Method of isolating mesenchymal stem cells from umbilical cord amniotic membrane using cell culture medium

Abstract

To provide a method of isolating a mesenchymal stem cell population from the amniotic membrane of the umbilical cord, and a highly uniform population of the mesenchymal stem cell isolated from the amniotic membrane of the umbilical cord.SOLUTION: The present invention relates to a method of isolating a mesenchymal stem cell population from an amniotic membrane of an umbilical cord, the method comprising cultivating umbilical cord tissue in a culture medium comprising DMEM (Dulbecco's modified eagle medium), F12 (Ham's F12 Medium), M171 (Medium 171) and FBS (Fetal Bovine Serum). The present invention also relates to a mesenchymal stem cell population isolated from the amniotic membrane of the umbilical cord, at least about 90% or more cells of the stem cell population expressing each of markers, CD73, CD90 and CD105, and lacking expression of markers, CD34, CD45 and HLA-DR. The invention also relates to a pharmaceutical composition of this mesenchymal stem cell population.SELECTED DRAWING: Figure 6c

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 62 / 404,582, filed on October 5, 2016, the content of which is hereby incorporated by reference in its entirety for all purposes.

[0002] Field of the Invention The present invention relates to a method for isolating mesenchymal stem cells (or a population of such stem cells) from the amnion of the umbilical cord, and to a population of mesenchymal stem cells isolated from the amnion of the umbilical cord. The present invention also relates to a cell culture medium for isolating mesenchymal stem cells from the amnion of the umbilical cord. The present invention also relates to a pharmaceutical composition and use of the isolated population of mesenchymal stem cells. The present invention also relates to a method of treating a disease or disorder comprising administering to a subject in need thereof the mesenchymal stem cell population of the present invention or a pharmaceutical composition containing such a population of mesenchymal stem cells.

Background Art

[0003] Background of the Invention Mesenchymal stem cells isolated from the amnion of the umbilical cord were first reported in US Patent Application No. 2006 / 0078993 (Patent Document 1) (leading to the registered US Patent No. 9,085,755 (Patent Document 2) and US Patent No. 9,737,568 (Patent Document 3)) as well as the corresponding international patent application WO2006 / 019357 (Patent Document 4). Since then, umbilical cord tissue has attracted attention as a source of pluripotent cells; the umbilical cord, and specifically the stem cells isolated from the amnion of the umbilical cord (also referred to as "cord lining stem cells"), are considered an excellent alternative source of cells for regenerative medicine because they are widely available. 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 (Non-Patent Document 1).

[0004] In subsequent studies, the phenotypes, proliferation rates, migration, immunogenicity, and immunomodulatory capabilities of human mesenchymal stem cells (MSCs) derived from the amnion of the umbilical cord (cord lining (CL-MSC)), umbilical cord blood (CB-MSC), placenta (P-MSC), and Wharton's jelly (WJ-MSC) were compared (Stubbendorf et al, Immunological Properties of Extraembryonic Human Mesenchymal Stromal Cells Derived from Gestational Tissue, STEM CELLS AND DEVELOPMENT Volume 22, Number 19, 2013, 2619-2629 (Non-Patent Document 2)). Stubbendorf et al concluded that MSC populations derived from extraembryonic gestational tissues exhibit diverse abilities to evade immune responses and exert immunomodulatory effects. The authors also found that these cells show the most promising potential for cell-based therapies because CL-MSCs exhibit low immunogenicity and enhanced proliferative and migratory capabilities. Therefore, future research should focus on the best disease models in which CL-MSCs can be administered.

[0005] Mesenchymal stem cells of the amnion can be easily obtained using the protocols described in US Patent Application No. 2006 / 0078993 (Patent Document 1) and International Patent Application WO2006 / 019357 (Patent Document 4). Having a method at hand to isolate a population of these cord lining MSCs that is highly homogeneous and thus can be used in clinical trials is advantageous for clinical trials using these cord lining MSCs.

[0006] Therefore, an object of the present invention is to provide a method for isolating a population of mesenchymal stem cells derived from the amnion of the umbilical cord that meets this requirement. Accordingly, it is also an object of the present invention to provide a highly homogeneous population of mesenchymal stem cells isolated from the amnion of the umbilical cord.

Prior Art Documents

Patent Documents

[0007] [Patent Document 1] U.S. Patent Application No. 2006 / 0078993 [Patent Document 2] U.S. Patent No. 9,085,755 [Patent Document 3] U.S. Patent No. 9,737,568 [Patent Document 4] International Patent Application WO2006 / 019357 [Non-Patent Document]

[0008] [Non-Patent Document 1] 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 [Non-Patent Document 2] Stubbendorf et al, Immunological Properties of Extraembryonic Human Mesenchymal Stromal Cells Derived from Gestational Tissue, STEM CELLS AND DEVELOPMENT Volume 22, Number 19, 2013, 2619-2629 [Summary of the Invention]

[0009] This object is achieved by a method, a mesenchymal stem cell population, each pharmaceutical composition, and a cell culture solution having the features of the independent claims.

[0010] In a first aspect, the present invention provides a method for isolating a mesenchymal stem cell population from the amnion of the umbilical cord, the method comprising culturing umbilical cord tissue in a culture medium comprising DMEM (Dulbecco's Modified Eagle Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and FBS (fetal bovine serum).

[0011] In a second aspect, the present invention provides an isolated mesenchymal stem cell population from the amnion 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 the expression of the following markers: CD34, CD45, and HLA-DR. In a plurality of embodiments 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. Additionally, 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 preferably lack the expression of the 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.

[0012] In a third aspect, the present invention provides a pharmaceutical composition containing the mammalian cells of the present invention (of the second aspect).

[0013] In a fourth aspect, the present invention provides a method for preparing a culture medium for isolation, the method comprising mixing the following to obtain a culture medium with a final volume of 500 ml: i. 250 ml of DMEM ii. 118 ml of M171 iii. 118 ml of DMEM / F12 iv. 12.5 ml of fetal bovine serum (FBS) to obtain a final concentration of 2.5% (v / v).

[0014] In a fifth aspect, the present invention provides a cell culture medium obtainable by the method of the fourth aspect.

[0015] In a sixth aspect, the present invention provides a method for isolating mesenchymal stem cells from the amnion of the umbilical cord, the method comprising culturing amniotic tissue in a culture medium prepared by the method of the fourth aspect.

[0016] In a seventh aspect, the present invention provides a cell culture medium comprising: - DMEM at a final concentration of about 55-65% (v / v), - F12 at a final concentration of about 5-15% (v / v), - M171 at a final concentration of about 15-30% (v / v), and - FBS at a final concentration of about 1-8% (v / v).

[0017] In an eighth aspect, the present invention provides the use of the cell culture medium of the seventh aspect for isolating mesenchymal stem cells from the amnion of the umbilical cord.

