion.

[0053] The isolation / culture of the mesenchymal umbilical cord lining stem cell population can be carried out under standard conditions for culturing mammalian cells. Typically, the method of isolating the mesenchymal umbilical cord lining stem cell population of the present invention is typically carried out under the following conditions (temperature, atmosphere): the conditions are generally used to culture cells of the species from which the cells are derived. For example, human umbilical cord tissue and mesenchymal umbilical cord lining stem cells are typically cultured at 37° C. in an air atmosphere containing 5% CO2, respectively. In this context, it is noted that in the present invention, mesenchymal cells can be derived from any mammalian species, such as mice, rats, guinea pigs, rabbits, goats, horses, dogs, cats, sheep, monkeys or humans, wherein in one embodiment, mesenchymal stem cells of human origin are preferred.

[0054] Once the desired / appropriate number of mesenchymal umbilical cord lining stem cells are obtained from the subculture, the mesenchymal stem cells are harvested by removing them from the culture container for subculture. The mesenchymal stem cells are usually harvested again by enzyme treatment, including trypsin digestion of cells. The separated mesenchymal stem cells are then collected and used directly or stored for further use. They are usually preserved by cryopreservation. The term "cryopreservation" is used in this article with its conventional meaning to describe the process of preserving mesenchymal stem cells by cooling to low subzero temperatures (such as (usually) -80°C or -196°C (boiling point of liquid nitrogen)). Cryopreservation can be performed as known to those skilled in the art, and may include the use of cryoprotectants such as dimethyl sulfoxide (DMSO) or glycerol, which slow down the formation of ice crystals in umbilical cord cells.

[0055] The isolated mesenchymal umbilical cord lining stem cell population obtained by the separation method of the present invention is highly defined and homogenous. In a typical embodiment of the method, at least about 90% or more, about 91% or more, about 92% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the isolated mesenchymal stem cells express the following markers: CD73, CD90 and CD105. In addition, in these embodiments, at least about 90% or more, about 91% or more, about 92% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the isolated mesenchymal stem cells may lack the expression of the following markers: CD34, CD45 and HLA-DR. In certain embodiments, about 97% or more, about 98% or more, or about 99% or more of the isolated mesenchymal stem cell population express CD73, CD90, and CD105, while lacking expression of CD34, CD45, and HLA-DR.

[0056] Therefore, according to the above disclosure, the present invention also relates to a population of mesenchymal stem cells isolated from the amniotic membrane of the umbilical cord, wherein at least about 90% or more of the cells of the stem cell population express each of the following markers: CD73, CD90 and CD 105. In a preferred embodiment, at least about 91% or more, about 92% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the cells of the isolated mesenchymal stem cell population are CD73+, CD90+ and CD105+, which means that the percentage of the isolated cell population expresses each of CD73, CD90 and CD105 (see the experimental part of this application). In addition, at least about 90% or more, about 91% or more, about 92% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the separated mesenchymal stem cells may lack the expression of the following markers. In a specific embodiment, about 97% or more, about 98% or more or about 99% or more of the separated mesenchymal stem cell population express CD73, CD90 and CD105, while lacking the expression of CD34, CD45 and HLA-DR. Such highly homogenous mesenchymal stem cell populations derived from the amniotic membrane of the umbilical cord are reported for the first time herein, and the cell populations meet the criteria for mesenchymal stem cells for cell therapy (see also the experimental section and, for example, Sensebe et al., "Production of mesenchymal stromal / stem cells according to good manufacturing practices: a review", supra). In this context it should be noted that the mesenchymal stem cell population can be obtained both by the isolation method of the present invention but, if necessary, also by a different method such as cell sorting.

[0057] As described above, the present invention also relates to a pharmaceutical composition comprising a population of mesenchymal stem cells isolated from the amniotic membrane of the umbilical cord, wherein at least about 90% or more of the cells of the stem cell population express each of the following markers: CD73, CD90 and CD105, and optionally lack the expression of CD34, CD45 and HLA-DR. The pharmaceutical composition may contain any pharmaceutically acceptable excipient and may be formulated for any desired pharmaceutical route of administration. The pharmaceutical composition may, for example, be suitable for systemic or local application.

[0058] In another aspect, the present invention relates to a method for preparing a culture medium for isolation, the method comprising mixing the following to obtain a final volume of 500 ml of culture medium:

[0059] i. 250 ml of DMEM

[0060] ii.118ml M171

[0061] iii.118ml DMEM / F12

[0062] iv. 12.5 ml fetal bovine serum (FBS) to give a final concentration of 2.5% (v / v).

[0063] As mentioned above, DMEM / F12 medium is a 1:1 mixture of DMEM and Ham's F12 medium. Therefore, 118 ml of DMEM / F12 medium contains 59 ml of DMEM and 59 ml of F12. Therefore, when this method of preparing the medium is used, the final concentration (v / v) in a total volume of 500 ml is as follows:

[0064] DMEM: 250 ml + 59 ml = 309 ml, corresponding to 309 / 500 = 61.8% (v / v)

[0065] M171: 118 ml, corresponding to 118 / 500 = 23.6% (v / v)

[0066] F12: 59 ml, corresponding to 59 / 500=11.8% (v / v).

[0067] Embodiments of the method of preparing a culture medium further comprise adding:

[0068] v. 1 ml of EGF stock solution (5 μg / ml) to give a final EGF concentration of 10 ng / ml, and

[0069] vi. Insulin 0.175 ml stock solution (14.28 mg / ml) to give a final insulin concentration of 5 μg / ml.

[0070] It should be noted here that in these embodiments, the volume of these components i to vi above-mentioned obtains the culture medium that final volume is 499.675ml.If no longer add other components in the culture medium, then remaining 0.325ml (volume added to 500ml) can be for example any one among component i to iv, and this means can be DMEM, M171, DMEM / F12 or FBS.Alternatively, certainly can regulate the concentration of EGF or insulin storing solution so that the cumulative volume of culture medium is 500ml.In addition, also note that component i to iv need not add according to the order that they are listed, but certainly can use any order to make these components mix to obtain culture medium of the present invention. This means, for example, that M171 and DMEM / F12 can be mixed together and then combined with DMEM and FBS to achieve final concentrations as described herein, i.e., a final concentration of about 55 to 65% (v / v) of DMEM, a final concentration of about 5 to 15% (v / v) of F12, a final concentration of about 15 to 30% (v / v) of M171 and a final concentration of about 1 to 8% (v / v) of FBS.

[0071] In other embodiments, the method further comprises adding one or more of the following supplements: adenine, hydrocortisone and 3,3',5-triiodo-L-thyronine sodium salt (T3) to DMEM in a volume of 0.325 ml, thereby obtaining a total volume of 500 ml of culture medium. In this embodiment, the final concentrations of these supplements in DMEM may be as follows:

[0072] about 0.05 to 0.1 μg / ml adenine, for example about 0.025 μg / ml adenine,

[0073] About 1 to 10 μg / ml hydrocortisone,

[0074] About 0.5 to 5 ng / ml 3,3',5-triiodo-L-thyronine sodium salt (T3), for example 1.36 ng / ml 3,3',5-triiodo-L-thyronine sodium salt (T3).

