RU2783992C2 - Method for isolation of mesenchymal stem cells from amniotic membrane of umbilical cord, using cell cultural medium - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method for isolation of a population of mesenchymal stem cells from amniotic membrane of the umbilical cord is proposed, including cultivation of umbilical cord tissue in a cultural medium containing Dulbecco’s modified eagle medium (hereinafter – DMEM), Ham’s nutrient mixture F-12 (hereinafter – F12), medium 171 (hereinafter – M171), and fetal bovine serum (hereinafter – FBS). The invention also relates to a population of mesenchymal stem cells, isolated from amniotic membrane of the umbilical cord, in which at least about 97% or more of cells of the stem cell population express each of following markers: CD73, CD90, and CD105, and do not express following markers: CD34, CD45, and HLA-DR. The invention also relates to a pharmaceutical composition of this population of mesenchymal stem cells for cell therapy; a method for the production of a cultural medium suitable for isolation of the population of mesenchymal stem cells from amniotic membrane of the umbilical cord, including mixing of following components: 250 ml of DMEM, 118 ml of M171, 118 ml of DMEM/F12, and 12.5 ml of FBS (the final concentration is 2.5%) to obtain a volume of 498.5 ml of the cultural medium. A cell cultural medium is proposed, containing: DMEM at the final concentration from about 55 to 65 % (vol./vol.), F12 at the final concentration from about 5 to 15% (vol./vol.), M171 at the final concentration from about 15 to 30% (vol./vol.), and FBS at the final concentration from about 1 to 8% (vol./vol.). The use of a cell cultural medium is proposed for isolation of mesenchymal stem cells from amniotic membrane of the umbilical cord, as well as for cultivation of mesenchymal stem cells from amniotic membrane of the umbilical cord.

EFFECT: invention provides the creation of a highly homogenous population of mesenchymal stem cells isolated from amniotic membrane of the umbilical cord.

59 cl, 12 dwg, 1 ex

Description

This application claims priority under U.S. Provisional Application No. 62/404,582, filed October 5, 2016, the contents of which are hereby incorporated by reference in their entirety for all purposes.

FIELD OF TECHNOLOGY

The present invention relates to a method for isolating mesenchymal stem cells (or such a population of stem cells) from an umbilical cord amniotic membrane, as well as a population of mesenchymal stem cells isolated from an umbilical cord amniotic membrane. The invention also relates to a cell culture medium (cell culture medium) for isolating mesenchymal stem cells from the umbilical cord amniotic membrane. The invention also relates to pharmaceutical compositions and uses of an isolated population of mesenchymal stem cells. The invention also relates to methods for treating a disease or disorder comprising administering a mesenchymal stem cell population or a pharmaceutical composition comprising such a mesenchymal stem cell population of the invention to a subject in need thereof.

BACKGROUND OF THE INVENTION

Mesenchymal stem cells isolated from the amniotic membrane of the umbilical cord were first described in US Patent Application 2006/0078993 (resulting in US Patents 9,085,755 and 9,737,568) and the corresponding International Patent Application WO 2006/019357. Since then, umbilical cord tissue has attracted attention as a source of multipotent cells; Because of its wide availability, the umbilical cord, and in particular stem cells isolated from the amniotic cord membrane (also referred to as "line cord stem cells"), are considered an excellent alternative source of cells for regenerative medicine. See Jeschke et al. Umbilical Cord Lining Membrane and Wharton's Jelly-Derived Mesenchymal Stem Cells: the Similarities and Differences; The Open Tissue Engineering and Regenerative Medicine Journal, 2011, 4, 21-27.

A follow-up study compared the phenotype, proliferation rate, migration, immunogenicity, and immunomodulatory abilities of human mesenchymal stem cells (MSCs) derived from the amniotic cord membrane (umbilical cord lining (CL-MSC)), cord blood (CB-MSC), placenta ( P-MSC) and Wharton's jelly (WJ-MSC) (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). Stubbendorf et al. concluded that populations of MSCs derived from extra-fetal gestational tissues show varying potential to evade immune response as well as exert immunomodulatory effects. The authors also found that CL-MSCs showed the most promising potential for cell therapy, as these cells showed low immunogenicity, but they also showed an increased proliferative and migratory potential, so future research should focus on the best disease models that can be applied. CL-MSC.