[0018] In a ninth aspect, the present invention provides the use of the cell culture medium of the seventh aspect for culturing mesenchymal stem cells derived from the amnion of the umbilical cord. [Invention 1001] A method for isolating a mesenchymal stem cell population from the amnion of the umbilical cord, the method comprising culturing umbilical cord tissue in a culture medium comprising DMEM (Dulbecco's Modified Eagle Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and FBS (fetal bovine serum). [Invention 1002] The method of the present invention 1001, wherein the culture medium contains DMEM at a final concentration of about 55-65% (v / v), F12 at a final concentration of about 5-15% (v / v), M171 at a final concentration of about 15-30% (v / v), and FBS at a final concentration of about 1-8% (v / v). [The present invention 1003] The method of the present invention 1002, wherein the culture medium contains DMEM at a final concentration of about 57.5-62.5% (v / v), F12 at a final concentration of about 7.5-12.5% (v / v), M171 at a final concentration of about 17.5-25.0% (v / v), and FBS at a final concentration of about 1.75-3.5% (v / v). [The present invention 1004] The method of the present invention 1003, wherein the culture medium contains 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). [The present invention 1005] The method according to any one of the present inventions 1001 to 1004, wherein the culture medium further contains epidermal growth factor (EGF) at a final concentration of about 1 ng / ml to about 20 ng / ml. [The present invention 1006] The method according to any one of the present inventions 1001 to 1005, wherein the culture medium contains EGF at a final concentration of about 10 ng / ml. [The present invention 1007] The method according to any one of the present inventions 1001 to 1006, wherein the culture medium contains insulin at a final concentration of about 1 μg / ml to 10 μg / ml. [The present invention 1008] The method according to any one of the present inventions 1001 to 1007, wherein the culture medium contains insulin at a final concentration of about 5 μg / ml. [The present invention 1009] The method according to any one of the above-mentioned present inventions, wherein the culture medium further contains at least one of the supplementary substances adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). [The present invention 1010] Any method of the present invention, wherein the culture medium contains all three of adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). [Invention 1011] Any method of Invention 1011 or 1012, wherein the culture medium contains adenine at a final concentration of about 0.01 to about 0.1 μg / ml, hydrocortisone at a final concentration of about 0.1 to about 10 μg / ml, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of about 0.5 to about 5 ng / ml. [Invention 1012] Any method of the present invention, including the step of culturing umbilical cord tissue until the cell proliferation of amniotic mesenchymal stem cells reaches a confluence density of about 70 - 80%. [Invention 1013] Any method of Invention 1012, including the step of removing mesenchymal stem cells from the culture vessel used for culturing. [Invention 1014] Any method of Invention 1013, wherein the step of removing mesenchymal stem cells from the culture vessel is performed by enzymatic treatment. [Invention 1015] Any method of Invention 1014, wherein the enzymatic treatment includes trypsin treatment. [Invention 1016] Any method of Invention 1013 - 1015, wherein the mesenchymal stem cells are transferred to a culture vessel for subculture for subculture. [Invention 1017] Any method of Invention 1016, wherein the mesenchymal cells are suspended at a concentration of 1.0×10 6 cells / ml for subculture. [Invention 1018] Any method of Invention 1017, wherein the mesenchymal stem cells are subcultured in a culture medium defined in any of Invention 1001 - 1010. [Invention 1019] Any method of Invention 1018, wherein the mesenchymal stem cells are subcultured until they reach a confluence density of about 70 - 80%. [Invention 1020] The method according to any one of the present inventions 1016 to 1019, wherein subculture is performed in a self - contained bioreactor. [The present invention 1021] The method according to the present invention 1020, wherein the bioreactor is selected from the group consisting of a parallel - plate bioreactor, a hollow - fiber bioreactor, and a microfluidic bioreactor. [The present invention 1022] Any of the methods of the present invention, wherein the umbilical cord tissue is a small piece from the whole umbilical cord or the amnion of the umbilical cord. [The present invention 1023] The culture is carried out in a cell culture incubator at a temperature of 37 °C in CO 2 Any of the methods of the present invention, wherein the culture is performed in a cell culture incubator. [The present invention 1024] The method according to the present invention 1023, comprising the step of removing mesenchymal stem cells from the culture vessel used for subculture. [The present invention 1025] The method according to the present invention 1024, wherein the step of removing mesenchymal stem cells from the culture vessel is performed by enzymatic treatment. [The present invention 1026] The method according to the present invention 1025, wherein the enzymatic treatment includes trypsin treatment. [The present invention 1027] The method according to the present invention 1026, further comprising the step of collecting the isolated mesenchymal stem cells. [The present invention 1028] 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 markers CD73, CD90, and CD105. Any of the methods of the present invention. [The present invention 1029] 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 lack the expression of the markers CD34, CD45, and HLA-DR (human leukocyte antigen - antigen D related), any method of the present invention as described above. [Inventive concept 1030] About 97% or more, about 98% or more, about 99% or more of the isolated mesenchymal stem cells express CD73, CD90, and CD105 and lack the expression of CD34, CD45, and HLA-DR, any method of either Inventive concept 1028 or 1029 of the present invention. [Inventive concept 1031] Any method of the present invention as described above, further comprising the step of storing the isolated stem / progenitor cells for further use. [Inventive concept 1032] The method of Inventive concept 1031, wherein the storing step is performed by cryopreservation. [Inventive concept 1033] An isolated mesenchymal stem cell population of umbilical cord amnion, wherein at least about 90% or more of the cells of the stem cell population express each of the markers CD73, CD90, and CD105, said mesenchymal stem cell population. [Inventive concept 1034] At least about 90% or more of the cells of the stem cell population lack the expression of the markers CD34, CD45, and HLA-DR, the mesenchymal stem cell population of Inventive concept 1033. [Inventive concept 1035] 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 in the isolated mesenchymal stem cell population express each of CD73, CD90, and CD105 and lack the expression of each of CD34, CD45, and HLA-DR, the mesenchymal stem cell population of the present invention 1034. [The present invention 1036] A mesenchymal stem cell population according to any one of the present inventions 1033 to 1035, which can be obtained by the method defined in any one of the present inventions 1001 to 1030. [The present invention 1037] A mesenchymal stem cell population according to any one of the present inventions 1033 to 1035, which is obtained by the method defined in any one of the present inventions 1001 to 1030. [The present invention 1038] A pharmaceutical composition comprising an isolated mesenchymal stem cell population of umbilical cord amnion, wherein at least about 90% or more of the cells of the stem cell population express each of the markers CD73, CD90, and CD105 and lack the expression of each of the markers CD34, CD45, and HLA-DR, said pharmaceutical composition. [The present invention 1039] A pharmaceutical composition according to the present invention 1038, which is suitable for systemic or local application. [The present invention 1040] A pharmaceutical composition according to the present invention 1038 or 1039, further comprising a pharmaceutically acceptable excipient. [The present invention 1041] A method for preparing a culture solution suitable for isolating a mesenchymal stem cell population from the amnion of the umbilical cord, comprising, to obtain a culture solution with a final volume of 500 ml, i. 250 ml of DMEM ii. 118 ml of M171 iii. 118 ml of DMEM / F12 iv. 12.5 ml of fetal bovine serum (FBS) (final concentration 2.5%) said method comprising the step of mixing. [The present invention 1042] v. Adding 1 ml of an EGF storage solution (5 μg / ml) to achieve a final concentration of 10 ng / ml vi. Adding 0.175 ml of an insulin storage solution (14.28 mg / ml) to achieve a final concentration of 5 μg / ml The method of the present invention 1041, further comprising the step of adding [The present invention 1043] The method of the present invention 1041 or 1042, further comprising the step of adding one or more of the supplementary substances adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3) to DMEM to make a culture solution with a total volume of 500 ml [The present invention 1044] The final concentration of the supplementary substances in DMEM is About 0.05 - 0.1 μg / ml of adenine, for example, about 0.025 μg / ml of adenine, About 1 - 10 μg / ml of hydrocortisone, About 0.5 - 5 ng / ml of 3,3',5-triiodo-L-thyronine sodium salt (T3), for example, 1.36 ng / ml of 3,3',5-triiodo-L-thyronine sodium salt (T3) The method of the present invention 1043, wherein the final concentration is [The present invention 1045] A cell culture solution obtainable by any of the methods of the present invention 1041 - 1044 [The present invention 1046] A method for isolating mesenchymal stem cells from the amniotic membrane of the umbilical cord, comprising the step of culturing the amniotic tissue in a culture solution prepared by the method defined in any of the present invention 1041 - 1044 [The present invention 1047] - DMEM with a final concentration of about 55 - 65% (v / v), - F12 with a final concentration of about 5 - 15% (v / v), - M171 with a final concentration of about 15 - 30% (v / v), and - FBS with a final concentration of about 1 - 8% (v / v) A cell culture solution comprising [The present invention 1048] The cell culture medium of the present invention 1047, containing DMEM at a final concentration of about 57.5 - 62.5% (v / v), F12 at a final concentration of about 7.5 - 12.5% (v / v), M171 at a final concentration of about 17.5 - 25.0% (v / v), and FBS at a final concentration of about 1.75 - 3.5% (v / v). [The present invention 1049] The cell culture medium of the present invention 1048, containing 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). [The present invention 1050] The cell culture medium of any one of the present inventions 1047 - 1049, further containing epidermal growth factor (EGF) at a final concentration of about 1 ng / ml to about 20 ng / ml. [The present invention 1051] The cell culture medium of any one of the present inventions 1007 - 1050, containing EGF at a final concentration of about 10 ng / ml. [The present invention 1052] The cell culture medium of any one of the present inventions 1047 - 1051, containing insulin at a final concentration of about 1 μg / ml to 10 μg / ml. [The present invention 1053] The cell culture medium of the present invention 1052, containing insulin at a final concentration of about 5 μg / ml. [The present invention 1054] The cell culture medium of any one of the present inventions 1047 - 1053, further containing at least one of the supplementary substances adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). [The present invention 1055] The cell culture medium of the present invention 1054, containing all three of adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). [The present invention 1056] A cell culture solution of the present invention 1054 or 1055 containing adenine at a final concentration of about 0.05 to about 0.1 μg / ml, hydrocortisone at a final concentration of about 1 to about 10 μg / ml, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of about 0.5 to about 5 ng / ml. [The present invention 1057] A cell culture solution of any one of the present inventions 1047 to 1056, wherein 500 ml of the cell culture solution contains i. 250 ml of DMEM ii. 118 ml of M171 iii. 118 ml of DMEM / F12 iv. 12.5 ml of fetal bovine serum (FBS) (final concentration 2.5%) [The present invention 1058] v. EGF at a final concentration of 10 ng / ml vi. Insulin at a final concentration of 5 μg / ml vi. 0.175 ml of insulin (final concentration 5 μg / ml) A cell culture solution of the present invention 1057, further containing [The present invention 1059] A cell culture solution of the present invention 1057 or 1058, further containing adenine at a final concentration of about 0.05 to about 0.1 μg / ml, hydrocortisone at a final concentration of about 1 to about 10 μg / ml, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of about 0.5 to about 5 ng / ml. [The present invention 1060] Use of a cell culture solution as defined in any one of the present inventions 1047 to 1059 for isolating mesenchymal stem cells from the amnion of the umbilical cord. [The present invention 1061] Use of a cell culture solution as defined in any one of the present inventions 1047 to 1059 for culturing mesenchymal stem cells derived from the amnion of the umbilical cord.

Brief Description of the Drawings

[0019] The present invention will be better understood with reference to the detailed description when considered in conjunction with non-limiting examples and the drawings.

Figure 1-1

Figure 1-2

Figure 2

Figure 3

Figure 4-1

Figure 4-2

Figure 5

Figure 6a

[0183] of WO2006 / 019357), and c) the culture medium PPT-6 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 for each of the three culture media used. The results are shown in FIGS. 6a-6c. More specifically, FIG. 6a shows the percentage of isolated mesenchymal umbilical cord lining stem cells expressing the stem cell markers CD73, CD90, and CD105 after isolation and culture from umbilical cord tissue in DMEM / 10% FBS.

Figure 6b

[0183] of WO2006 / 019357), and c) the culture medium PPT-6 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 for each of the three culture media used. The results are shown in FIGS. 6a - 6c. More specifically, FIG. 6b shows the percentage of isolated mesenchymal umbilical cord lining stem cells expressing the stem cell markers CD73, CD90, and CD105 after isolation and culture from umbilical cord tissue in PTT-4.

Figure 6c

[0183] of WO2006 / 019357), and c) culture medium PPT-6 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 for each of the three culture media used. The results are shown in FIGS. 6a - 6c. More specifically, FIG. 6c shows the percentage of isolated mesenchymal umbilical cord lining stem cells expressing the stem cell markers CD73, CD90, and CD105 after isolation and culture from umbilical cord tissue in PTT-6.

Figure 7a

Figure 7b

Mode for Carrying Out the Invention

[0020] Detailed Description of the Invention As described above, in a first aspect, the present invention is directed to a method of isolating a mesenchymal stem cell population from the amnion of the umbilical cord, the method comprising culturing umbilical cord tissue in a culture medium containing DMEM (Dulbecco's Modified Eagle Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and FBS (Fetal Bovine Serum). By using such a medium, a mesenchymal stem cell population is isolated from the amnion of the umbilical cord, with more than 90% or even 99% or more of the cells being positive for three mesenchymal stem cell markers, CD73 and CD90, while at the same time these stem cells lack the expression of CD34, CD45, and HLA-DR (see the experimental section), which means that 99% or more of the cells in this population express the stem cell markers CD73, CD90, and CD105 while not expressing the markers CD34, CD45, and HLA-DR, which has surprisingly been found in the present application.Such an extremely uniform and well-defined cell population fully meets the generally accepted criteria for human mesenchymal stem cells to be used in 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, and is thus an ideal candidate for clinical trials and cell-based therapies. Additionally, by using a bioreactor such as the Quantum cell expansion system, it is possible to obtain a large number of mesenchymal stem cells, such as 300 million to 700 million mesenchymal stem cells per run (see also the experimental section). Therefore, the present invention enables the provision of the amount of stem cells required for therapeutic applications, such as use in wound healing, in a cost-effective manner. In addition, all components used to prepare the culture medium of the present invention are commercially available in GMP quality. Thus, the present invention opens a route for GMP production of this highly uniform mesenchymal stem cell population from the amniotic membrane of the umbilical cord.