[0075] In light of the above disclosure, the present invention also relates to a cell culture medium obtainable or obtained by the method for preparing a culture medium as described herein.

[0076] In addition, the present invention also relates to a method for isolating mesenchymal stem cells from the amniotic membrane of the umbilical cord, wherein the method comprises culturing the amniotic membrane tissue in a culture medium prepared by the method described herein.

[0077] Therefore, the present invention also relates to a cell culture medium comprising:

[0078] - DMEM at a final concentration of about 55 to 65% (v / v),

[0079] - F12 at a final concentration of about 5 to 15% (v / v),

[0080] - M171 at a final concentration of about 15 to 30% (v / v) and

[0081] - FBS at a final concentration of about 1 to 8% (v / v).

[0082] In some embodiments of the culture medium described herein, the culture medium comprises DMEM at a final concentration of about 57.7 to 62.5% (v / v), F12 at a final concentration of about 7.5 to 12.5% ​​(v / v), M171 at a final concentration of about 17.5 to 25.0% (v / v), and FBS at a final concentration of about 1.75 to 3.5% (v / v). In other embodiments, the culture medium may comprise DMEM at a final concentration of about 61.8% (v / v), F12 at a final concentration of about 11.8% (v / v), M171 at a final concentration of about 23.6% (v / v), and FBS at a final concentration of about 2.5% (v / v).

[0083] In addition, the culture medium may further comprise epidermal growth factor (EGF) at a final concentration of about 1 ng / ml to about 20 ng / ml. In some embodiments, the culture medium comprises EGF at a final concentration of about 10 ng / ml. The culture medium described herein may further comprise insulin at a final concentration of about 1 μg / ml to about 10 μg / ml. In such embodiments, the culture medium may comprise insulin at a final concentration of about 5 μg / ml.

[0084] The cell culture medium of the present invention may further comprise at least one of the following supplements: adenine, hydrocortisone and 3,3',5-triiodo-L-thyronine sodium salt (T3). In some embodiments, the culture medium comprises all three of adenine, hydrocortisone and 3,3',5-triiodo-L-thyronine sodium salt (T3). If present, the culture medium may comprise adenine at a final concentration of about 0.01 to about 0.1 μg / ml adenine or about 0.05 to about 0.1 μg / ml adenine, comprise hydrocortisone at a final concentration of about 0.1 to about 10 μg / ml hydrocortisone or about 1 to about 10 μg / ml hydrocortisone, and / or comprise 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of about 0.5 to about 5 ng / ml.

[0085] In an embodiment of the cell culture medium, 500 ml of the cell culture medium of the present invention comprises:

[0086] i. 250 ml of DMEM

[0087] ii.118ml M171

[0088] iii.118ml DMEM / F12

[0089] iv. 12.5 ml fetal bovine serum (FBS) (final concentration 2.5%).

[0090] In further embodiments, the cell culture medium may further comprise:

[0091] v. EGF at a final concentration of 10 ng / ml, and

[0092] vi. Insulin at a final concentration of 5 μg / ml.

[0093] Insulin and EGF can be added to the culture medium using selected stock solutions so that the total volume of the culture medium does not exceed 500 ml.

[0094] In a specific embodiment, components i to vi of the culture medium of the present invention are Figure 5 The components shown in Figure 5 The catalog numbers shown were obtained from the respective manufacturers. Figure 5 The medium obtained by mixing the components i to vi shown is also referred to herein as "PTT-6". It is also noted in this context that components i to vi and any other components (such as antibiotics from any other commercial supplier) can be used to prepare the medium of the present invention.

[0095] In addition, the cell culture medium of the present invention can contain adenine at a final concentration of about 0.01 to about 0.1 μg / ml adenine or about 0.05 to about 0.1 μg / ml adenine, hydrocortisone at a final concentration of about 0.1 to about 10 μg / ml, about 0.5 to about 10 μg / ml, or about 1 to about 10 μg / ml hydrocortisone, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of about 0.1 to about 5 ng / ml or about 0.5 to about 5 ng / ml.

[0096] Finally, the present invention also provides a method for treating a patient suffering from a disease, the method comprising administering to the patient the mesenchymal umbilical cord lining stem cells disclosed herein or a pharmaceutical composition containing stem cells disclosed herein. The disease may be any of the diseases described above. In order to treat the subject, the mesenchymal stem cell population of the present invention may be administered in any suitable manner, including, but not limited to, topical administration, by implantation or by injection. The stem cell population may, for example, be placed directly on a wound such as a burn or diabetic wound (see International Patent Application WO2007 / 046775). Alternatively, the stem cell population may also be implanted subcutaneously, for example, directly under the skin, in body fat or peritoneum.

[0097] The present invention is further illustrated by the following non-limiting experimental examples.

[0098] Experimental Examples

[0099] 1. Cryopreservation of umbilical cord tissue before mesenchymal stem cell isolation

[0100] As described below, umbilical cord tissue (the cord was donated with the mother's informed consent) was processed to allow for the subsequent isolation of mesenchymal stem cells from the amniotic membrane of the cord.

[0101] 1.1 Washing of umbilical cord tissue samples:

[0102] a. Remove the scalpel from the protective cover.

[0103] b. Clamp the umbilical cord with forceps and cut it into 10 cm long sections with a scalpel. Place the unusable umbilical cord back into the original tissue cup.

[0104] c. Transfer the 10 cm long umbilical cord segment to a new 150 mm Petri dish. A 150 mm Petri dish can be used instead of the cup.

[0105] d. Use the lid of a 150 mm culture dish as a place to place forceps and a scalpel.

[0106] e. Use a 30 ml syringe to remove 25 ml of Plasmalyte A (Baxter, catalog #2B2543Q). Hold the syringe at a 45° angle with one hand and dispense Plasmalyte A directly onto the umbilical cord tissue.

[0107] f. Tilt the culture dish slightly and remove Plasmalyte A using a 30 ml syringe and blunt needle.

[0108] g. Collect the used Plasmalyte A in a 300 ml transfer bag which serves as a waste container and dispose of it in the biohazard bin.

[0109] h. Repeat the washing procedure, using a new dish for each wash if necessary. Make sure all blood clots on the surface have been removed. If washing the tissue is necessary, use more Plasmalyte A.

[0110] i. Place the tissue into a new labeled tissue culture dish to continue cutting the tissue. Add 20 ml of Plasmalyte A to the dish so that the tissue does not dry out while cutting.

[0111] j. Cut the umbilical cord into equal length pieces of about 1 cm, a total of 10 pieces.

[0112] k. Then cut each 1 cm block into smaller pieces, each block is about 0.3 cm x 0.3 cm to 0.5 cm x 0.5 cm.

[0113] l. Remove any Plasmalyte A from the culture dish.

[0114] m. Use a 30 ml syringe to remove 25 ml of Plasmalyte A from the original Plasmalyte A bag and dispense directly onto the umbilical cord tissue piece.