Although amniotic membrane mesenchymal stem cells can be easily obtained using the protocol described in US Patent Application 2006/0078993 and International Patent Application WO 2006/019357, for clinical trials with these MSCs derived from the umbilical lining, it would be useful to have a method is available to isolate a population of these MSCs from the umbilical cord lining that is highly homogeneous and thus can be used for clinical trials.

Accordingly, it is an object of the invention to provide a method for isolating a population of mesenchymal stem cells from the umbilical cord amniotic membrane that satisfies this need. Thus, it is also an object of the invention to create a highly homogeneous population of mesenchymal stem cells isolated from the amniotic membrane of the umbilical cord.

BRIEF DESCRIPTION OF THE INVENTION

This problem is solved using methods, a population of mesenchymal stem cells, an appropriate pharmaceutical composition and a cell culture medium, characterized by features of independent claims.

In the first aspect, the invention relates to a method for isolating a population of mesenchymal stem cells from the amniotic membrane of the umbilical cord, a method comprising culturing umbilical cord tissue in a culture medium containing DMEM (Dulbecco's Modified Eagle's medium), F12 (Ham's F-12 medium), M171 (medium 171) and FBS (fetal bovine serum).

In a second aspect, the invention relates to an isolated umbilical cord amniotic membrane mesenchymal stem cell population, wherein at least about 90% or more of the cells in this stem cell population express each of the following markers: CD73, CD90, and CD105. Preferably, this isolated population of mesenchymal stem cells does not express the following markers: CD34, CD45 and HLA-DR. In 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 population of mesenchymal stem cells express each of CD73, CD90 and CD105. In addition, in these embodiments of the second aspect, at least about 91% or more, about 92% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96 % or more, about 97% or more, about 98% or more, about 99% or more of the cells of the isolated population of mesenchymal stem cells preferably do not express CD34, CD45 and HLA-DR markers. This population of mesenchymal stem cells can be obtained by the method of isolating the population of mesenchymal stem cells according to the first aspect.

In a third aspect, the invention relates to a pharmaceutical composition containing a mammalian cell (according to the second aspect) according to the invention.

In a fourth aspect, the invention relates to a method for making a culture medium for isolation, comprising mixing the following components to obtain a final volume of 500 ml of culture medium:

i. 250 ml DMEM

ii. 118 ml М171

iii. 118 ml DMEM/F12

iv. 12.5 ml fetal bovine serum (FBS) to obtain a final concentration of 2.5% (v/v).

In a fifth aspect, the invention relates to a cell culture medium obtainable by the method of the fourth aspect.

In a sixth aspect, the invention relates to a method for isolating stem cells from an umbilical cord amniotic membrane, comprising culturing amniotic membrane tissue in a culture medium prepared by the method of the fourth aspect.

In a seventh aspect, the invention relates to a cell culture medium containing:

- DMEM at a final concentration of approximately 55 to 65% (v/v),

- F12 at a final concentration of approximately 5 to 15% (v/v),

- M171 at a final concentration of approximately 15 to 30% (v/v) and

- PBS at a final concentration of approximately 1 to 8% (v/v).

In an eighth aspect, the invention relates to the use of the cell culture medium according to the seventh aspect for the isolation of mesenchymal stem cells from the amniotic cord membrane.

In a ninth aspect, the invention relates to the use of the cell culture medium according to the seventh aspect for culturing mesenchymal stem cells from an umbilical cord amniotic membrane.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be better understood by reference to the detailed description, taken in conjunction with non-limiting examples and drawings, in which:

On FIG. 1 shows the Lonza data sheet for Dulbecco's Modified Eagle's Medium, including the DMEM catalog number used to create an illustrative example of the medium of the invention (PTT-6) in the Experimental Examples section;

On FIG. 2 shows the Lonza data sheet for the Ham F-12 medium;

On FIG. 3 shows the Lonza datasheet for DMEM: F12 (1:1) including the DMEM: F12 (1:1) catalog number used to create an illustrative example of the inventive medium (PTT-6) in the Experimental Examples section;

On FIG. 4 shows the Life Technologies Corporation data sheet for M171 media, including the catalog number of the M171 media used to create an illustrative example of the media of the invention (PTT-6) in the Experimental Examples section;

On FIG. 5 shows a list of ingredients, including commercial suppliers and part number, used in the Experimental Examples section to prepare PTT-6 medium.