[0021] In this regard, it is noted that the culture solution of the present invention enables the isolation of a mesenchymal stem cell population (also referred to herein as "mesenchymal stem cells") from amniotic membrane under conditions that allow for the cell proliferation of mesenchymal stem / progenitor cells without differentiating the mesenchymal stem / progenitor cells. Thus, after isolating mesenchymal stem cells from amniotic membrane as described herein, the isolated mesenchymal stem / progenitor cell population has the ability to differentiate into multiple cell types, as described, for example, in U.S. Patent Application No. 2006 / 0078993, U.S. Patent No. 9,085,755, International Patent Application WO2006 / 019357, U.S. Patent No. 8,287,854, or WO2007 / 046775. For example, as described in U.S. Patent Application No. 2006 / 0078993, mesenchymal stem cells of the amniotic membrane of the umbilical cord have a spindle shape, express the genes POU5f1, Bmi-1, leukemia inhibitory factor (LIF), and secrete activin A and follistatin. The mesenchymal stem cells isolated in the present invention can differentiate into any type of mesenchymal cells, including but not limited to adipocytes, dermal fibroblasts, chondrocytes, osteoblasts, tendon cells, ligament fibroblasts, cardiomyocytes, smooth muscle cells, skeletal muscle cells, adipocytes, mucin-producing cells, cells derived from endocrine glands such as insulin-producing cells (e.g., β-islet cells), or neuroectodermal cells. The stem cells isolated in the present invention can be differentiated in vitro for later use of differentiated cells for medical purposes. An example of such an approach is the differentiation of mesenchymal stem cells into insulin-producing β-islet cells, which can then be administered to patients suffering from insulin deficiency such as diabetes, for example, by transplantation (in this regard, also see WO2007 / 046775). Alternatively, the mesenchymal stem cells of the present invention can be used in an undifferentiated state for cell-based therapy for the purpose of wound healing, such as the treatment of burns or chronic diabetic wounds. In these therapeutic applications, the mesenchymal stem cells of the present invention can help promote wound healing by interacting with the surrounding diseased tissue or can also differentiate into respective skin cells (for example, see again WO2007 / 046775).

[0022] In this regard, it is noted that the mesenchymal stem cell population described herein can be isolated and cultured from any umbilical cord tissue as long as the umbilical cord tissue contains amnion (also referred to as "umbilical cord lining"). Thus, the mesenchymal stem cell population can be isolated from the whole umbilical cord (small pieces therefrom), as described in the experimental section of this application. Therefore, this umbilical cord tissue may contain, in addition to the amnion, any other tissue / component of the umbilical cord. For example, as shown in FIG. 16 of U.S. Patent Application No. 2006 / 0078993 or International Patent Application WO2006 / 019357, the amnion of the umbilical cord is the outermost part of the umbilical cord that covers the umbilical cord. In addition, the umbilical cord contains one vein (carrying oxygenated and nutrient-rich blood to the fetus) and two arteries (carrying deoxygenated and nutrient-depleted blood away from the fetus). For protection and mechanical support, these three blood vessels are mostly embedded in Wharton's jelly, a gelatinous substance made of mucopolysaccharide. Thus, the umbilical cord tissue used in the present invention may also include this one vein, two arteries, and Wharton's jelly. The use of such whole (intact) parts of the umbilical cord has the advantage that it is not necessary to separate the amnion from the other components of the umbilical cord. This reduces the isolation steps, and thus the method of the present invention becomes simpler, faster, less error-prone, and more economical - all of which are important aspects of GMP production required for the therapeutic application of mesenchymal stem cells. Therefore, the isolation of mesenchymal stem cells can start from tissue explants, and subsequently, if a larger amount of mesenchymal stem cells is desired, for example, for use in clinical trials, the isolated mesenchymal stem cells can be subsequently passaged (cultured). Alternatively, it is also possible to isolate mesenchymal umbilical cord lining stem cells from the amnion by first separating the amnion from the other components of the umbilical cord and culturing the amnion in the culture medium of the present invention. This culture can also be performed with tissue explants, and optionally, subsequent passaging of the isolated mesenchymal stem cells is performed.In this regard, the term "tissue explant" or "tissue explant method" is used in its ordinary meaning in the art and refers to a method in which tissue or pieces thereof, once harvested, are placed in a cell culture dish containing a culture medium (growth medium) and over time, stem cells migrate from the tissue onto the surface of the dish. These primary stem cells can then be further expanded by micropropagation (subculture), as also described herein, and transferred to fresh dishes. In this regard, it is noted that in the first step of isolating amniotic mesenchymal stem cells from the umbilical cord, from a therapeutic generation-of-cells perspective, a master cell bank of the isolated mesenchymal stem cells can be obtained, and a working cell bank can be obtained in subsequent subcultures. When the mesenchymal stem cell population of the present invention (specifically, a population of mesenchymal stem cells in which at least about 98% or 99% thereof expresses each of the markers CD73, CD90, and CD105 and lacks the expression of each of the markers CD34, CD45, and HLA-DR) is used for clinical trials or as an approved therapy, for this purpose, the cell population of the working cell bank is typically used. The stem cell population at the isolation stage (which may constitute the master cell bank) and the stem cell population at the subculture stage (which may constitute the working cell bank) can both be stored, for example, in cryopreserved form.

[0023] As described above, this method of isolating mesenchymal stem cells from the amnion of the umbilical cord has the advantage that all the components used in the culture medium of the present invention are available in GMP quality, thus presenting the possibility that the mesenchymal stem cells are isolated under GMP conditions for subsequent therapeutic administration.

[0024] "DMEM" refers to Dulbecco's Modified Eagle Medium, which was developed in 1969 and is a modification of Basal Medium Eagle (BME) (see Figure 1 showing the data sheet of DMEM available from Lonza). The first DMEM formulation contained 1000 mg / L glucose and was first reported for the culture of embryonic mouse cells. Since then, DMEM has become a standard medium for cell culture and is commercially available from various sources, such as ThermoFisher Scientific (catalog number 11965-084), Sigma Aldrich (catalog number D5546), or Lonza. Therefore, any commercially available DMEM can be used in the present invention. In a preferred embodiment, the DMEM used herein is the DMEM medium available from Lonza under catalog number 12-604F. This medium is DMEM supplemented with 4.5 g / L glucose and L-glutamine. In another preferred embodiment, the DMEM used herein is the DMEM medium of Sigma Aldrich catalog number D5546, which contains 1000 mg / L glucose and sodium bicarbonate but does not contain L-glutamine.

[0025] The "F12" medium refers to Ham's F12 medium. This medium is also a standard cell culture medium and was originally designed as a nutrient mixture that can culture a wide variety of mammalian cells and hybridoma cells when used with serum in combination with hormones and transferrin (see Figure 2 showing the data sheet of Ham's F12 medium from Lonza). Any commercially available Ham's F12 medium (e.g., from ThermoFisher Scientific (catalog number 11765-054), Sigma Aldrich (catalog number 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.

[0026] "DMEM / F12" or "DMEM:F12" means a 1:1 mixture of DMEM and Ham's F12 medium (see Figure 3 showing the data sheet of DMEM:F12 (1:1) medium from Lonza). DMEM / F12 (1:1) medium is also a basal medium widely used to support the growth of many different mammalian cells and is commercially available from various suppliers such as ThermoFisher Scientific (catalog number 11330057), Sigma Aldrich (catalog number D6421), or Lonza. Any commercially available DMEM:F12 medium can be used in the present invention. In a preferred embodiment, the DMEM:F12 medium used herein is DMEM / F12 (1:1) medium (DMEM:F12 with L-glutamine, 15 mM HEPES, and 3.151 g / L glucose) available from Lonza under catalog number 12-719F.

[0027] "M171" means culture medium 171 developed as a basal medium for culturing the growth of normal human mammary epithelial cells (see Figure 4 showing the data sheet of M171 medium from Life Technologies Corporation). This basal medium is also widely used and is commercially available from suppliers such as ThermoFisher Scientific or 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 M171 medium available from Life Technologies Corporation under catalog number M171500.

[0028] "FBS" refers to fetal bovine serum (also known as fetal calf serum), which means the blood fraction remaining after natural blood clotting and subsequent removal of any residual red blood cells by centrifugation. Fetal bovine serum is the most widely used serum supplement for in vitro cell culture of eukaryotic cells because it has very low levels of antibodies, contains more growth factors, and enables versatility in many different cell culture applications. FBS is preferably obtained from members of the International Serum Industry Association (ISIA) whose main focus is on the proper traceability management of the origin, the authenticity of the labeling, and the safety and safe use of sera and animal-derived products through appropriate standardization and monitoring. Suppliers of FBS that are ISIA members include, to name just a few, Abattoir Basics Company, Animal Technologies Inc., Biomin Biotechnologia LTDA, GE Healthcare, Gibco by Thermo Fisher Scientific, and Life Science Production. In a presently preferred embodiment, FBS is obtained from GE Healthcare under catalog number A15-151.

[0029] Turning now to the culture medium of the present invention, the culture medium can contain DMEM at a final concentration of about 55-65% (v / v), F12 at a final concentration of about 5-15% (v / v), M171 at a final concentration of about 15-30% (v / v), and FBS at a final concentration of about 1-8% (v / v) for the isolation or culture of mesenchymal umbilical lining stem cells. As used herein, the value "% (v / v)" refers to the volume of an individual constituent relative to the final volume of the culture medium. This means that if DMEM is present in the culture medium at a final concentration of about 55-65% (v / v), for example, 1 liter of the culture medium contains about 550-650 ml of DMEM.

[0030] In other embodiments, the culture medium may contain DMEM at a final concentration of about 57.5 - 62.5% (v / v), F12 at a final concentration of about 7.5 - 12.5% (v / v), M171 at a final concentration of about 17.5 - 25.0% (v / v), and FBS at a final concentration of about 1.75 - 3.5% (v / v). In a further embodiment, the culture medium may contain 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).

[0031] In addition to the above components, the culture medium may contain a supplement that is advantageous for culturing mesenchymal umbilical cord lining stem cells. The culture medium of the present invention may contain, for example, epidermal growth factor (EGF). When present, EGF may be present in the culture medium at a final concentration of about 1 ng / ml to about 20 ng / ml. In some of these embodiments, the culture medium may contain EGF at a final concentration of about 10 ng / ml.

[0032] The culture medium of the present invention may also contain insulin. When present, insulin may be present at a final concentration of about 1 μg / ml to 10 μg / ml. In some of these embodiments, the culture medium may contain insulin at a final concentration of about 5 μg / ml

[0033] The culture medium may further contain 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 contain all three of adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). In these embodiments, the culture medium may contain adenine at a final concentration of about 0.05 - about 0.1 μg / ml, hydrocortisone at a final concentration of about 1 - about 10 μg / ml, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of about 0.5 - about 5 ng / ml.