[0115] n. Hold the dish at an angle so that all the Plasmalyte A used to wash the tissue is collected on one side and then removed using a syringe and blunt needle.

[0116] o. Repeat the wash once more. No clots should remain.

[0117] NOTE: If the umbilical cord is not frozen immediately, store the cord tissue in Plasmalyte A until ready to freeze.

[0118] 1.2 Cryopreservation of umbilical cord tissue:

[0119] a. Preparation of cryopreservation solution:

[0120] i. Prepare 50 ml of a freezing solution consisting of 60% Plasmalyte A, 30% 5% human serum albumin and 10% dimethyl sulfoxide (DMSO).

[0121] ii. Label a 150 ml transfer bag "Tissue Freeze" and connect the plasma transfer device to the port using aseptic technique.

[0122] iii. Use a 30 ml syringe to remove 30 ml of Plasmalyte A from the original Plasmalyte A bag and transfer it to a transfer bag labeled "Tissue Freezing Solution" with the date and time the solution was prepared.

[0123] iv. Use a 20 ml syringe to take 15 ml of 5% human serum albumin and transfer it to the labeled transfer bag.

[0124] v. Add 5 ml DMSO to the transfer bag.

[0125] vi. Mix well and record the mixing status of the freezing liquid.

[0126] b. Remove Plasmalyte A from tissue before adding freezing solution.

[0127] c. Using a 60 ml syringe, draw all 50 ml of the cryopreserved solution into the syringe and add approximately 30 ml of the cryopreserved solution to the 150 mm cell culture dish containing the umbilical cord tissue. Place a blunt needle on the syringe to maintain sterility.

[0128] d. Spin the dish containing tissue and cryovial every 1 minute for 10 minutes.

[0129] e. Use forceps to select 8 randomly selected pieces and place them into each of 4 4 ml cryovials. Select 4 randomly selected pieces and place them into one 1.8 ml cryovial. These pieces should be free of blood clots.

[0130] f. Add the remaining cryovial to each cryovial containing umbilical cord tissue, filling the 4 ml tube to the 3.6 ml line and the 1.8 ml Nunc vial to the 1.8 ml line.

[0131] g. Label one Bactec Lytic / 10-Anaerobic / F bottle and one Bactec PlusAerobic / F bottle with the tissue ID.

[0132] h. Use a syringe and blunt needle to take out 20ml of frozen liquid from the culture dish, wipe the Bactec vial with an alcohol swab, convert the blunt needle into an 18g needle, and inoculate the aerobic and anaerobic Bactec bottles, 10ml per bottle.

[0133] i. Start the controlled rate freezer.

[0134] j. After controlled rate freezing is complete, place the device in a liquid nitrogen freezer with continuous temperature monitoring until further use.

[0135] 2. Isolation of Mesenchymal Umbilical Cord Lining Stem Cells from Umbilical Cord Tissue

[0136] 2.1 Preparation of culture medium for treating MSCs from umbilical cord tissue:

[0137] a. Prepare 500 ml of PTT6 (culture / growth medium) by mixing the following in the order listed:

[0138] i.DMEM, 250ml

[0139] ii.M171 118ml

[0140] iii.DMEM F12 118ml

[0141] iv. FBS 12.5 ml (final concentration 2.5%)

[0142] v. EGF 1ml (final concentration 10ng / ml)

[0143] vi. Insulin 0.175ml (final concentration 5μg / ml)

[0144] The volume of above-mentioned components i to vi obtains the culture medium that final volume is 499.675ml.If other components are no longer added in the culture medium, the remaining 0.325ml (volume added to 500ml) can be any one of, for example, components i to iv, which means that it can be DMEM, M171, DMEM / F12 or FBS.Alternatively, the concentration of EGF or insulin stock solution can certainly be adjusted so that the cumulative volume of the culture medium is 500ml.Alternatively, the stock solution of antibiotic (such as penicillin-streptomycin-amphotericin) can be added to obtain a final volume of 500ml.One or more of the following supplements of 0.325ml volume can also be added to the culture medium: adenine, hydrocortisone and 3,3', 5-triiodo-L-thyronine sodium salt (T3), thus obtaining a culture medium that cumulative volume is 500ml.

[0145] vii. Label the bottle with "PTT6", the date the medium was prepared, the operator's initials, and the expiration date followed by the phrase "Expires". The expiration date is the earliest expiration date of any component or 1 month from the preparation date, whichever comes first.

[0146] b. In a 50 ml centrifuge tube, add 2.5 ml FBS to 47.5 ml HBSS to prepare rinse medium (Hank's buffered saline solution (HBSS) with 5% FBS without calcium or magnesium). Label the tube "Rinse Medium", the operator's initials, and the date the medium was prepared.

[0147] c. All culture media were sterile tested using Bactec Lytic / 10–Anaerobic / F (Becton Dickinson & Company) and Bactec Pluc+Aerobic / F (Becton Dickinson & Company). 20 ml of the prepared culture medium was injected into each bottle.

[0148] 2.2 Thawing of umbilical cord tissue for MSC harvesting:

[0149] a. When the operator is ready to process the samples in the clean room, begin thawing. Do not thaw more than 1 vial at a time unless the vials are from the same donor.

[0150] b. Wipe the water bath with disinfectant, then wipe with 70% isopropyl alcohol, and fill the water bath with 1L of sterile water. Heat the water bath to 36-38°C.

[0151] c. Prepare 10 mL of rinse medium consisting of 70% to 90% PlasmaLyte A in a clean room under a biosafety cabinet. Sterile filter the solution using a 0.2-μm syringe filter attached to a 10 ml syringe and keep the solution refrigerated until use.

[0152] d. Attach treatment labels to 50 ml conical tubes.

[0153] e. Make sure the water bath temperature is 36-38°C.

[0154] f. Remove tissue vials from liquid nitrogen storage and thaw quickly in a 37°C water bath filled with 1L sterile water. The vial holder for the Mr.Frosty NalgeneCryo 1°C Freezing Container floats with the vials and can be used as a floating stand while thawing samples.

[0155] g. Remove the vial from the water bath and spray it with 70% isopropyl alcohol solution. A good time to remove the vial from the water bath is when small ice can be seen floating in the vial - it is recommended that the internal temperature of the vial is below 37°C.

[0156] h. Place vial into transfer channel and alert cleanroom processing technician.

[0157] 2.3 Preparation of tissue processing:

[0158] a. Umbilical cord tissue processing should be performed in an environmentally monitored (EM) cleanroom. The entire room and hood should be cleaned at the end of each shift.

[0159] b. Prepare / clean the biosafety cabinet.

[0160] c. Perform presence particle counts while working in a biosafety cabinet.

[0161] d. Assemble all necessary supplies in a biosafety cabinet, inspecting each supply for damage to packaging and expiration dates. When handling syringes, serological pipettes, sterile forceps, scalpels, tissue plates, and needles, make sure not to touch any surfaces that come in contact with sterile products. Only the outside of the syringe barrel, tubing, plunger tip, and / or needle cap or sheath are safe to handle. If the surfaces have been touched or come into contact with a non-sterile surface, discard the supply.