On FIG. 6 shows the results of flow cytometry experiments in which umbilical cord-derived mesenchymal stem cells were examined for expression of the mesenchymal stem cell markers CD73, CD90, and CD105. For these experiments, mesenchymal stem cells were isolated from umbilical cord tissue by culturing the umbilical cord tissue in three different culture media, followed by subculturing the mesenchymal stem cells in the appropriate medium. The following three media were used in these experiments: a) 90% (v/(v/v/DMEM) supplemented with 10% FBS (v/(v), b) PTT-4 culture medium described in US Patent Application 2006 /0078993 and the corresponding international patent application WO 2006/019357, which consists of 90% (vol./(vol.) CMRL1066 and 10% (vol./vol.) PBS (see paragraph [0183] in WO 2006/019357 and c), the culture medium of the present invention PPT-6, the composition of which is described in this application. In this flow cytometry analysis, two different samples of the umbilical cord lining mesenchymal stem cell (CLMC) population were examined for each of the three culture media used. The results are shown in Fig. Fig. 6a - Fig. 6c In more detail, Fig. 6a shows the percentage of isolated cord lining mesenchymal stem cells expressing CD73, CD90 and CD105 stem cell markers after isolation from cord tissue and cultured in DMEM/10% PBS, Figs. 6b shows the percentage of mesenchymal stem cells isolated from the umbilical lining. nas expressing stem cell markers CD73, CD90, and CD105 after isolation from umbilical cord tissue and cultured in PTT-4, and in FIG. 6c shows the percentage of isolated umbilical cord lining mesenchymal stem cells expressing CD73, CD90 and CD 105 stem cell markers after isolation from umbilical cord tissue and cultured in PTT-6.

On FIG. 7 shows the results of flow cytometry experiments in which mesenchymal stem cells isolated from the umbilical cord were examined for the expression of stem cell markers (CD73, CD90 and CD105, CD34, CD45 and HLA-DR (human leukocyte antigen - D-related antigen) that used to determine the suitability of multipotent human mesenchymal stem cells for cell therapy and compared with the expression of these markers by bone marrow mesenchymal stem cells.For this experiment, umbilical cord amniotic membrane mesenchymal stem cells were isolated from umbilical cord tissue by culturing umbilical cord tissue in the culture medium of the present invention PPT-6 , and bone marrow mesenchymal stem cells were isolated from human bone marrow using a standard protocol.Figure 7a shows the percentage of isolated umbilical cord lining mesenchymal stem cells that express stem cell markers CD73, CD90, and CD105 and do not express ss CD34, CD45, and HLA-DR after isolation from umbilical cord tissue and cultivation in PTT-6 medium, while in Fig. 7b shows the percentage of isolated bone marrow mesenchymal stem cells that express CD73, CD90 and CD 105 and do not express CD34, CD45 and HLA-DR.

DETAILED DESCRIPTION OF THE INVENTION

As explained above, in a first aspect, the invention relates to a method for isolating a population of mesenchymal stem cells from an amniotic cord membrane, comprising culturing cord tissue in a culture medium containing DMEM (Dulbecco's Modified Eagle's Medium), F12 (Ham's Medium F-12), M171 (Wednesday 171) and FBS (fetal bovine serum). In the present application, it was unexpectedly found that the use of such a medium provides the isolation of a population of mesenchymal stem cells from the amniotic membrane of the umbilical cord, of which more than 90% or even 99% or more of the cells are positive for the three mesenchymal stem cell markers CD73, CD90 and, while At the same time, these stem cells lack the expression of CD34, CD45 and HLA-DR (see the Experimental Examples section), which means that 99% or even more of the cells in this population express the CD73, CD90 and CD105 stem cell markers without expressing CD34, CD45 and HLA-DR markers. Such an extremely homogeneous and well-defined cell population is an ideal candidate for clinical trials and cell therapy, since these cells, for example, fully meet the criteria generally accepted for human mesenchymal stem cells for use 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. In addition, using a bioreactor such as the Quantum Cell Expansion System, a large number of mesenchymal stem cells can be obtained, for example, from 300 to 700 million mesenchymal stem cells in one cycle (see also the Experimental Examples section). Thus, the present invention makes it possible to provide the quantities of stem cells that are needed for therapeutic applications, such as their 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 (Good Medical Practice) quality. Accordingly, the present invention opens the way to the production of this highly homogeneous population of mesenchymal stem cells from the umbilical cord amniotic membrane in accordance with GMP standards.