[0034] In the method of the present invention, the umbilical cord tissue can be cultured until an appropriate number of (primary) mesenchymal umbilical cord lining stem cells grow out from the tissue. In a typical embodiment, the umbilical cord tissue is cultured until the cell growth of the mesenchymal stem cells of the amnion reaches a confluence density of about 70% to about 80%. As used herein, the terms "confluence density" or "confluent" are used in their ordinary meaning in the art of cell culture and refer to an estimated value / index of the number of adherent cells in a culture dish or flask, with reference to the percentage of the surface covered by cells. It is noted that, for example, 50 percent confluence means that approximately half of the surface is covered and there is still room for the cells to grow. 100 percent confluence means that the surface is completely covered by cells and there is no room left for the cells to grow as a monolayer.

[0035] Once an appropriate number of primary cells (mesenchymal umbilical cord lining stem cells) are obtained from the umbilical cord lining tissue by tissue explants, the mesenchymal stem cells are removed from the culture vessel used for culturing. By doing so, a master cell bank containing (primary) isolated mesenchymal stem cells of the amnion can be obtained. Typically, since mesenchymal stem cells are adherent cells, the removal is performed using standard enzymatic treatment. For example, the enzymatic treatment may include trypsin treatment as described in International US Patent Application No. 2006 / 0078993, International Patent Application WO2006 / 019357, or International Patent Application WO2007 / 046775, which means that the growing cells can be recovered by trypsin treatment (0.125% trypsin / 0.05% EDTA) for further expansion. If the recovered mesenchymal stem cells are used, for example, to create a master cell bank, the cells can be cryopreserved and stored for further use as described below herein.

[0036] Once collected, the mesenchymal stem cells can be transferred to a culture vessel for subculture. Subculture can also be started from cryopreserved primary cells, i.e., from the master cell bank. For subculture, any suitable amount of cells can be seeded into a culture vessel such as a cell culture plate. For this purpose, mesenchymal cells can be suspended, for example, at a concentration of about 0.5×10 6 cells / ml to about 5.0×10 6 cells / ml in a suitable medium for subculture (most conveniently, the culture solution of the present invention). In one embodiment, the cells are about 1.0×10 6Suspend at a concentration of cells / ml. Subculture can be carried out by culturing in a simple culture flask, but can also be carried out, for example, by culturing in a multi-layer system such as CellStack (Corning, Corning, NY, USA) or Cellfactory (Nunc, part of Thermo Fisher Scientific Inc., Waltham, MA, USA) that can be stacked in an incubator. Alternatively, subculture can also be carried out in a closed self-contained system such as a bioreactor. Bioreactors of various designs are well known to those skilled in the art, for example, there are parallel plate, hollow fiber, or microfluidic bioreactors. For example, see Sensebe et al., "Production of mesenchymal stromal / stem cells according to good manufacturing practices: a review". An example of a commercially available hollow fiber bioreactor is, for example, the Quantum® cell expansion system (Terumo BCT, Inc) used for the expansion of 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 for the subculture of the mesenchymal stem cell population of the present invention is the Xuri cell expansion system available from GE Heathcare. The culture of mesenchymal stem cells in an automated system such as the Quantum® cell expansion system should generate a working cell bank under GMP conditions for therapeutic applications and is particularly effective when a large number of cells are required.

[0037] The subculture of the mesenchymal umbilical cord lining stem cells of the present invention is carried out in the culture medium of the present invention. Therefore, the culture medium of the present invention can be used for both the isolation of mesenchymal stem cells from amniotic membrane and the subsequent culture of the isolated primary cells by subculture. Similarly for subculture, the mesenchymal stem cells can be cultured until an appropriate amount of cells has proliferated. In an illustrative embodiment, the mesenchymal stem cells are subcultured until they reach a confluence density of about 70% to about 80%.

[0038] The isolation / culture of the population of mesenchymal umbilical cord lining stem cells can be carried out under standard conditions for culturing mammalian cells. Typically, the method of the present invention for isolating a population of mesenchymal umbilical cord lining stem cells is typically carried out under the conditions (temperature, atmosphere) normally used for culturing the cells of the species from which the cells are derived. For example, human umbilical cord tissue and mesenchymal umbilical cord lining stem cells are each typically cultured at 37°C in an air atmosphere containing 5% CO 2 This is relevant, in the present invention, the mesenchymal cells may be derived from any mammalian species such as mouse, rat, guinea pig, rabbit, goat, horse, dog, cat, sheep, monkey, or human, and in one embodiment, mesenchymal stem cells of human origin are preferably noted.

[0039] Once the desired / appropriate number of mesenchymal umbilical cord lining stem cells have been obtained from subculture, they are recovered by removing the mesenchymal stem cells from the culture vessel used for subculture. The recovery of mesenchymal stem cells is typically also carried out by enzymatic treatment including trypsinization of the cells. The isolated mesenchymal stem cells are then collected and used directly or stored for further use. Typically, the storage is carried out by cryopreservation. The term "cryopreservation" is used herein in its ordinary meaning to represent the process by which mesenchymal stem cells are stored by cooling to sub-zero temperatures such as (typically) -80°C or -196°C (the boiling point of liquid nitrogen). Cryopreservation can be carried out as known to those skilled in the art and may include the use of cryoprotective agents such as dimethyl sulfoxide (DMSO) or glycerol to slow the formation of ice crystals in the cells of the umbilical cord.

[0040] The isolated population of mesenchymal umbilical cord lining stem cells obtained by the isolation method of the present invention is highly defined and highly homogeneous. 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 the expression of CD34, CD45, and HLA-DR.

[0041] Accordingly, in accordance with the above disclosure, the present invention also relates to a population of mesenchymal stem cells isolated from the amnion 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. 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 isolated mesenchymal stem cell population are CD73+, CD90+, and CD105+, which means that this proportion of the isolated cell population expresses each of CD73, CD90, and CD105 (see the experimental section 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 isolated mesenchymal stem cells may lack the expression of the following markers. 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 the expression of CD34, CD45, and HLA-DR. Such a highly homogeneous population of mesenchymal stem cells derived from the amnion of the umbilical cord is reported herein for the first time and meets the criteria for mesenchymal stem cells to be used in 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"). In this regard, it is noted that this mesenchymal stem cell population can be obtained by the isolation method of the present invention, but can also be obtained by different methods such as cell sorting if desired.

[0042] Consistent with the above, the present invention also relates to a pharmaceutical composition comprising a population of mesenchymal stem cells isolated from the amnion of the umbilical cord, wherein at least about 90% or more of the cells of the stem cell population express each of the markers CD73, CD90, and CD105, and optionally lack the expression of CD34, CD45, and HLA-DR. The pharmaceutical composition may comprise any pharmaceutically acceptable excipient and may be formulated for any desired pharmaceutical administration method. The pharmaceutical composition may be suitable for, for example, systemic or topical application.

[0043] In a further aspect, the present invention relates to a method of preparing a culture medium for isolation, the method comprising the step of mixing the following to obtain a culture medium with a final volume of 500 ml: i. 250 ml of DMEM ii. 118 ml of M171 iii. 118 ml of DMEM / F12 iv. 12.5 ml of fetal bovine serum (FBS) to reach a final concentration of 2.5% (v / v).

[0044] As described above, the DMEM / F12 medium is a 1:1 mixture of DMEM and Ham's F12 culture medium. Thus, 118 ml of DMEM / F12 medium contains 59 ml of DMEM and 59 ml of F12. Therefore, when this method of preparing the culture medium is used, the final concentration (v / v) in a total volume of 500 ml is as follows: DMEM: 250 ml + 59 ml = 309 ml, corresponding to 309 / 500 = 61.8% (v / v). M171: 118 ml, corresponding to 118 / 500 = 23.6% (v / v). F12: 59 ml, corresponding to 59 / 500 = 11.8% (v / v).

[0045] Aspects of this method of preparing the culture medium further comprise the step of adding the following: v. 1 ml of an EGF stock solution (5 μg / ml) to achieve a final EGF concentration of 10 ng / ml, and vi. 0.175 ml of insulin storage solution (14.28 mg / ml) to achieve a final insulin concentration of 5 μg / ml.

[0046] It is noted herein that in these embodiments, the above volumes of these components i-vi result in a culture solution with a final volume of 499.675 ml. If no additional components are added to the culture solution, the remaining 0.325 ml (to make a total volume of 500 ml) may be any of components i-iv, which means, for example, any of DMEM, M171, DMEM / F12, or FBS. Alternatively, the concentration of the EGF or insulin storage solution can of course be adjusted so that the total volume of the culture solution is 500 ml. In addition, components i-vi do not necessarily have to be added in the order in which they are listed, and it is of course possible to mix these components in any order to reach the culture solution 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 obtain the final concentrations described herein, i.e., a final concentration of DMEM of about 55-65% (v / v), a final concentration of F12 of about 5-15% (v / v), a final concentration of M171 of about 15-30% (v / v), and a final concentration of FBS of about 1-8% (v / v).

[0047] In other embodiments, the method further includes adding one or more of the supplement substances adenine, hydrocortisone, and sodium 3,3',5-triiodo-L-thyronine (T3) in a volume of 0.325 ml to DMEM, thereby making a culture solution with a total volume of 500 ml. In this embodiment, the final concentrations of these supplement substances in DMEM may be as follows: about 0.05-0.1 μg / ml of adenine, for example about 0.025 μg / ml of adenine, about 1-10 μg / ml of hydrocortisone, About 0.5 to 5 ng / ml of 3,3',5-triiodo-L-thyronine sodium salt (T3), for example 1.36 ng / ml of 3,3',5-triiodo-L-thyronine sodium salt (T3).

[0048] In accordance with the above disclosure, the present invention also targets cell culture fluids that can be obtained or derived by the method of preparing the culture media described herein.

[0049] In addition, the present invention also relates to a method for isolating mesenchymal stem cells from the amnion of the umbilical cord, which method includes culturing the amnion tissue in a culture fluid prepared by the method described herein.

[0050] Accordingly, the present invention also targets cell culture fluids comprising: - 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 - FBS at a final concentration of about 1 to 8% (v / v).

[0051] In certain embodiments of the culture fluids described herein, the medium comprises 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 other embodiments, the culture fluid 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).

[0052] In addition, the culture medium may further contain epidermal growth factor (EGF) at a final concentration of about 1 ng / ml to about 20 ng / ml. In certain embodiments, the culture medium contains EGF at a final concentration of about 10 ng / ml. The culture medium described herein may further contain insulin at a final concentration of about 1 μg / ml to 10 μg / ml. In such embodiments, the culture medium may contain insulin at a final concentration of about 5 μg / ml.

[0053] The cell culture medium of the present invention may further contain at least one of the following supplementary substances: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). In certain embodiments, the culture medium contains all three of adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). When present, the culture medium may contain adenine at a final concentration of about 0.01 to about 0.1 μg / ml 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 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 0.5 to about 5 ng / ml.