[0162] e. Record the lot numbers and expiration dates (if applicable) of all reagents and supplies to be used.

[0163] f. Receive the thawed vials by cleaning the vials with a lint-free cloth soaked in 70% alcohol and then transferring to a biosafety cabinet.

[0164] g. Using the aspiration needle attached to the syringe, remove as much fluid as possible from the vial. Avoid aspirating tissue.

[0165] h. Using sterile forceps, transfer the tissue to a sterile 100 mm culture dish.

[0166] i. Add an aliquot of 5 ml of rinse medium to the tissue fragments.

[0167] j. Spin the contents for 15-30 seconds, then remove the rinse medium using a pipette or syringe with an aspiration needle. Repeat this rinse process twice

[0168] k. Add 2 mL of rinsing medium to the tissue to prevent the tissue from drying out.

[0169] 2.4 Beginning of MSC self-organized outgrowth:

[0170] a. Label the bottom of the 6-well plate with "Outgrowth 1", the MSC lot number or umbilical cord tissue ID, and the date the outgrowth was started. If using a 60 mm tissue culture dish, divide the plate into 4 quadrants by drawing a grid on the bottom of the dish.

[0171] b. Using sterile disposable forceps, place one 3 x 3 mm to 5 x 5 mm piece of tissue into each well. If using a 60 mm tissue culture dish, place the tissue in the middle of each quadrant to keep the tissues separate (more than 1 cm from each other).

[0172] c. Add 3 ml of PTT6 to each well.

[0173] d. Using a pipette needle attached to a 30 ml syringe, draw up enough medium to barely cover the tissue. Do not tilt the plate. Do not touch the bottom of the well with the pipette needle.

[0174] e. Using an inverted optical microscope, observe the cell outgrowth every day (24 ± 6 hours). A real-time cell culture imaging system can be used instead of an optical microscope.

[0175] f. Change the culture medium every day. Always ensure that the culture medium is equilibrated to room temperature before use.

[0176] i. Aspirate the medium.

[0177] ii. Add 3 ml of PTT6.

[0178] iii. Aspirate until the tissue is almost submerged in the medium.

[0179] g. When outgrowth of cells from the tissue is observed, transplant the tissue into a new 6-well plate using the same procedure as 4.a to 4.e above, except that the plate is labeled "Outgrowth 2". The cell outgrowth in the "Outgrowth 1" plate is maintained by adding 2 ml of PTT6 to each well. Observe confluence daily. Change the medium every 2-3 days (be sure to equilibrate the medium to room temperature before use).

[0180] h. When cell outgrowth is observed in the "Outgrowth 2" plate, repeat steps 4.a to 4.e, except label the plate "Outgrowth 3". Maintain cell outgrowth in the "Outgrowth 2" plate by adding 2 ml of PTT6 to each well. Observe confluence daily. Change the medium every 2-3 days (make sure the medium is equilibrated to room temperature before use).

[0181] i. When outgrowth is observed in the "Outgrowth 3" plate, discard the tissue. If the tissue is very small and does not appear to interfere with cell growth, dispose of it when subculturing.

[0182] j. When cells reach 40-50% confluency, observe cells daily to prevent overexpansion.

[0183] k. When cells reach 70-80% confluence, subculture the cells. Do not allow cells to expand beyond 80% confluence.

[0184] The size of the tissue fragments is about 1-3 mm, and the tissue fragment method / cell culture is performed in a 175 square millimeter culture dish, so the average number of mesenchymal stem cells harvested from the fragments is generally about 4,000-6,000 cells / fragment. Thus, when mesenchymal stem cells grow from 48 fragments at the same time, about 300,000 cells can be obtained at the time of harvest. Then, by inoculating these 300,000 cells in a 175 cm2 culture dish as described in Example 2.5 below, 2 The 300,000 mesenchymal stem cells collected from the separation block are used for subculture in a cell culture flask (this can be referred to as passage 1). The mesenchymal stem cells obtained from this passage 1 can then be seeded again in a 175 cm 2 Cells are cultured in a cell culture flask (passage 2) and expanded as described in Example 2.5 below. Cells obtained from passages 1 and 2 can be cryopreserved as a "bank", wherein the mesenchymal stem cells obtained after passage 2 are considered to represent a master cell bank, which will be used, for example, to further expand the mesenchymal stem cells in a bioreactor as described in Example 2.7 below.

[0185] 2.5. Subculturing MSCs in cell culture dishes

[0186] a. Perform particle counts while working in a biosafety cabinet. Equilibrate all media to room temperature before use.

[0187] b. When the cell outgrowth reaches approximately 70-80% confluence, subculture the cells.

[0188] i. Remove PTT6 from the culture dish.

[0189] ii. Rinse with HBSS without calcium or magnesium.

[0190] iii. Add 0.2 ml 1X TrypLE-EDTA and rotate for 1-2 minutes.

[0191] iv. Tilt the dish 30-45° to allow the cells to move downward by gravity flow. Gently tap on the side of the plate to accelerate detachment.

[0192] v. Add 1 ml of PTT6. Gently pipette up and down, then transfer the cells to a 15 ml centrifuge tube. Use a clean pipette tip for each well. Cells from all 6 wells can be combined into a single 15 ml tube.

[0193] vi. Centrifuge at 1200 rpm for 10 minutes.

[0194] vii. Remove supernatant and resuspend cells in 5 ml PTT6.

[0195] c. Subculturing MSCs.

[0196] i. Aliquot 50 μl of the cell suspension and analyze TNC and viability by Trypan Blue Exclusion Assay.

[0197] ii. Count the cells using a hemocytometer. Expect a count of 20-100 cells / square. If the count is above 100, dilute the original sample 1:5 and repeat the trypan blue method using a hemocytometer.

[0198] iii. Calculate the viable cells / ml and total viable cells:

[0199] 1. Viable cells / ml = viable cell count × dilution factor × 10 4

[0200] 2. Total viable cells = viable cell count × dilution factor × total volume × 10 4

[0201] iv. Calculate % survival:

[0202] 1. % survival rate = viable cell count × 100 / (viable cell count + dead cell count)

[0203] v. Dilute the cell suspension to 1.0×10 6 cells / ml

[0204] 1. "X" volume = total viable cells / 10 6 cells / ml

[0205] 2. For example, if the total number of viable cells is 1.0×10 7 ;

[0206] 3. “X” = 10 7 / 10 6 cells / ml or 10ml, therefore by adding 5ml to the cell suspension (i.e. 5ml), the total cell volume can be brought to 10ml.

[0207] vi. If the cell suspension is less than 10 6 / ml, then determine each 150mm culture dish or 175cm 2 The bottle was inoculated with 2×10 6 The volume required for each cell.

[0208] 1.2×10 6 The volume required for cells = 2×10 6 cells ÷ viable cells / ml

[0209] 2. For example, if the viable cells / ml is 8×10 5 cells / ml, 2×10 6 cells÷8×10 5 cells / ml or 2.5ml.

[0210] vii. Set aside 0.5 ml for MSC marker analysis.