In this context, it should be noted that the culture medium of the present invention allows the isolation of a population of mesenchymal stem cells (also referred to herein as "mesenchymal stem cells") from the amniotic membrane under conditions that allow the proliferation of mesenchymal stem/progenitor cells without differentiation of mesenchymal stem/progenitor cells. Thus, after the isolation of mesenchymal stem cells from the amniotic membrane, as described in this application, the population of isolated mesenchymal stem/progenitor cells can differentiate into several cell types, as described, for example, in US patent application 2006/0078993, US patent 9085755, international patent application WO 2006/019357, US patent 8287854 or WO 2007/046775. As described in US Patent Application 2006/0078993, umbilical cord amniotic membrane mesenchymal stem cells are spindle shaped and express the following genes: POU5f1, Bmi-1, leukemia inhibitory factor (LIF), and secrete activin A and follistatin. The mesenchymal stem cells isolated in the present invention can, for example, be differentiated into any type of mesenchymal cell such as, but not limited to, adipocyte, skin fibroblasts, chondrocytes, osteoblasts, tenocytes, ligamentous fibroblasts, cardiomyocytes, smooth muscle cells, skeletal muscle cells, adipocytes, mucin-producing cells, cells derived from endocrine glands, such as insulin-producing cells (eg, β-islet cells), or neuroectodermal cells. The stem cells isolated in the present invention can be differentiated in vitro to subsequently use the differentiated cell for medical purposes. An illustrative example of this approach is the differentiation of mesenchymal stem cells into insulin-producing β-islet cells, which can then be introduced, for example, by implantation, into a patient suffering from insulin deficiency, for example, diabetes mellitus (in this context, cf., also WO 2007/046775) . Alternatively, the mesenchymal stem cells of the invention can be used in an undifferentiated state for cell therapy, for example for wound healing purposes such as burns or chronic diabetic wounds. In these therapeutic applications, the mesenchymal stem cells of the invention can either promote wound healing by interacting with surrounding diseased tissue, or can also differentiate into corresponding skin cells (cf., for example, WO 2007/046775).

In this context, it should be noted that the population of mesenchymal stem cells described here can be isolated and cultured (i.e. obtained) from any tissue of the umbilical cord, as long as the tissue of the umbilical cord contains an amniotic membrane (which is also called the "lining of the umbilical cord (umbilical cord)" ). Accordingly, a population of mesenchymal stem cells can be isolated from (pieces of) the entire umbilical cord, as described in the "Experimental Examples" section of this application. Thus, this umbilical cord tissue may contain, in addition to the amniotic membrane, any other tissue/component of the umbilical cord. As shown, for example, in FIG. 16 of US Patent Application 2006/0078993 or International Patent Application WO 2006/019357, the amniotic membrane of the umbilical cord is the outermost part of the umbilical cord covering the umbilical cord. In addition, the umbilical cord contains one vein (which carries oxygenated, nutrient-rich blood to the fetus) and two arteries (which carry oxygen-poor, depleted blood away from the fetus). For protection and mechanical support, these three blood vessels are encapsulated in wharton jelly, a jelly-like substance composed primarily of mucopolysaccharides. Accordingly, the umbilical cord tissue used in the present invention may also contain one vein, two arteries, and a jelly wharton. The use of such a whole (intact) section of the umbilical cord has the advantage that the amniotic membrane does not need to be separated from the other components of the umbilical cord. This reduces the number of isolation steps and thus makes the method of the present invention simpler, faster, less error prone and more economical - all important aspects for GMP production, which is necessary for the therapeutic use of mesenchymal stem cells. Thus, isolation of mesenchymal stem cells may begin with tissue explantation, followed by subsequent subculturing (culturing) of isolated mesenchymal stem cells if more mesenchymal stem cells are required, for example, for use in clinical trials. Alternatively, it is also possible to first separate the amniotic membrane from other umbilical cord components and isolate lining mesenchymal stem cells from the amniotic membrane by culturing the amniotic membrane in the culture medium of the present invention. This culture can also be carried out with a tissue explant, optionally followed by subculture of the isolated mesenchymal stem cells. In this context, the term "tissue explant" or "tissue explant method" is used in its usual meaning in the art to refer to a method in which the collected tissue or a piece of such tissue is placed on a cell culture dish containing culture (growth) medium, and due to which stem cells eventually migrate from the tissue to the surface of the dish. These primary stem cells can then be expanded and transferred to fresh dishes by micropropagation (subculture), as also described here. In this context, it should be noted that from the point of view of cell production for therapeutic purposes, in the first step of isolating the mesenchymal stem cells of the amniotic membrane from the umbilical cord, a master cell bank is obtained from the isolated mesenchymal stem cells, while a working cell bank can be obtained in subsequent subculturing. If the population of mesenchymal stem cells of the present invention (in particular, the population of mesenchymal stem cells, of which at least about 98% or 99% or more express each of the markers CD73, CD90 and CD 105 and do not express each of the markers: CD34, CD45 and HLA-DR) is used for clinical trials or as an approved therapeutic agent, a population of cells from a working bank will usually be used for this purpose. Both the stem cell population at the isolation stage (which may constitute the main cell bank) and the stem cell population at the subculturing stage (which may constitute the working cell bank) may, for example, be stored in cryopreserved form.