[0054] In an embodiment of the cell culture medium, 500 ml of the cell culture medium of the present invention contains the following: i. 250 ml of DMEM ii. 118 ml of M171 iii. 118 ml of DMEM / F12 iv. 12.5 ml of fetal bovine serum (FBS) (final concentration 2.5%).

[0055] In a further embodiment, the cell culture medium may further contain the following: v. EGF at a final concentration of 10 ng / ml, and vi. Insulin at a final concentration of 5 μg / ml.

[0056] Both insulin and EGF can be added to the culture medium using an optimal preservation solution so that the total volume of the culture medium does not exceed 500 ml.

[0057] In certain examples, the constituents i-vi of the culture medium of the present invention are the constituents shown in FIG. 5, which means that they are obtained from each manufacturer using the catalog numbers shown in FIG. 5. The medium obtained by mixing constituents i-vi as shown in FIG. 5 is also referred to herein as "PTT-6". In this regard, it is again noted that any other commercial supplier's constituent substances i-vi and any other components such as antibiotics can be used in preparing the medium of the present invention.

[0058] In addition, the cell culture medium of the present invention may contain adenine at a final concentration of about 0.01 to about 0.1 μg / ml or about 0.05 to about 0.1 μg / ml adenine, hydrocortisone at a final concentration of about 0.1 to 10 μg / ml, about 0.5 to about 10 μg / ml, or about 1 to about 10 μg / ml, 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.

[0059] Finally, the present invention also provides a method for treating a patient having a disease, the method comprising administering to the patient a pharmaceutical composition containing mesenchymal umbilical lining stem cells or stem cells as disclosed herein. The disease can be any of the diseases as described above. To treat a subject, the mesenchymal stem cell population of the present invention can be administered by any suitable method including, but not limited to, topical administration, transplantation, or injection. The stem cell population may be placed directly on a wound such as a burn wound or a diabetic wound (see International Patent Application WO2007 / 046775). Alternatively, the stem cell population may be transplanted subcutaneously, for example, directly under the skin, into body fat, or intraperitoneally.

[0060] The present invention will be further illustrated by the following non-limiting experimental examples.

Example

[0061] Experimental Examples 1. Cryopreservation of umbilical cord tissue before isolation of mesenchymal stem cells Umbilical cord tissue (the umbilical cord was donated after obtaining the mother's informed consent) was processed as follows to isolate mesenchymal stem cells from the amnion of the umbilical cord.

[0062] 1.1 Washing of umbilical cord tissue samples: a. Remove the surgical scalpel from the protective cover. b. Hold the umbilical cord firmly with forceps and cut the umbilical cord into 10-cm-long pieces using the surgical scalpel. Discard any unusable tissue back into the original tissue cup. c. Transfer the 10-cm-long umbilical cord pieces to a new 150-mm culture dish. A 150-mm culture dish can also be used in place of the cup. d. Use the cover of the 150-mm culture dish as a place to hold the forceps and surgical scalpel. e. Withdraw 25 ml of Plasmalyte A (Baxter, catalog # 2B2543Q) using a 30-ml syringe. Hold the syringe at a 45° angle with one hand and dispense the Plasmalyte A directly onto the umbilical cord tissue. f. Remove the Plasmalyte A using a 30-ml syringe and a blunt needle while holding the culture dish at a slight angle. g. Collect the used Plasmalyte A in a 300-ml transfer bag serving as a waste container and dispose of it in a biohazard trash can. h. Repeat the washing procedure using a new culture dish for each wash as needed. Confirm that all blood clots on the surface have been removed. Additional Plasmalyte A can be used if further tissue cleaning is required. i. Place the tissue in a new labeled tissue culture dish and continue cutting the tissue. Put 20 ml of Plasmalyte A into the dish to prevent the tissue from drying during cutting. j. Cut the umbilical cord into approximately 1-cm sections of equal length, for a total of 10 sections. k. Further cut each 1-cm section into smaller pieces, approximately 0.3 cm×0.3 cm to 0.5 cm×0.5 cm per piece. l. Remove all of the Plasmalyte A from the dish. m. Withdraw 25 ml of Plasmalyte A from the original Plasmalyte A bag using a 30-ml syringe and dispense it directly onto the umbilical cord tissue pieces. n. Hold the culture dish at an angle and collect all of the Plasmalyte A used for tissue washing on one side, and remove it with a syringe and blunt needle. o. Repeat the washing one more time. No blood clots should remain.

[0063] Note: If the umbilical cord is not frozen immediately, maintain the umbilical cord tissue in Plasmalyte A until just before freezing.

[0064] 1.2 Cryopreservation of umbilical cord tissue: a. Prepare the cryopreservation solution: i. Prepare 50 ml of a cryopreservation solution consisting of 60% Plasmalyte A, 30% of 5% human serum albumin, and 10% dimethyl sulfoxide (DMSO). ii. Label a 150-ml transfer bag with "Tissue Cryopreservation Solution" and attach a plasma transfer set to the port using aseptic technique. iii. Withdraw 30 ml of Plasmalyte A from the original Plasmalyte A bag using a 30-ml syringe and transfer it into the transfer bag labeled with "Tissue Cryopreservation Solution" along with the date and time of solution preparation. iv. Withdraw 15 ml of 5% human serum albumin using a 20-ml syringe and transfer it into the labeled transfer bag. v. Add 5 ml of DMSO to the transfer bag. vi. Mix well and record the mixing of the cryopreservation solution. b. Remove Plasmalyte A from the tissue before adding the freezing solution. c. Using a 60 ml syringe, withdraw all 50 ml of the freezing solution into the syringe and add approximately 30 ml of the freezing solution to the 150 mm cell culture dish containing the umbilical cord tissue. Attach a blunt needle to the syringe and keep it sterile. d. Swirl the culture dish containing the tissue and the freezing solution for 1 minute intervals over a period of 10 minutes. e. Using forceps, select 8 randomly chosen sections and place each of them into each of 4 four - ml cryovials. Select 4 randomly chosen sections and place them into one 1.8 - ml cryovial. These sections must not contain blood clots. f. Fill each cryovial containing umbilical cord tissue with the remaining freezing solution up to the 3.6 - ml fill line for the 4 - ml tubes and up to the 1.8 - ml line for the 1.8 - ml Nunc vials. g. Label one Bactec Lytic / 10 - Anaerobic / F bottle and one Bactec Pluc Aerobic / F bottle with the tissue ID. h. Using the syringe and blunt needle, remove 20 ml of the freezing solution from the culture dish. After wiping the Bactec vials with an alcohol swab, replace the blunt needle with an 18 - g needle and inoculate 10 ml each into the aerobic and anaerobic Bactec bottles. i. Start the controlled - rate freezer. j. After the controlled - rate freezer is complete, place the unit in a liquid nitrogen freezer with continuous temperature monitoring until further use.

[0065] 2. Isolation of mesenchymal umbilical cord lining stem cells from umbilical cord tissue 2.1 Preparation of medium for treating MSCs from umbilical cord tissue: a. To prepare 500 ml of PTT6 (culture medium / growth medium), add the following in the order listed: i. 250 ml of DMEM ii. 118 ml of M171 iii. 118 ml of DMEM F12 iv. 12.5 ml of FBS (final concentration 2.5%) v. 1 ml of EGF (final concentration 10 ng / ml) vi. 0.175 ml of insulin (final concentration 5 μg / ml).

[0066] The above volumes of components i - vi result in a culture medium with a final volume of 499.675 ml. If no further components are added to the culture medium, the remaining 0.325 ml (to make a total volume of 500 ml) may be any of components i - iv, for example, DMEM, M171, DMEM / F12, or FBS. Alternatively, the concentration of the EGF or insulin stock solution can of course be adjusted so that the total volume of the culture medium is 500 ml. Alternatively, a stock solution of an antibiotic such as penicillin - streptomycin - amphotericin can be added so that the final volume is 500 ml. It is also possible to add one or more of the supplementary substances adenine, hydrocortisone, and sodium 3,3',5 - triiodo - L - thyronine (T3) in a volume of 0.325 ml to the culture medium, thereby making a culture medium with a total volume of 500 ml.

[0067] vii. Label the bottle with "PTT6", along with the preparation date of the medium, the operator's initials, and the phrase "Expiration Date" followed by the expiration date. The expiration date is the earlier of either the earliest expiration date of any of the components or one month after the preparation date.

[0068] b. To prepare a rinse medium (calcium - and magnesium - free Hank's balanced salt solution (HBSS) containing 5% FBS), add 2.5 ml of FBS to 47.5 ml of HBSS in a 50 ml centrifuge tube. Label the tube with the operator's initials and the preparation date of the medium as "Rinse Medium". c. Test all media for sterility using Bactec Lytic / 10 - Anaerobic / F (Becton Dickinson & Company) and Bactec Plus + Aerobic / F (Becton Dickinson & Company). Inject 20 ml of the prepared medium into each bottle.

[0069] 2.2 Thawing of umbilical cord tissue for MSC recovery: a. Start thawing when the operator is ready to process the samples in the clean room. Do not thaw more than two vials at a time, except when the vials are from the same donor. b. Wipe the water bath with a disinfectant and then with 70% isopropanol, and fill it with 1 L of sterile water. Heat the water bath to 36 - 38 °C. c. Prepare 10 ml of a rinse medium consisting of 70% - 90% PlasmaLyte A under the biosafety cabinet in the clean room. Sterile filter this solution through a 0.2 - μm syringe filter attached to a 10 ml syringe, and maintain the solution refrigerated until use. d. Label a 50 ml conical tube with the processing label. e. Confirm that the water bath temperature is 36 - 38 °C. f. Remove the tissue vials from liquid nitrogen storage and quickly thaw them in a 37 °C water bath filled with 1 L of sterile water. The vial holder of the Mr. Frosty Nalgene Cryo 1 °C freezing container holds the vials in place and floats, and can be used as a floating rack when thawing the samples. g. Remove the vials from the water bath and spray them with a 70% isopropanol solution. The appropriate timing to lift the vials out of the water bath is when small ice can be seen floating in the vial - indicating that the internal temperature of the vial is less than 37 °C. h. Place the vials in the pass - through and notify the clean room processing technician.