[0211] viii. In a 150 mm dish or 175 cm 2 The bottle was inoculated with 2×10 6 cells.

[0212] ix. Observe cells for attachment, colony formation, and confluence every three days. When cells reach 40-50% confluence, observe cells every 1-2 days to prevent overexpansion. Do not allow cells to expand beyond 80% confluence. A real-time cell culture monitoring system can be used instead of an optical microscope.

[0213] x. Change the culture medium every 2-3 days.

[0214] 2.6 Cryopreservation of MSCs

[0215] a. Perform presence particle counts while working in a biosafety cabinet.

[0216] b. When the cells reach about 70-80% confluence, 2 The flask was filled with 2 ml of 1X Tryp LE-EDTA to detach the cells.

[0217] i. Remove PTT6 from the culture dish.

[0218] ii. Wash with 5 ml of HBSS or PBS without calcium or magnesium.

[0219] iii. Add 2 ml 1X TrypLE-EDTA and rotate for 1-2 minutes.

[0220] iv. Tilt the dish 30-45° to allow the cells to move downward by gravity flow. Gently tap the side of the dish to help speed up the separation.

[0221] v. Add 10 ml of PTT6 to inactivate TrypLE and mix thoroughly to separate cell clumps.

[0222] vi. Use a Pasteur pipette to transfer the cells to a 15 ml centrifuge tube.

[0223] vii. Centrifuge at 1200 rpm for 10 minutes.

[0224] viii. Aspirate the medium and resuspend with 10 ml PTT6.

[0225] ix. Aliquot 50 μl and determine total viable cells and % viability as above. Cell counts should be completed within 15 minutes as cells may begin to clump.

[0226] c. Prepare cells for cryopreservation.

[0227] i. Prepare cell suspension medium and cryopreservation medium, and freeze cells

[0228] 2.7. Subculturing (expansion) of MSCs in a Quantum bioreactor (Terumo BTC, Inc.)

[0229] A Quantum bioreactor can also be used, which can be used to expand MSCs. The starting cell count for expansion in a Quantum bioreactor should be 20 million to 30 million cells per run. The yield per run is typically 300 million to 700 million MSCs during harvest. The bioreactor is operated according to the manufacturer's instructions. The mesenchymal stem cells thus obtained are typically cryopreserved (see below) and used as a working cell bank.

[0230] Materials / Reagents:

[0231] 1. Quantum Amplification Device

[0232] 2. Quantum waste bag

[0233] 3. Quantum culture medium bag

[0234] 4. Quantum enters the bag

[0235] 5.PTT6

[0236] 6. PBS

[0237] 7. Fibronectin

[0238] 8. TrypLE

[0239] 9. 3ml syringe

[0240] 10. Glucose test strips

[0241] 11. Lactic acid test paper

[0242] 12. 60ml cell culture plate or equivalent

[0243] 13. Medical grade 5% CO2 gas mixture

[0244] 14. 50ml Combi-tip

[0245] equipment:

[0246] 1. Biological safety cabinet

[0247] 2. Blood glucose meter (Bayer Healthcare / Ascensia Contour blood glucose meter)

[0248] 3. Lactate Plus (Nova Biomedical)

[0249] 4. Peristaltic pump with head

[0250] 5. Centrifuge, Eppendorf 5810

[0251] 6. Sterile tube connector

[0252] 7. M4 Repeating Pipette

[0253] 8. RF sealer

[0254] process:

[0255] 1. Prepare the Quantum Bioreactor

[0256] a) Start-up of Quantum Bioreactor

[0257] b) Coating bioreactor:

[0258] 1) Prepare fibronectin solution in a biosafety cabinet.

[0259] 1) Allow the freeze-dried fibronectin to adapt to room temperature (≥15 min at room temperature)

[0260] 2) Add 5 ml of sterile distilled water; do not rotate or stir

[0261] 3) Allow fibronectin to form a solution for 30 minutes.

[0262] 4) Using a 10 ml syringe with an 18 g needle attached, transfer the fibronectin solution into the cell access bag containing 95 ml PBS.

[0263] 2) Connect the bag to the "reagent" line

[0264] 3) Check for air bubbles (air bubbles can be removed by using Remove IC Air or Remove EC Air and using Scrub as the entry source.

[0265] 4) Open or set up the coating bioreactor program ( Figure 1 , steps 3-5).

[0266] 5) Run the program

[0267] 6) While the program is running to coat the bioreactor, prepare a media bag with 4 L of PTT6 media.

[0268] 7) Connect the media bag to the IC media line using a sterile tubing connector.

[0269] 8) When the bioreactor coating step is complete, separate the cells for the fibronectin solution into the bag using a RF sealer.

[0270] c) Wash off excess fibronectin

[0271] d) Conditioning the bioreactor with culture medium.

[0272] 2. Cultivating cells in a Quantum bioreactor

[0273] a) Loading and attaching cells with a homogenous suspension:

[0274] b) Feeding and culturing cells

[0275] 1) Choose the medium flow rate to feed the cells.

[0276] 2) Lactate and glucose were sampled daily.

[0277] 3) As lactate levels increase, adjust the flow rate of the medium. The actual maximum tolerated lactate concentration will be determined by the culture flask culture from which the cells were derived. Determine if there is enough PTT6 medium in the medium bag. If necessary, replace the PTT6 medium bag with a new one.

[0278] 4) When the flow rate reaches the expected value, measure lactate levels every 8-12 hours. If lactate levels do not decrease or if lactate levels continue to increase, harvest the cells.

[0279] 3. Harvesting cells from the Quantum bioreactor

[0280] a) Harvest cells after the last lactate and glucose sampling when lactate concentration has not decreased.

[0281] b) Harvesting cells:

[0282] 1) Connect the cell access bag containing 100 ml of TrypLE to the "reagent" line using a sterile tubing connector.

[0283] 2) Confirm that there is enough PBS in the PBS bag. If not, connect a new bag with at least 1.7 liters of PBS to the "Wash" line using a sterile tubing connector.

[0284] 3) Run the Harvest program

[0285] 4. Cryopreservation of cells

[0286] 1) Once the cells are harvested, transfer the cells to a 50 ml centrifuge tube to pellet the cells.

[0287] 2) Resuspend with 25 ml of cold cell suspension buffer. Count cells using a Sysmex or Biorad cell counter. Attach the cell count report to the corresponding Quantum processing batch record.

[0288] 3) Adjust the cell concentration to 2×10 7 / ml

[0289] 4) Add an equal volume of cryopreservation solution and mix thoroughly (do not shake or vortex)

[0290] 5) Using a repeating pipette, add 1 ml of the cell suspension in cryopreservation solution to each 1.8 ml vial. Cryopreserve using the CRF procedure as described in SOP D6.100 CB Cryopreservation Using Controlled Rate Freezers.

[0291] 6) Store the vials in a designated liquid nitrogen storage space.

[0292] 7) Attach the CRF run report to the form of the corresponding MSC P3-Quantum process batch record.