As mentioned above, the present method for isolating mesenchymal stem cells from the umbilical cord amniotic membrane has the advantage that all components used in the culture medium of the present invention are available in GMP quality and thus allow mesenchymal stem cells to be isolated under conditions , corresponding to GMP, for subsequent therapeutic use.

By "DMEM" is meant Dulbecco's Modified Eagle's Medium, which was developed in 1969 and is a modification of Eagle's Basal Medium (BME) (see Fig. 1 showing the specification of DMEM supplied by Lonza). The original DMEM formula contains 1000 mg/L glucose and was first proposed for culturing mouse embryonic cells. Since then, DMEM has become the standard cell culture medium that is commercially available from various sources such as ThermoFisher Scientific (p/n 11965-084), Sigma Aldrich (p/n D5546), or Lonza, to name a few. Thus, any commercially available DMEM can be used in the present invention. In preferred embodiments, the DMEM used here is the DMEM available from Lonza under part number 12-604F. This medium is DMEM supplemented with 4.5 g/l glucose and L-glutamine). In another preferred embodiment, the DMEM used in this application is DMEM under Sigma Aldrich catalog number D5546, which contains 1000 mg/l glucose and sodium bicarbonate, but does not contain L-glutamine.

"F12" refers to Ham's F-12. This medium is also a standard cell culture medium and is a nutrient mixture originally designed for culturing a wide range of mammalian cells and hybridoma cells when used with serum in combination with hormones and transferrin (see Fig. 2 showing the specification of Ham's F-12 medium from Lonza). Any commercially available Ham F-12 medium (for example, from ThermoFisher Scientific (p/n 11765-054), Sigma Aldrich (p/n N4888), or Lonza, to name but a few) may be used in the present invention. In preferred embodiments, Ham's F-12 medium from Lonza is used.

By "DMEM/F12" or "DMEM:F12" is meant a 1:1 mixture of DMEM with Ham's F12 culture medium (see Fig. 3 for the specification for DMEM:F12 (1:1) from Lonza). Also, DMEM/F12 (1:1) is a widely used basal medium to support the growth of many different mammalian cells and is commercially available from various vendors such as ThermoFisher Scientific (p/n 11330057), Sigma Aldrich (p/n D6421), or Lonza . Any commercially available DMEM medium: F12 can be used in the present invention. In preferred embodiments, the DMEM:F12 medium used in this application is DMEM/F12 (1:1) supplied by Lonza under catalog number 12-719F (which is DMEM:F12 with L-Glutamine, 15 mM HEPES and 3.151 g/l glucose).

By "M171" is meant culture medium 171, which was developed as a basal medium for culturing (culture medium) normal human breast epithelial cells (see Fig. 4 showing the specification for M171 medium from Life Technologies Corporation). Also, this basal medium is widely used and commercially available, for example, from a supplier such as ThermoFisher Scientific or Life Technologies Corporation (part number M171500). Any commercially available M171 medium may be used in the present invention. In preferred embodiments, the M171 medium used herein is the M171 medium supplied by Life Technologies Corporation under catalog number M171500.

By "FBS" is meant fetal bovine serum (also called "fetal calf serum"), that is, the fraction of blood that remains after natural blood clotting followed by centrifugation to remove any remaining red blood cells. Fetal bovine serum is the most widely used serum supplement for in vitro eukaryotic cell culture because it has very low levels of antibodies and contains more growth factors, providing versatility in many different cell culture applications. PBS is preferably obtained from a member of the International Whey Industry Association (ISIA), whose main concern is the safety and safe use of whey and animal products, through proper traceability, labeling validity, and appropriate standardization and oversight. PBS suppliers that are ISIA members include Abattoir Basics Company, Animal Technologies Inc., Biomin Biotechnologia LTDA, GE Healthcare, Gibco from Thermo Fisher Scientific and Life Science Production, just to name a few. In presently preferred embodiments, PBS is obtained from GE Healthcare under catalog number A15-151.