[0070] 2.3 Preparation for tissue treatment: a. Umbilical cord tissue processing must be performed in an environmental monitor (EM) clean room. At the end of each shift, complete cleaning of the room and the hood is carried out. b. Prepare / clean the biosafety cabinet. c. During work in the biosafety cabinet, perform biological particle counting. d. While checking for damaged packaging and expiration dates respectively, collect all necessary items into the biosafety cabinet. When handling syringes, serum pipettes, sterilized forceps, surgical scalpels, tissue plates, and needles, never touch the surfaces that will come into contact with the sterilized products. Only the outside of the syringe barrel, tubing, plunger tip, and / or needle cap or case may be safely handled. If the surface is touched or the surface touches a non-sterile surface, discard the item. e. Record the lot number and expiration date (if applicable) of all reagents and items used. f. Clean the vial with a lint-free wipe moistened with 70% alcohol, and then transfer it into the biosafety cabinet to receive the thawed vial. g. Using a suction needle attached to a syringe, withdraw as much liquid as possible from the vial. Avoid aspirating the tissue. h. Using sterilized forceps, transfer the tissue to a sterilized 100 mm Petri dish. i. Add 5 ml of rinse medium in a fixed divided amount to the tissue fragments. j. Swirl the contents for 15 - 30 seconds, and then remove the rinse medium with a pipette or a syringe with a suction needle. Repeat this rinsing process twice. k. Add 2 mL of rinse medium to the tissue so that the tissue does not dry out.

[0071] 2.4 Initiation of MSC proliferation from tissue: a. Label the bottom of the 6-well plate with the label "Proliferation 1", along with the MSC lot number or umbilical cord tissue ID and the start date of proliferation. When using a 60 mm tissue culture dish, draw a grid on the bottom of the dish to divide the plate into four sections. b. Using sterile disposable forceps, place one piece of tissue measuring 3×3 mm to 5×5 mm into each well. When using a 60 mm tissue culture dish, place the tissue in the center of each section and leave the tissues separated (more than 1 cm apart from each other). c. Fill each well with 3 ml of PTT6. d. Using a suction needle connected to a 30 ml syringe, withdraw enough medium to just barely cover the tissue. Do not tilt the plate. Do not touch the bottom of the well with the suction needle. e. Observe cell proliferation daily (24±6 hours) using an inverted optical microscope. Instead of an optical microscope, a real-time cell culture imaging system may be used. f. Change the medium daily. Always equilibrate the medium to room temperature before use. i. Aspirate and remove the medium. ii. Add 3 ml of PTT6. iii. Aspirate until the tissue is just barely immersed in the medium. g. When cell proliferation is observed from the tissue, transfer the tissue to a new 6-well plate using the same procedure as in 4.a - 4.e above, except label the plate with "Proliferation 2". Maintain cell proliferation in the "Proliferation 1" plate by adding 2 ml of PTT6 to each well. Observe the confluence density daily. Replace the medium every 2 - 3 days (always equilibrate the medium to room temperature before use). h. When cell proliferation is observed in the "Proliferation 2" plate, repeat steps 4.a - 4.e except label the plate with "Proliferation 3". Maintain cell proliferation in the "Proliferation 2" plate by adding 2 ml of PTT6 to each well. Observe the confluence density daily. Replace the medium every 2 - 3 days (always equilibrate the medium to room temperature before use). i. Dispose of the tissue when growth is observed in the "Proliferation 3" plate. If the tissue is very small and does not appear to interfere with cell growth, dispose of the tissue during subculture. j. Observe the cells daily to prevent excessive growth when the cell confluence reaches 40 - 50%. k. Subculture the cells when the cell confluence reaches 70 - 80%. Do not allow the cells to grow beyond 80% confluence.

[0072] When the size of the tissue explant is about 1 - 3 mm and the culture of the tissue explant / cells is carried out in a 175 mm square culture dish, the average number of mesenchymal stem cells recovered from the explant is typically about 4,000 - 6,000 cells / explant. Thus, when simultaneously growing mesenchymal stem cells from 48 explants, about 300,000 cells can be obtained at the time of recovery. These 300,000 mesenchymal stem cells collected from the explants can then be seeded into a 175 cm 2 cell culture flask with such 300,000 cells and can be used for subculture (this can be referred to as passage 1). Then, using the mesenchymal stem cells obtained from this passage 1, as described in Example 2.5 below, seed them again into a 175 cm 2 flask (passage 2) and the cells can be grown. The cells obtained from both passage 1 and passage 2 can be "banked" by cryopreservation, and the mesenchymal stem cells obtained after passage 2 are regarded as representing the master cell bank, which, as described in Example 2.7 below, is for further expansion of mesenchymal stem cells, for example, in a bioreactor.

[0073] 2.5 Subculture of MSCs in cell culture dishes a. Perform biological particle counting during work inside the biosafety cabinet. Equilibrate all media to room temperature before use. b. Subculture the cells when cell growth reaches a confluence of about 70 - 80%. i. Remove PTT6 from the Petri dish. ii. Rinse with HBSS without calcium and magnesium. iii. Add 0.2 ml of 1× TrypLE-EDTA and swirl for 1 - 2 minutes. iv. Tilt the dish at 30 - 45° to allow the cells to move downward by gravity flow. Gently tap the side of the plate to promote detachment. 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. Pool the cells from all 6 wells into a single 15 ml tube. vi. Centrifuge at 1200 rpm for 10 minutes. vii. Remove the supernatant and resuspend the cells in 5 ml of PTT6. c. Passage the MSCs. i. Aliquot 50 μl of the cell suspension and assay for TNC and viability by trypan blue exclusion assay. ii. Count the cells using a hemocytometer. Predict to count 20 - 100 cells / section. If the number is more than 100, dilute the original sample 1:5 and repeat the trypan blue method using a hemocytometer. iii. Count viable cells / ml and total viable cells: 1. Viable cells / ml = number of viable cells × dilution factor × 10 4 2. Total viable cells = number of viable cells × dilution factor × total volume × 10 4 iv. Count % viability: 1. % viability = (number of viable cells × 100) / (number of viable cells + number of dead cells) v. Dilute the cell suspension to 1.0×10 6 cells / ml: 1. "X" volume = total viable cells / 10 6 cells / ml 2. For example, if the total number of viable cells is 1.0×10 7 cells; 3. "X" = 10 7 / 10 6cells / ml, i.e., 10 ml, and thus, the total cell volume is made 10 ml by adding 5 ml to the cell suspension (which is 5 ml). vi. when the cell suspension is less than 10 6 cells / ml, determine the volume required to seed 2×10 2 cells into each 150 mm Petri dish or 175 cm 6 flask. 1. Volume for 2×10 6 cells = 2×10 6 cells ÷ viable cells / ml 2. For example, when the viable cells / ml is 8×10 5 cells / ml, 2×10 6 cells ÷ 8×10 5 cells / ml, i.e., 2.5 ml is required. vii. Set aside 0.5 ml for MSC marker analysis. viii. Seed 2×10 6 cells into each 150 mm Petri dish or 175 cm 2 flask with 30 ml of PTT6. ix. Observe every 3 days for attachment, colony formation, and confluence. When the cells reach 40 - 50% confluence, observe the cells daily to every 2 days to prevent overgrowth. Do not allow the cells to grow beyond 80% confluence. Instead of an optical microscope, a real-time cell culture monitoring system can be used. x. Replace the medium every 2 - 3 days.

[0074] 2.6 Cryopreservation of MSC cells a. Perform bioparticle counting during work in the biosafety cabinet. b. When the cells reach 70 - 80% confluence, detach the cells from each 150 mm Petri dish or 175 cm 2 flask using 2 ml of 1×TrypLE-EDTA. i. Remove PTT6 from the Petri dish. ii. Wash with 5 ml of calcium- and magnesium-free HBSS or PBS. iii. Add 2 ml of 1× TrypLE-EDTA and swirl for 1 - 2 minutes. iv. Tilt the dish at 30 - 45° to allow the cells to move downward by gravity flow. Gently tap the side of the Petri dish to assist in detachment. v. Add 10 ml of PTT6 to inactivate TrypLE. Mix well to dissociate cell clumps. vi. Transfer the cells to a 15 ml centrifuge tube using a Pasteur pipette. vii. Centrifuge at 1200 rpm for 10 minutes. viii. Aspirate the medium and resuspend in 10 ml of PTT6. ix. Aliquot 50 μl and determine the total viable cell count and % viability as described above. Cell counting should be performed within 15 minutes as the cells may start to aggregate. c. Prepare cells for cryopreservation. i. Prepare cell suspension medium and cryopreservation medium and freeze the cells.

[0075] 2.7. Subculture (Expansion) of MSCs in a Quantum Bioreactor (Terumo BTC, Inc.) It is also possible to expand MSCs using a Quantum bioreactor. The starting cell number for expansion in the Quantum bioreactor should be 20 - 30 million cells per run. A typical yield per run is 300 - 700 million MSCs at harvest. The bioreactor is operated according to the manufacturer's protocol. The mesenchymal stem cells obtained in this way are typically cryopreserved (see below) and become a working cell bank.

[0076] Materials / Reagents: 1. Quantum Expansion Set 2. Quantum Waste Bag 3. Quantum Medium Bag 4. Quantum Inlet Bag 5. PTT6 6. PBS 7. Fibronectin 8. TrypLE 9. 3 ml Syringe 10. Glucose Test Strip 11. Lactate Test Strip 12. 60 ml Cell Culture Plate or Equivalent 13. Medical Grade 5% CO 2 Gas Mixture 14. 50 ml Combichip

[0077] Equipment: 1. Biosafety Cabinet 2. Glucose Meter (Bayer Healthcare / Ascensia Contour Blood Glucose Meter) 3. Lactate Plus (Nova Biomedical) 4. Peristaltic Pump with Head 5. Centrifuge, Eppendorf 5810 6. Sterile Tube Connector 7. M4 Continuous Pipettor 8. RF Sealer