[0293] 3. Using different culture media to analyze the stem cells in the mesenchymal umbilical cord lining stem cell population isolated from umbilical cord tissue Expression of markers

[0294] Flow cytometry experiments were performed to analyze the expression of mesenchymal stem cell markers CD73, CD90, and CD105 in mesenchymal stem cells isolated from umbilical cord.

[0295] For these experiments, mesenchymal stem cells were isolated from umbilical cord tissue by culturing the tissue in three different culture media and then subcultured in the corresponding culture media as described in Example 2.

[0296] In these experiments, the following three culture media were used: a) 90% (v / v) DMEM supplemented with 10% FBS (v / v), b) PTT-4 culture medium described in U.S. Patent Application 2006 / 0078993 and the corresponding International Patent Application WO2006 / 019357, which consists of 90% (v / v) CMRL1066 and 10% (v / v) FBS (see paragraph

[0183] of WO2006 / 019357), and c) PPT-6 culture medium of the present invention, the composition of which is described herein. In this flow cytometric analysis, two different samples of the umbilical cord lining mesenchymal stem cell (CLMC) population were analyzed in each of the three culture media used.

[0297] The flow cytometry analysis employed the following protocol.

[0298] Materials and methods

[0299] Instrument Name Company Name Series Name BD FACS CANDO BD V07300367 Inverted microscope, CKX41SF Olympus 4K40846 Centrifuge, microspinning tabletop Biosan 010213-1201-0003

[0300]

[0301]

[0302]

[0303]

[0304] process:

[0305] a) Isolation and culture of cells from the umbilical cord lining

[0306] 1. The excised tissue samples were incubated in a cell culture plate and immersed in the corresponding culture medium, and then maintained in a CO2 incubator at 37°C as described in Example 2.

[0307] 2. Replace the culture medium every 3 days.

[0308] 3. Monitor cell outgrowth from tissue culture explants under a light microscope.

[0309] 4. At approximately 70% confluence, cells were detached from the culture dishes by trypsinization (0.0125% trypsin / 0.05% EDTA) and used for flow cytometry experiments.

[0310] b) Trypsinization of cells used for experiments

[0311] 1. Remove the culture medium from the cell culture plate.

[0312] 2. Gently rinse with sterile 1X PBS to remove traces of FBS, as FBS will interfere with the enzymatic action of trypsin.

[0313] 3. Add 1X trypsin to the cell culture plate and incubate at 37°C for 3-5 minutes.

[0314] 4. Observe the cells under a microscope and ensure that the cells have detached. Neutralize the trypsin by adding complete medium containing FBS (DMEM with 10% FBS).

[0315] 5. Use a pipette to break up the cell clumps by pumping the cells in the culture medium against the wall of the culture plate. Collect the cell suspension and transfer it to a 50 ml centrifuge tube.

[0316] 6. Add sterile 1X PBS to the plate and rinse, collecting the cell suspension into the same centrifuge tube.

[0317] 7. Centrifuge at 1800 rpm for 10 minutes.

[0318] 8. Discard the supernatant and resuspend the cell pellet with PBA medium.

[0319] c) Count cells

[0320] 1. Ensure that the hemocytometer and its cover glass are clean and dry. Preferably, wash with 70% ethanol and dry, then wipe with Kim paper towel (dust-free paper).

[0321] 2. Aliquot a small amount of suspended cells into microcentrifuge tubes and remove from the BSC hood.

[0322] 3. Stain the suspended cells with an equal volume of Trypan blue, for example, add 500 μl of Trypan blue to 500 μl of suspension (dilution factor = 2X, to obtain 0.2% Trypan blue solution).

[0323] 4. Avoid exposing cells to trypan blue for more than 30 minutes because trypan blue is toxic and will lead to an increase in non-viable cells, thus giving an erroneous cell count.

[0324] 5. Add 20 μl of the cell suspension mixture to each well of the hemacytometer and observe under an optical microscope.

[0325] a. Count the number of viable cells (bright cells; non-viable cells readily absorb trypan blue and therefore appear dark) in each quadrant of the hemacytometer, for a total of 8 quadrants in the upper and lower chambers.

[0326] The total cell count was expressed as (average number of cells / quadrant) × 10 4 Cells / ml are given.

[0327] d) Staining cells

[0328] i. Preparation of cells before staining

[0329] - Aliquot the cell suspension in duplicate into 3 tubes (CD73, CD90, CD105) and 2 tubes (negative control), each containing 50,000 cells.

[0330] ii. Staining with primary antibody (Ab)

[0331] Add 1 μl of primary antibody (0.5 mg / ml Ab) to 100 μl of cell suspension and incubate at 4°C for 45 min.

[0332] · Use PBA to make up to 1 ml.

[0333] Centrifuge at 8000 rpm for 5 min at 4°C.

[0334] Remove the supernatant.

[0335] - Add 1 ml PBA and resuspend the pellet.

[0336] Centrifuge at 8000 rpm for 5 min at 4°C.

[0337] Remove the supernatant.

[0338] Resuspend in 100ul PBA.

[0339] iii. Staining with secondary antibody -In the dark

[0340] Add 1ul [0.5mg / ml ab] secondary antibody to 100ul cell suspension and incubate at 4°C for 30min.

[0341] · Use PBA to make up to 1 ml.

[0342] Centrifuge at 8000 rpm for 5 min at 4°C.

[0343] Remove the supernatant.

[0344] - Add 1 ml PBA and resuspend the pellet.

[0345] Centrifuge at 8000 rpm for 5 min at 4°C.

[0346] Remove the supernatant.

[0347] • Resuspend in 200-300ul PBA for flow cytometry analysis.

[0348] • Transfer cells to FACS tubes for reading in a BD FACS CANDO flow cytometer.

[0349] The results of flow cytometry analysis are shown in Figures 6a to 6c . Figure 6a Shown is the percentage of isolated mesenchymal umbilical cord lining stem cells expressing the stem cell markers CD73, CD90 and CD105 following isolation from umbilical cord tissue and culture in DMEM / 10% FBS. Figure 6b The percentage of isolated mesenchymal umbilical cord lining stem cells expressing stem cell markers CD73, CD90 and CD105 after isolation from umbilical cord tissue and culture in PTT-4 is shown, and Figure 6c Shown is the percentage of isolated mesenchymal umbilical cord lining stem cells expressing stem cell markers CD73, CD90 and CD105 following isolation from umbilical cord tissue and culture in PTT-6. Figure 6a As can be seen in Figure 2, the isolated cell population cultured with DMEM / 10% FBS as the medium had approximately 75% CD73+ cells, 78% CD90+ cells, and 80% CD105+ cells (average of two sets of experiments), while the isolated cell population cultured with PPT-4 medium (see Figure 2) had approximately 75% CD73+ cells, 78% CD90+ cells, and 80% CD105+ cells (average of two sets of experiments). Figure 6b) after isolation / culture of umbilical cord tissue from the present invention, the number of CD73-positive, CD90-positive and CD105-positive mesenchymal stem cells was about 87% (CD73+ cells), 93% (CD90+ cells) and 86% (CD105+ cells), which is the average of two sets of experiments. With respect to all three markers (CD73, CD90, CD105), the purity of the mesenchymal stem cell population obtained by the culture means in the PTT-6 medium of the present invention is at least 99.0%, meaning that the purity of the cell population is significantly higher than that of the culture using PPT-4 medium or DMEM / 10% FBS. Moreover, even more importantly, the mesenchymal stem cell population obtained by the culture means in PTT-6 is essentially a 100% pure and defined stem cell population. This makes the stem cell population of the present invention an ideal candidate for stem cell-based therapies. Therefore, this mesenchymal umbilical cord lining stem cell population may become the gold standard for such stem cell-based therapeutic methods.