With respect to the culture medium of the present invention, it may contain DMEM at a final concentration of approximately 55 to 65% (v/v), F12 at a final concentration of approximately 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). As used herein, "% (vol./vol.)" refers to the volume of the individual component relative to the final volume of the culture medium. This means that if DMEM, for example, is present in the culture medium at a final concentration of about 55 to 65% (v/v), 1 liter of culture medium contains about 550 to 650 ml of DMEM.

In other embodiments, the culture medium may contain DMEM at a final concentration of approximately 57.5 to 62.5% (v/v), F12 at a final concentration of approximately 7.5 to 12.5% (v/v). ), M171 at a final concentration of approximately 17.5 to 25.0% (v/v) and PBS at a final concentration of 1.75 to 3.5% (v/v). In other embodiments, the culture medium may contain DMEM at a final concentration of approximately 61.8% (v/v), F12 at a final concentration of approximately 11.8% (v/v), M171 at a final concentration of approximately 23.6 % (v/v) and FBS at a final concentration of approximately 2.5% (v/v).

In addition to the above components, the culture medium may contain supplements that are beneficial to the mesenchymal stem cells of the umbilical cord lining. The culture medium of the present invention may, for example, contain epidermal growth factor (EGF). If EGF is present in the culture medium, it may be at a final concentration of from 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 approximately 10 ng/mL.

The culture medium of the present invention may also contain insulin. If insulin is present, its final concentration may be from about 1 µg/ml to 10 µg/ml. In some of these embodiments, the culture medium may contain insulin at a final concentration of approximately 5 μg/ml.

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

In the method of the invention, umbilical cord tissue can be cultured until a suitable amount of lining (primary) mesenchymal stem cells has grown from the tissue. In typical embodiments, umbilical cord tissue is cultured until amniotic membrane mesenchymal stem cell outgrowth reaches approximately 70-80% confluency. It should be noted that the term "confluence" is used in its usual meaning in the field of cell culture and is understood as an estimate/indicator of the number of adherent cells in a culture dish or flask, which refers to the proportion of the surface covered with cells. For example, 50% confluency means that approximately half of the surface is covered and there is still room for cell growth. 100% confluence means that the surface is completely covered with cells, leaving no room for cells to grow as a monolayer.

After the required number of primary cells (lining mesenchymal stem cells) were obtained from the umbilical cord lining tissue by tissue explantation, the mesenchymal stem cells were recovered from the culture container used for culture. Thus, a master cell bank containing (primary) isolated amniotic membrane mesenchymal stem cells can be obtained. As a rule, since mesenchymal stem cells are adherent cells, the extraction is carried out using standard enzymatic processing. For example, an enzymatic treatment may include trypsin treatment as described in US 2006/0078993, WO 2006/019357, or WO 2007/046775, which means that overgrown cells can be harvested by trypsinization ( 0.125% trypsin/0.05% EDTA) for further propagation. If the collected mesenchymal stem cells are used, for example, to create a master cell bank, the cells can also be cryopreserved and stored for further use, as explained below in this application.

After harvesting, the mesenchymal stem cells can be transferred to a culture container for subculture. Subculturing can also be started from frozen primary cells, i.e. from the master cell bank. For subculture, any suitable number of cells can be seeded into a culture container, such as a cell culture plate. To do this, mesenchymal cells can be suspended in a suitable medium (most conveniently in the culture medium of the present invention) for subculturing at a concentration of, for example, from about 0.5×10 ⁶ cells/ml to about 5.0×10 ⁶ cells/ml . In one embodiment, cells are suspended for subculture at a concentration of approximately 1.0 x 10 ⁶ cells/mL. Subculturing can be done by culturing either in simple culture flasks, but also, for example, in a multilayer system such as Cell Stacks (Corning, Corning, NY, USA) or Cellfactory (Nunc, a subsidiary of Thermo Fisher Scientific Inc., Waltham, Massachusetts). , USA) that can be stacked in incubators. Alternatively, subculturing can also be carried out in a closed offline system such as a bioreactor. Various bioreactor designs are known to those skilled in the art, such as parallel plate, hollow fiber, or microfluidic bioreactors. See, for example, Sensebe et al. "Production of mesenchymal stromal/stem cells according to good manufacturing practices: a review", supra. An illustrative example of a commercially available hollow fiber bioreactor is the Quantum® Cell Expansion System (Terumo BCT, Inc), which has been used, for example, to expand bone marrow mesenchymal stem cells for clinical trials (cf., 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 commercially available bioreactors that can be used to subculture a population of mesenchymal stem cells of the present invention is the Xuri Cell Expansion System manufactured by GE Heathcare. Cultivation of a population of mesenchymal stem cells in an automated system such as the Quantum® Cell Expansion System is of particular advantage if a working cell bank for therapeutic use is to be produced under GMP conditions and a large number of cells are required.