[0078] Procedure: 1. Preparation of Quantum Bioreactor a) Pre - preparation of Quantum Bioreactor b) Coating of the Bioreactor: 1) Prepare fibronectin solution in a biosafety cabinet. 1) Equilibrate lyophilized fibronectin to room temperature (≥15 minutes at room temperature). 2) Add 5 ml of sterile distilled water; do not swirl or stir. 3) Make fibronectin into solution over 30 minutes. 4) Transfer the fibronectin solution to the Ccell inlet bag containing 95 ml of PBS using a 10 ml syringe with an 18g needle. 2) Connect the bag to the "reagent" line. 3) Check for air bubbles (air bubbles can be removed by using "IC air removal" or "EC air removal" and by using "washing" as the inlet source). 4) Open or set the program for coating the bioreactor (Figure 1, steps 3 - 5). 5) Run the program. 6) While the program is running to coat the bioreactor, prepare a media bag of 4 L of PTT6 medium. 7) Connect the media bag to the IC media line using a sterile tube connector. 8) When the coating step of the bioreactor is completed, remove the cell inlet bag used for the fibronectin solution using an RF sealer. c) Wash and remove excess fibronectin d) Acclimatize the bioreactor with the medium 2. Cell culture in the Quantum bioreactor a) Cell loading and adhesion using a homogeneous suspension: b) Cell feeding and culture 1) Select the medium flow rate to feed the cells. 2) Sample daily for lactate and glucose. 3) As the lactate level rises, adjust the medium flow rate. The actual maximum allowable lactate concentration is defined by the flask culture from which the cells are derived. Check that sufficient PTT6 medium is in the medium bag. If necessary, replace the PTT6 medium bag with a new one. 4) When the flow rate reaches the desired value, measure the lactate level every 8 - 12 hours. If the lactate level does not decrease or continues to rise, harvest the cells. 3. Recovery of Cells from the Quantum Bioreactor a) After sampling for lactic acid and glucose for the last time when the lactic acid concentration does not decrease, recover the cells. b) Cell recovery: 1) Using a sterile tube connector, connect the cell inlet bag filled with 100 ml of TrypLE to the "reagent" line. 2) Ensure that sufficient PBS is in the PBS bag. If not, using a sterile tube connector, connect a new bag containing at least 1.7 liters of PBS to the "washing" line. 3) Run the recovery program. 4. Cryopreservation of Cells 1) Once the cells are recovered, transfer the cells to a 50 ml centrifuge tube and pellet the cells. 2) Resuspend with 25 ml of cold cell suspension solution. Count the cells using a Sysmex or Biorad cell counter. Attach the cell count report to each Quantum processing batch record. 3) Adjust the cell concentration to 2×10 7 cells / ml. 4) Add an equal volume of cryopreservation solution and mix well (do not shake or vortex). 5) Using a continuous pipettor, add 1 ml of the cell suspension in the cryopreservative to each 1.8 ml vial. Using a controlled rate freezer, cryopreserve using the CRF program as described in SOP D6.100 CB Cryopreservation. 6) Store the vials in the designated liquid nitrogen storage space. 7) Attach the CRF execution report to the sheet of each MSC P3 - Quantum processing batch record.

[0079] 3. Analysis of stem cell marker expression in mesenchymal umbilical cord lining stem cell populations isolated from umbilical cord tissue using different culture media 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.

[0080] For these experiments, mesenchymal stem cells were isolated from umbilical cord tissue by culturing the umbilical cord tissue in three different culture media as described in Example 2, and subsequently the mesenchymal stem cells were subcultured in each medium.

[0081] In these experiments, the following three culture media were used: a) 90% (v / v) DMEM supplemented with 10% FBS (v / v), b) a culture medium PTT-4 consisting of 90% (v / v) CMRL1066 and 10% (v / v) FBS, as described in US Patent Application No. 2006 / 0078993 and corresponding International Patent Application WO2006 / 019357 (see paragraph

[0183] of WO2006 / 019357), and c) the culture medium PPT-6 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 for each of the three culture media used.

[0082] The following protocol was used for the flow cytometry analysis.

[0083] Materials and Methods TIFF2025083555000002.tif195152

[0084] Procedures a) Isolation and culture of cells from the umbilical cord lining membrane 1. As described in Example 2, the explant tissue samples were incubated in a cell culture plate, immersed in each medium, and then maintained in a CO 2 incubator at 37°C. 2. The medium was changed every three days. 3. Cell growth from the tissue culture explants was monitored under an optical microscope. 4. At the time of approximately 70% confluence, the cells were separated from the dish by trypsin treatment (0.0125% trypsin / 0.05% EDTA) and used for the flow cytometry experiment. b) Trypsin treatment of experimental cells 1. Remove the medium from the cell culture plate. 2. Since FBS interferes with the enzymatic action of trypsin, gently rinse with sterile 1×PBS to remove trace amounts of FBS. 3. Add 1X trypsin to the cell culture plate and incubate at 37 °C for 3 - 5 minutes. 4. Observe the cells under a microscope to ensure they are detached. Neutralize the trypsin by adding complete medium containing FBS (DMEM containing 10% FBS). 5. Use a pipette to break up the cell clumps by pipetting the cells against the wall of the plate in the medium. Collect the cell suspension and transfer it to a 50 ml centrifuge tube. 6. Add sterile 1×PBS to the plate, rinse it, and collect the cell suspension into the same centrifuge tube. 7. Centrifuge this at 1800 rpm for 10 minutes. 8. Discard the supernatant and resuspend the cell pellet in PBA medium. c) Cell counting 1. Preferably, clean the hemocytometer and its cover glass with 70% ethanol, dry them, and then wipe with a Kimwipe (lint - free paper) to ensure they are clean and dry. 2. Aliquot a small amount of the suspended cells into a microcentrifuge tube and remove it from the BSC hood. 3. Stain the suspended cells with an equal volume of trypan blue. For example, add 500 μl of trypan blue to 500 μl of the suspension (dilution factor = 2X, resulting in a 0.2% trypan blue solution). 4. Since trypan blue is toxic and can lead to an increase in non - viable cells, causing false cell counts, do not expose the cells to trypan blue for more than 30 minutes. 5. Add 20 μl of the cell suspension mixture to each chamber of the hemocytometer and view it under an optical microscope. a. For a total of eight compartments in the upper and lower chambers, count the number of live cells (bright cells; non-live cells readily take up trypan blue and thus are darkly colored) in each compartment of the hemocytometer. The total cell count is given as (average cell count / compartment) × 10 4 cells / ml. d) Staining of cells i. Preparation before staining cells · Aliquot a cell suspension containing 50,000 cells each into three tubes (CD73, CD90, CD105) in duplicates and two tubes (negative control). ii. Staining with primary antibody (Ab) · Add 1 μl of primary antibody [0.5 mg / ml Ab] to 100 μl of the cell suspension and incubate at 4°C for 45 minutes. · Adjust to 1 ml with PBA. · Centrifuge at 8000 rpm for 5 minutes at 4°C. · Remove the supernatant. · Add 1 ml of PBA and resuspend the pellet. · Centrifuge at 8000 rpm for 5 minutes at 4°C. · Remove the supernatant. · Resuspend in 100 μl of PBA. iii. Staining with secondary Ab - In the dark · Add 1 μl of secondary antibody [0.5 mg / ml ab] to 100 μl of the cell suspension and incubate at 4°C for 30 minutes. · Adjust to 1 ml with PBA. · Centrifuge at 8000 rpm for 5 minutes at 4°C. · Remove the supernatant. · Add 1 ml of PBA and resuspend the pellet. · Centrifuge at 8000 rpm for 5 minutes at 4°C. · Remove the supernatant. · Resuspend in 200 - 300 μl of PBA for flow cytometry analysis. · Transfer the cells to a FACS tube for reading on a BD FACS CANDO flow cytometer.

[0085] The results of the flow cytometry analysis are shown in FIGS. 6a-6c. FIG. 6a shows the percentage of isolated mesenchymal umbilical lining stem cells expressing the stem cell markers CD73, CD90, and CD105 after isolation and culture from umbilical cord tissue in DMEM / 10% FBS, FIG. 6b shows the percentage of isolated mesenchymal umbilical lining stem cells expressing the stem cell markers CD73, CD90, and CD105 after isolation and culture from umbilical cord tissue in PTT-4, and FIG. 6c shows the percentage of isolated mesenchymal umbilical lining stem cells expressing the stem cell markers CD73, CD90, and CD105 after isolation and culture from umbilical cord tissue in PTT6-. As can be seen from FIG. 6a, the population isolated using DMEM / 10% FBS as the culture medium had approximately 75% CD73+ cells, 78% 90+ cells, and 80% CD105+ cells (average of two experiments), whereas the number of mesenchymal stem cells that are CD73 positive, CD90 positive, and CD105 positive after isolation / culture of umbilical cord tissue using the PPT-4 culture medium (see FIG. 6b) is approximately 87% (CD73+ cells), 93% / CD90+ cells), and 86% (CD105+ cells) on average of two experiments. The purity of the mesenchymal stem cell population obtained by culturing in the PTT-6 medium of the present invention is at least 99.0% for all three markers (CD73, CD90, CD105), which means that the purity of this cell population is significantly higher than that in the case of culturing using the PPT-4 medium or DMEM / 10% FBS. In addition, and even more importantly, the mesenchymal stem cell population obtained by culturing in PTT-6 is an essentially 100% pure and distinct stem cell population. Thereby, the stem cell population of the present invention is an ideal candidate for stem cell-based therapies. Therefore, this population of mesenchymal umbilical lining stem cells can be an optimal criterion for such stem cell-based therapeutic approaches.

[0086] The findings shown in FIG. 6 are further supported by the results of the flow cytometry analysis shown in FIGS. 7a and 7b. FIG. 7a shows the percentage of isolated mesenchymal umbilical lining stem cells (mesenchymal stem cells of the amnion of the umbilical cord) that express the stem cell markers CD73, CD90, and CD105 and lack the expression of CD34, CD45, and HLA-DR after isolation and culture from umbilical cord tissue in PTT-6 medium. As shown in FIG. 7a, the mesenchymal stem cell population contains 97.5% viable cells, 100% of which express each of CD73, CD90, and CD105 (see the columns for "CD73+CD90+" and "CD73+CD105+"), 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 the columns for "CD34-CD45-" and "CD34-HLA-DR-"). Thus, the mesenchymal stem cell population obtained by culturing in PTT-6 medium is an essentially 100% pure and distinct stem cell population that meets the criteria for enabling mesenchymal stem cells to be used in cell therapy (95% or more of the stem cell population expresses CD73, CD90, and CD105, while 98% or more of the stem cell population lacks the expression of CD34, CD45, and HLA-DR, see Sensebe et al., "Production of mesenchymal stromal / stem cells according to good manufacturing practices: a review"). It is noted that the amnion mesenchymal stem cells of the present invention are adherent to plastic under standard culture conditions, differentiate into osteoblasts, adipocytes, and chondroblasts in vitro, see U.S. Patent No. 9,085,755, U.S. Patent No. 8,287,854, or WO2007 / 046775, and thus meet the generally accepted criteria for the use of mesenchymal stem cells in cell therapy.

[0087] Figure 7b shows the percentage of isolated bone marrow mesenchymal stem cells that express CD73, CD90, and CD105 and lack the expression of CD34, CD45, and HLA-DR. As shown in Figure 7b, the bone marrow mesenchymal stem cell population contains 94.3% viable cells, 100% of which express each of CD73, CD90, and CD105 (see the columns for "CD73+CD90+" and "CD73+CD105+"), whereas only 62.8% of the bone marrow stem cell population lacks the expression of CD45 and 99.9% of the stem cell population lacks the expression of CD34 and HLA-DR (see the columns for "CD34-CD45-" and "CD34-HLA-DR-"). Thus, the bone marrow mesenchymal stem cells, considered to be the optimal criteria for mesenchymal stem cells, are much less homogeneous / pure with respect to stem cell markers than the mesenchymal stem cell population of the present application (amnion of umbilical cord). This finding also shows that the stem cell population of the present invention can be an ideal candidate for stem cell-based therapies and can be the optimal criteria for stem cell-based therapeutic approaches.