[0350] pass Figure 7a and Figure 7b The results of the flow cytometric analysis shown in further confirmed the findings shown in FIG6 . Figure 7a The percentage of isolated mesenchymal umbilical cord lining stem cells (umbilical cord amniotic membrane mesenchymal stem cells) expressing stem cell markers CD73, CD90 and CD105 and lacking expression of CD34, CD45 and HLA-DR after isolation from umbilical cord tissue and culture in PPT-6 medium is shown. Figure 7aAs shown, the mesenchymal stem cell population contains 97.5% of viable cells, 100% of which express each of CD73, CD90 and CD105 (see "CD73+CD90+" and "CD73+CD105+" rows), while 99.2% of the stem cell population does not express CD45, and 100% of the stem cell population does not express CD34 and HLA-DR (see "CD34-CD45-" and "CD34-HLA-DR-" rows). Therefore, the mesenchymal stem cell population obtained by culturing in PTT-6 medium is essentially a 100% pure and defined stem cell population that meets the standards of mesenchymal stem cells for cell therapy (95% or more of the stem cell population express CD73, CD90 and CD105, while 98% or more of the stem cell population lacks expression of CD34, CD45 and HLA-DR, see Sensebe et al., "Production of mesenchymal stromal / stem cells according to good manufacturing practices: a review", supra). It should be noted here that the amniotic mesenchymal stem cells of the present invention adhere to plastic and differentiate into osteoblasts, adipocytes and chondroblasts in vitro under standard culture conditions, see US Pat. No. 9,085,755, US Pat. No. 8,287,854 or WO 2007 / 046775, and therefore meet the generally accepted criteria for mesenchymal stem cells for cell therapy.

[0351] Figure 7b The percentage of isolated bone marrow mesenchymal stem cells expressing CD73, CD90 and CD105 and lacking expression of CD34, CD45 and HLA-DR is shown. Figure 7b As shown, the bone marrow mesenchymal stem cell population contains 94.3% of viable cells, of which 100% express each of CD73, CD90 and CD105 (see "CD73+CD90+" and "CD73+CD105+" rows), while only 62.8% of the bone marrow stem cell population lacks expression of CD45, and 99.9% of the stem cell population lacks expression of CD34 and HLA-DR (see "CD34-CD45-" and "CD34-HLA-DR-" rows). Therefore, compared to the (umbilical cord amniotic membrane) mesenchymal stem cell population of the present application, the bone marrow mesenchymal stem cells, which are considered to be the gold standard of mesenchymal stem cells, have much lower homology / purity in terms of stem cell markers. This finding also shows that the stem cell population of the present invention can be an ideal candidate for stem cell-based therapy, and that the stem cell population can become the gold standard for stem cell-based treatment methods.

[0352] It will be apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0353] All patents and publications mentioned in this specification represent the level of ordinary technicians in the field to which the invention belongs. All patents and publications are incorporated herein by reference to the same extent as if each individual publication was specifically and individually incorporated by reference.

[0354] The invention described in an illustrative manner herein can be implemented in the absence of any one or more elements, any one or more limitations not specifically disclosed herein. Thus, for example, the terms "include", "comprises" and "contains" etc. should be understood broadly and without limitation. In addition, the terms and expressions used herein have been used as descriptive words, not restrictive, and when using these terms and expressions, there is no intention to exclude any equivalent form of the features shown and described or parts thereof, but it is recognized that various modifications can be made within the scope of the invention claimed. Thus, it should be understood that although the present invention is specifically disclosed by preferred embodiments and optional features, those skilled in the art may make modifications and changes to the invention presented therein disclosed herein, and such modifications and changes are considered to be within the scope of the invention. The invention is described broadly and generally herein. Each narrower species and sub-generic grouping falling within the general disclosure also forms part of the present invention. In the case of removing any subject matter from the category premise or negative limitation, this includes the general description of the invention, regardless of whether the deleted content is described in detail herein. In addition, in the case where the features or aspects of the present invention are described in accordance with the Markush group, those skilled in the art will recognize that the present invention is also described in accordance with any single member or member subgroup of the Markush group. Other embodiments of the invention will be apparent from the following claims.

Claims

1. A method for isolating a population of mesenchymal stem cells from an amniotic membrane of an umbilical cord, the method comprising culturing the umbilical cord tissue in a culture medium comprising DMEM (Dulbecco's Modified Eagle's Medium), F12 (Ham's F12 Medium), M171 (Medium 171) and FBS (fetal bovine serum), The culture medium comprises DMEM at a final concentration of 55 to 65% (v / v), F12 at a final concentration of 5 to 15% (v / v), M171 at a final concentration of 15 to 30% (v / v), and FBS at a final concentration of 1 to 3.5% (v / v).

2. The method of claim 1, wherein the culture medium comprises DMEM at a final concentration of 57.5 to 62.5% (v / v), F12 at a final concentration of 7.5 to 12.5% ​​(v / v), M171 at a final concentration of 17.5 to 25.0% (v / v), and FBS at a final concentration of 1.75 to 3.5% (v / v).

3. The method of claim 2, wherein the culture medium comprises DMEM at a final concentration of 61.8% (v / v), F12 at a final concentration of 11.8% (v / v), M171 at a final concentration of 23.6% (v / v), and FBS at a final concentration of 2.5% (v / v). The method according to claim 1 , wherein the culture medium further comprises epidermal growth factor (EGF) at a final concentration of 1 ng / ml to 20 ng / ml. The method according to claim 4 , wherein the culture medium comprises EGF at a final concentration of 10 ng / ml. The method according to claim 1 , wherein the culture medium comprises insulin at a final concentration of 1 μg / ml to 10 μg / ml.

7. The method of claim 6, wherein the culture medium comprises insulin at a final concentration of 5 µg / ml.

8. The method of claim 1, wherein the culture medium further comprises at least one of the following supplements: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3).

9. The method of claim 8, wherein the culture medium comprises all three of adenine, hydrocortisone and 3,3',5-triiodo-L-thyronine sodium salt (T3).

10. The method according to claim 9, wherein the culture medium comprises adenine at a final concentration of 0.01 to 0.1 µg / ml, hydrocortisone at a final concentration of 0.1 to 10 µg / ml, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of 0.5 to 5 ng / ml.