Subculturing mesenchymal stem cells according to the invention is carried out in the culture medium according to the present invention. Accordingly, the culture medium of the present invention can be used both for the isolation of mesenchymal stem cells from the amniotic membrane and for the subsequent cultivation of the isolated primary cells by subculturing. Also, for subculturing, mesenchymal stem cells can be cultured until the desired number of cells has grown. In exemplary embodiments, the mesenchymal stem cells are subcultured until the mesenchymal stem cells reach about 70% to about 80% confluence. Isolation/culturing of the lining mesenchymal stem cell population can be performed under standard mammalian cell culture conditions. In general, the method for isolating a population of lining mesenchymal stem cells according to the invention is generally carried out under conditions (temperature, atmosphere) that are commonly used for culturing cells of the species from which the cells are derived. For example, human umbilical cord tissue and lining mesenchymal stem cells, respectively, are typically cultured at 37° C. in an air atmosphere with 5% CO ₂ . In this context, it should be noted that in the present invention, mesenchymal cells can be obtained from any mammalian species, such as mouse, rat, guinea pig, rabbit, goat, horse, dog, cat, sheep, monkey, or human, and in one implementation mesenchymal stem cells of human origin are preferred.

Once the desired/sufficient amount of lining mesenchymal stem cells has been obtained from the subculture, the mesenchymal stem cells are harvested by removing them from the culture container used for subculture. The collection of mesenchymal stem cells is usually carried out again by enzymatic treatment, including trypsinization of the cells. The isolated mesenchymal stem cells are subsequently harvested and either directly used or stored for later use. As a rule, preservation is carried out by cryopreservation. The term "cryopreservation" is used herein in its usual sense to describe the process in which mesenchymal stem cells are preserved by cooling to low 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 the person skilled in the art, and may include the use of cryoprotectants such as dimethyl sulfoxide (DMSO) or glycerol, which slow down the formation of ice crystals in the cells of the umbilical cord.

The isolated population of lining mesenchymal stem cells obtained by the isolation method of the present invention is well defined and homogeneous. In typical embodiments 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 isolated mesenchymal stem cells express the following markers: CD73, CD90, and CD 105. 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 specific 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 CD34, CD45, and HLA-DR expression.

Thus, in accordance with the above description, the present invention also relates to a population of mesenchymal stem cells isolated from the amniotic membrane of the umbilical cord, and at least about 90% or more of the cells of this population of stem cells express each of the following markers: CD73, CD90 and CD105 . In preferred embodiments, at least about 91% or more, about 92% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the cells of the isolated mesenchymal stem cell population are CD73 ⁺ , CD90 ⁺ and CD105 ⁺ , that is, this percentage of the isolated cell population expresses each of CD73, CD90 and CD105 (see. section "Experimental examples" 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 isolated mesenchymal stem cells may lack the expression of the following markers. In specific embodiments, about 97% or more, about 98% or more, or about 99% or more of an isolated population of mesenchymal stem cells express CD73, CD90, and CD105 while not expressing CD34, CD45, and HLA-DR. Such a highly homogeneous population of mesenchymal stem cells derived from the amniotic membrane of the umbilical cord is described here for the first time, and it meets the criteria for the use of mesenchymal stem cells for cell therapy (see also the section "Experimental examples" and, for example, Sensebe et al. "Production of mesenchymal stromal/stem cells according to good manufacturing practices: a review", see above). In this context, it should be noted that this population of mesenchymal stem cells can be obtained by the isolation method of the present invention, but also by another method, such as cell sorting, if desired.

In accordance with the foregoing, the present invention also relates to a pharmaceutical composition comprising a population of mesenchymal stem cells isolated from the amniotic membrane of the umbilical cord, in which at least about 90% or more of the cells of the stem cell population express each of the following markers: CD73, CD90 and CD105 and optionally lack CD34, CD45 and HLA-DR expression. The pharmaceutical composition may contain any pharmaceutically acceptable excipient and may be formulated for any desired pharmaceutical route of administration. The pharmaceutical composition may, for example, be adapted for systemic or topical use.