[0088] It will be readily 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.

[0089] All patents and publications mentioned herein are indicative of the level of those skilled in the art to which the present invention pertains. All patents and publications are hereby incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0090] The invention illustratively described in this specification can be appropriately carried out in the absence of any one or more elements and one or more limitations not specifically disclosed herein. Thus, for example, terms such as "comprising," "including," "containing," etc. shall be read comprehensively and non - restrictively. Further, the terms and expressions used in this specification are used as terms of explanation rather than terms of limitation, and the use of such terms and expressions is not intended to exclude any equivalents of the features shown and described or parts thereof, and it is recognized that various modifications are possible within the scope of the invention claimed. Accordingly, although the invention has been specifically disclosed by preferred embodiments and optional features, it should be understood that modifications and changes to the invention embodied therein disclosed herein may be entrusted to those skilled in the art, and that such modifications and changes are considered to be within the scope of the invention. The invention is described herein broadly and generically. Each of the narrower species and sub - generic groups that fall within the scope of the generic disclosure also forms part of the invention. This includes the generic description of the invention using conditional or negative limitations that exclude any subject matter, whether or not the excluded subject matter is specifically recited herein. In addition, when features or aspects of the invention are described from the perspective of a Markush group, those skilled in the art will recognize that the invention is also thereby described from the perspective of any individual member or subgroup of members of that Markush group. Further aspects of the invention will become apparent from the appended claims.

Claims

1. 1. A method for isolating a mesenchymal stem cell population from the amniotic membrane of an umbilical cord, 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).

2. 2. The method of claim 1, wherein the culture medium comprises DMEM at a final concentration of about 55-65% (v / v), F12 at a final concentration of about 5-15% (v / v), M171 at a final concentration of about 15-30% (v / v), and FBS at a final concentration of about 1-8% (v / v).

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

4. 4. The method of claim 3, wherein the culture medium comprises 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).

5. The method of any one of claims 1 to 4, wherein the culture medium further comprises epidermal growth factor (EGF) at a final concentration of about 1 ng / ml to about 20 ng / ml.

6. The method according to any one of claims 1 to 5, wherein the culture medium contains EGF at a final concentration of about 10 ng / ml.

7. The method according to any one of claims 1 to 6, wherein the culture medium contains insulin at a final concentration of about 1 µg / ml to 10 µg / ml.

8. The method according to any one of claims 1 to 7, wherein the culture medium contains insulin at a final concentration of about 5 µg / ml.

9. 10. 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).

10. 2. The method of claim 1, wherein the culture medium contains all three of adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3).

11. 13. The method of claim 11 or 12, wherein the culture medium comprises adenine at a final concentration of about 0.01 to about 0.1 μg / ml adenine, hydrocortisone at a final concentration of about 0.1 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.

12. 2. The method of any one of the preceding claims, comprising culturing the umbilical cord tissue until cell proliferation of the amniotic mesenchymal stem cells reaches about 70-80% confluency.

13. The method according to claim 12, further comprising the step of removing the mesenchymal stem cells from the culture vessel used for the culture.

14. The method according to claim 13, wherein the step of removing the mesenchymal stem cells from the culture vessel is carried out by enzymatic treatment.

15. The method of claim 14, wherein the enzyme treatment comprises trypsin treatment.

16. The method according to any one of claims 13 to 15, wherein the mesenchymal stem cells are transferred to a culture vessel for subculturing for subculturing.

17. Mesenchymal cells were cultured at 1.0 x 10 6 The method of claim 16, wherein the cells are suspended at a concentration of 1000 cells / ml.

18. The method according to claim 17, wherein the mesenchymal stem cells are subcultured in a culture medium as defined in any one of claims 1 to 10.

19. The method of claim 18, wherein the mesenchymal stem cells are subcultured until they reach about 70-80% confluency.

20. The method according to any one of claims 16 to 19, wherein the subcultivation is carried out in a self-contained bioreactor.

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

22. 2. The method of any one of the preceding claims, wherein the umbilical cord tissue is a piece from the whole umbilical cord or the amniotic membrane of the umbilical cord.

23. Incubate at 37 °C and CO 2 13. The method according to any one of the preceding claims, carried out in a cell culture incubator.

24. The method according to claim 23, comprising the step of removing the mesenchymal stem cells from the culture vessel used for the subculture.

25. The method according to claim 24, wherein the step of removing the mesenchymal stem cells from the culture vessel is carried out by enzymatic treatment.

26. 26. The method of claim 25, wherein the enzyme treatment comprises trypsin treatment.

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

28. 2. The method of any one of the preceding claims, wherein 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 markers CD73, CD90, and CD105.

29. 2. The method of any one of the preceding claims, wherein 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 lack expression of the markers CD34, CD45, and HLA-DR (human leukocyte antigen-antigen D related).

30. 30. The method of any one of claims 28 or 29, wherein about 97% or more, about 98% or more, about 99% or more of the isolated mesenchymal stem cells express CD73, CD90, and CD105, and lack expression of CD34, CD45, and HLA-DR.

31. 2. The method of any one of the preceding claims, further comprising storing the isolated stem / progenitor cells for further use.

32. 32. The method of claim 31 , wherein the preserving step is performed by cryopreservation.

33. 1. An isolated mesenchymal stem cell population of umbilical cord amniotic membrane, wherein at least about 90% or more of the cells of said stem cell population express each of the markers CD73, CD90, and CD105.

34. 34. The mesenchymal stem cell population of claim 33, wherein at least about 90% or more of the cells of said stem cell population lack expression of the markers CD34, CD45, and HLA-DR.

35. 35. The mesenchymal stem cell population of claim 34, wherein 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, and lack expression of each of CD34, CD45, and HLA-DR.

36. A mesenchymal stem cell population according to any one of claims 33 to 35, obtainable by a method as defined in any one of claims 1 to 30.

37. A mesenchymal stem cell population according to any one of claims 33 to 35, obtainable by a method as defined in any one of claims 1 to 30.

38. A pharmaceutical composition comprising an isolated mesenchymal stem cell population of umbilical cord amniotic membrane, wherein at least about 90% or more of the cells of the stem cell population express each of the markers CD73, CD90, and CD105, and lack expression of each of the markers CD34, CD45, and HLA-DR.

39. 39. The pharmaceutical composition of claim 38, adapted for systemic or local application.

40. 40. The pharmaceutical composition of claim 38 or 39, further comprising a pharma- ceutically acceptable excipient.

41. 1. A method for producing a culture medium suitable for isolating a mesenchymal stem cell population from the amniotic membrane of an umbilical cord, comprising the steps of: i. 250 ml of DMEM ii. M171 118ml iii. 118 ml of DMEM / F12 iv. Fetal Bovine Serum (FBS) 12.5 ml (final concentration 2.5%) The method further comprising the step of mixing

42. v. 1 ml of EGF stock solution (5 μg / ml) to achieve a final concentration of 10 ng / ml vi. 0.175 ml of insulin stock solution (14.28 mg / ml) to achieve a final concentration of 5 μg / ml 42. The method of claim 41, further comprising the step of adding:

43. 43. The method of claim 41 or 42, further comprising adding one or more of the following supplements to the DMEM, thereby bringing the total culture volume to 500 ml.

44. The final concentrations of supplements in DMEM were: about 0.05 to 0.1 μg / ml adenine, for example about 0.025 μg / ml adenine; about 1-10 μg / ml hydrocortisone, About 0.5 to 5 ng / ml of 3,3',5-triiodo-L-thyronine sodium salt (T3), e.g., 1.36 ng / ml of 3,3',5-triiodo-L-thyronine sodium salt (T3) 44. The method of claim 43, wherein:

45. A cell culture medium obtainable by the method according to any one of claims 41 to 44.

46. A method for isolating mesenchymal stem cells from the amniotic membrane of an umbilical cord, said method comprising culturing the amniotic tissue in a culture medium prepared by the method defined in any one of claims 41 to 44.

47. - DMEM at a final concentration of approximately 55-65% (v / v), - F12 at a final concentration of approximately 5-15% (v / v), - M171 at a final concentration of approximately 15-30% (v / v), and - Final concentration of approximately 1-8% (v / v) FBS A cell culture medium comprising:

48. 48. The cell culture medium of claim 47, comprising DMEM at a final concentration of about 57.5-62.5% (v / v), F12 at a final concentration of about 7.5-12.5% ​​(v / v), M171 at a final concentration of about 17.5-25.0% (v / v), and FBS at a final concentration of about 1.75-3.5% (v / v).

49. 49. The cell culture medium of claim 48, comprising 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).

50. 50. The cell culture medium of any one of claims 47 to 49, further comprising epidermal growth factor (EGF) at a final concentration of about 1 ng / ml to about 20 ng / ml.

51. 51. The cell culture medium of any one of claims 7 to 50, comprising EGF at a final concentration of about 10 ng / ml.

52. 52. The cell culture medium of any one of claims 47 to 51, comprising insulin at a final concentration of about 1 μg / ml to 10 μg / ml.

53. 53. The cell culture medium of claim 52, comprising insulin at a final concentration of about 5 μg / ml.

54. 54. The cell culture medium of any one of claims 47 to 53, further comprising at least one of the following supplements: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3).

55. 55. The cell culture medium of claim 54, comprising all three of adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3).

56. 56. The cell culture medium of claim 54 or 55, comprising adenine at a final concentration of about 0.05 to about 0.1 μg / ml adenine, hydrocortisone at a final concentration of 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 0.5 to about 5 ng / ml.

57. 500 ml of cell culture medium i. 250 ml of DMEM ii. M171 118ml iii. 118 ml of DMEM / F12 iv. Fetal Bovine Serum (FBS) 12.5 ml (final concentration 2.5%) The cell culture medium according to any one of claims 47 to 56, comprising:

58. v. EGF at a final concentration of 10 ng / ml vi. Insulin at a final concentration of 5 μg / ml vi. 0.175 ml of insulin (final concentration 5 μg / ml) 58. The cell culture medium of claim 57, further comprising:

59. 59. The cell culture medium of claim 57 or 58, further comprising adenine at a final concentration of about 0.05 to about 0.1 μg / ml adenine, hydrocortisone at a final concentration of 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 0.5 to about 5 ng / ml.

60. 60. Use of the cell culture medium as defined in any one of claims 47 to 59 for isolating mesenchymal stem cells from the amniotic membrane of the umbilical cord.

61. 60. Use of a cell culture medium as defined in any one of claims 47 to 59 for culturing mesenchymal stem cells derived from the amniotic membrane of an umbilical cord.

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