11. The method according to any one of claims 1 to 10, comprising culturing the umbilical cord tissue until the cell outgrowth of the amniotic mesenchymal stem cells reaches 70-80% confluence. 12 . The method according to claim 11 , comprising removing the mesenchymal stem cells from a culture container used for the culture. 13 . The method according to claim 12 , wherein the removing of the mesenchymal stem cells from the culture container is performed by enzyme treatment. The method of claim 13 , wherein the enzymatic treatment comprises trypsin digestion. 15 . The method according to any one of claims 12 to 14 , wherein the mesenchymal stem cells are transferred to a culture container for subculture for subculture.

16. The method according to claim 15, wherein the mesenchymal cells are expressed at 1.0 x 10 6 The cells were suspended at a concentration of 10 cells / ml for subculture. 17 . The method according to claim 16 , wherein the mesenchymal stem cells are subcultured in the culture medium defined in any one of claims 1 to 9 . 18 . The method according to claim 17 , wherein the mesenchymal stem cells are subcultured until the mesenchymal stem cells reach 70-80% confluence.

19. The method according to claim 15, wherein the subculturing is performed in a self-contained bioreactor.

20. The method of claim 19, wherein the bioreactor is selected from the group consisting of a parallel plate bioreactor, a hollow fiber bioreactor, and a microfluidic bioreactor.

21. The method of any one of claims 1 to 10, wherein the umbilical cord tissue is a portion of the entire umbilical cord or the amniotic membrane of the umbilical cord.

22. The method according to any one of claims 1 to 10, wherein the culturing is carried out at a temperature of 37°C in a CO2 cell culture incubator. 23 . The method according to claim 15 , comprising removing the mesenchymal stem cells from a culture container used for the subculture.

24. The method according to claim 23, wherein removing the mesenchymal stem cells from the culture container is performed by enzyme treatment.

25. The method of claim 24, wherein the enzymatic treatment comprises trypsin digestion.

26. The method of claim 25, further comprising collecting the isolated mesenchymal stem cells.

27. The method of claim 1, wherein at least 90% or more of the isolated mesenchymal stem cells express the following markers: CD73, CD90, and CD105.

28. The method of claim 27, wherein at least 90% or more of the isolated mesenchymal stem cells lack expression of the following markers: CD34, CD45, and HLA-DR (human leukocyte antigen-antigen D-related).

29. The method of claim 27 or 28, wherein 97% or more of the isolated mesenchymal stem cells express CD73, CD90 and CD105 and lack expression of CD34, CD45 and HLA-DR.

30. The method of claim 29, wherein 98% or more of the isolated mesenchymal stem cells express CD73, CD90 and CD105 and lack expression of CD34, CD45 and HLA-DR.

31. The method of claim 30, wherein 99% or more of the isolated mesenchymal stem cells express CD73, CD90 and CD105 and lack expression of CD34, CD45 and HLA-DR.

32. The method of claim 26, further comprising preserving the isolated mesenchymal stem cells for further use.

33. The method of claim 32, wherein the preservation is by cryopreservation.

34. A method for preparing a culture medium suitable for isolating a population of mesenchymal stem cells from the amniotic membrane of an umbilical cord, the method comprising mixing the following to obtain a final volume of 500 ml of culture medium: i. 250 ml of DMEM ii. 118 ml M171 iii. 118 ml DMEM / F12 iv. 12.5 ml fetal bovine serum (FBS) to give a final concentration of 2.5%.

35. The method of claim 34, further comprising adding: v. 1 ml of 5 µg / ml EGF stock solution to give a final concentration of 10 ng / ml, vi. 0.175 ml of 14.28 mg / ml insulin stock solution to give a final concentration of 5 µg / ml.

36. The method of claim 34 or 35, further comprising adding one or more of the following supplements to DMEM: adenine, hydrocortisone and 3,3',5-triiodo-L-thyronine sodium salt (T3), thereby obtaining a total volume of 500 ml of medium.

37. The method of claim 36, wherein the final concentration of the supplement in DMEM is as follows: 0.05 to 0.1 µg / ml adenine, 1 to 10 µg / ml hydrocortisone, 0.5 to 5 ng / ml 3,3',5-triiodo-L-thyronine sodium salt (T3).

38. A cell culture medium obtainable by the method of any one of claims 34 to 37.

39. A method for isolating mesenchymal stem cells from amniotic membrane of an umbilical cord, the method comprising culturing amniotic membrane tissue in a culture medium prepared by the method defined in any one of claims 34 to 37.

40. A cell culture medium comprising: - DMEM at a final concentration of 55 to 65 % (v / v), - F12 at a final concentration of 5 to 15 % (v / v), - Final concentration of 15 to 30 % (v / v) of M171 and - FBS at a final concentration of 1 to 3.5 % (v / v).

41. The cell culture medium of claim 40, wherein the cell culture medium comprises DMEM at a final concentration of 57.7 to 62.5% (v / v), F12 at a final concentration of 7.5 to 12.5% ​​(v / v), M171 at a final concentration of 17.5 to 25.0% (v / v), and FBS at a final concentration of 1.75 to 3.5% (v / v).

42. The cell culture medium of claim 41, wherein the cell culture medium comprises DMEM at a final concentration of 61.8% (v / v), F12 at a final concentration of 11.8% (v / v), M171 at a final concentration of 23.6% (v / v), and FBS at a final concentration of 2.5% (v / v).

43. The cell culture medium of claim 40, wherein the culture medium further comprises epidermal growth factor (EGF) at a final concentration of 1 ng / ml to 20 ng / ml.

44. The cell culture medium of claim 43, wherein the culture medium comprises EGF at a final concentration of 10 ng / ml.

45. The cell culture medium of claim 40, wherein the culture medium comprises insulin at a final concentration of 1 µg / ml to 10 µg / ml.

46. ​​The cell culture medium of claim 45, wherein the culture medium comprises insulin at a final concentration of 5 µg / ml.

47. The cell culture medium of claim 40, wherein the cell culture medium further comprises at least one of the following supplements: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3).

48. The cell culture medium of claim 47, wherein the culture medium comprises all three of adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3).

49. The cell culture medium of claim 47, wherein the culture medium comprises adenine at a final concentration of 0.05 to 0.1 µg / ml, hydrocortisone at a final concentration of 1 to 10 µg / ml, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of 0.5 to 5 ng / ml.

50. The cell culture medium of claim 40, wherein 500 ml of the cell culture medium comprises: i. 250 ml of DMEM ii. 118 ml M171 iii. 118 ml DMEM / F12 iv. 12.5 ml fetal bovine serum (FBS).

51. The cell culture medium of claim 50, further comprising: v. EGF at a final concentration of 10 ng / ml vi. Insulin at a final concentration of 5µg / ml vi. Insulin 0.175 ml to give a final concentration of 5µg / ml.

52. The cell culture medium according to claim 50 or 51, further comprising adenine at a final concentration of 0.05 to 0.1 µg / ml, hydrocortisone at a final concentration of 1 to 10 µg / ml, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of 0.5 to 5 ng / ml.

53. Use of a cell culture medium as defined in any one of claims 40 to 52 for isolating mesenchymal stem cells from the amniotic membrane of the umbilical cord.

54. Use of a cell culture medium as defined in any one of claims 40 to 52 for culturing mesenchymal stem cells from the amniotic membrane of the umbilical cord.

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