In a further aspect, the invention relates to a method for preparing culture medium for isolation, comprising mixing the following components to obtain a final volume of 500 ml of culture medium:

i. 250 ml DMEM

ii. 118 ml М171

iii. 118 ml DMEM/F12

iv. 12.5 ml fetal bovine serum (FBS) to obtain a final concentration of 2.5% (v/v).

As explained above, DMEM/F12 is a 1:1 mixture of DMEM and Ham's F-12. Thus, 118 ml of DMEM/F12 medium contains 59 ml of DMEM and 59 ml of F12. Accordingly, when using this culture medium preparation method, the final concentrations (v/v) for a total volume of 500 ml are as follows:

DMEM: 250 ml + 59 ml = 309 ml corresponds to 309/500=61.8% (v/v)

M171: 118 ml, corresponds to 118/500=23.6% (v/v)

F12: 59 ml corresponds to 59/500=11.8% (v/v).

Embodiments of this culture medium preparation method further include adding

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

vi. 0.175 ml stock insulin solution (14.28 mg/ml) to obtain a final insulin concentration of 5 µg/ml.

It should be noted that in these embodiments, the aforementioned volumes of these i.-vi components result in a final volume of 499.675 ml of culture medium. If no additional components are added to the culture medium, the remaining 0.325 ml (to reach a total volume of 500 ml) can, for example, be any of components i-iv, i.e. DMEM, M171, DMEM/F12, or PBS. 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. In addition, it should also be noted that the components of i.-iv. it is not necessary to add in the order in which they are listed, but, of course, any order can also be used to mix these components to obtain a growth medium of the present invention. This means that, for example, M171 and DMEM/F12 can be mixed together and then combined with DMEM and FBS to achieve final concentrations as described here, i.e. a final DMEM concentration of approximately 55 to 65% (v/v) , a final F12 concentration of approximately 5 to 15% (v/v), a final M171 concentration of approximately 15 to 30% (v/v), and a final PBS concentration of approximately 1 to 8% (v/v). ).

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

from about 0.05 to 0.1 µg/ml adenine, for example about 0.025 µg/ml adenine,

from 1 to 10 mcg / ml of hydrocortisone,

approximately 0.5 to 5 ng/ml 3,3',5-triiodo-L-thyronine sodium (T3), e.g. 1.36 ng/ml 3,3',5-triiodo-L-thyronine sodium ( T3).

In accordance with the above description, the invention also relates to a cell culture medium, which can be obtained or which is obtained by the method of preparing the medium described here.

Furthermore, the invention also relates to a method for isolating mesenchymal stem cells from an umbilical cord amniotic membrane, which method comprises culturing amniotic membrane tissue in a culture medium obtained by the method described herein.

Thus, the present invention also relates to a cell culture medium containing:

- DMEM at a final concentration of approximately 55 to 65% (v/v),

- F12 at a final concentration of approximately 5 to 15% (v/v),

- M171 at a final concentration of approximately 15 to 30% (v/v) AND

- PBS at a final concentration of approximately 1 to 8% (v/v).

In some embodiments of the culture medium described herein, the medium contains DMEM at a final concentration of approximately 57.5 to 62.5% (v/v), F12 at a final concentration of approximately 7.5 to 12.5% (v/v). ./v), M171 at a final concentration of about 17.5 to 25.0% (v/v) and PBS at a final concentration of about 1.75 to 3.5% (v/v). In other embodiments, the culture medium may contain DMEM at a final concentration of approximately 61.8% (v/v), F12 at a final concentration of approximately 11.8% (v/v), M171 at a final concentration of approximately 23.6 % (v/v) v) and FBS at a final concentration of approximately 2.5% (v/v).

In addition, the culture medium may further contain epidermal growth factor (EGF) at a final concentration of from about 1 ng/ml to about 20 ng/ml. In certain embodiments, the culture medium contains EGF at a final concentration of approximately 10 ng/mL. The culture medium described in this application may additionally contain insulin at a final concentration of from about 1 μg/ml to 10 μg/ml. In such embodiments, the culture medium may contain insulin at a final concentration of approximately 5 μg/mL.

The cell culture medium of the present invention may further contain at least one of the following additives: adenine, hydrocortisone and 3,3',5-triiodo-L-thyronine (T3) sodium salt. In some embodiments, the culture medium includes all three of the following additives: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine (T3) sodium salt. With these additives, the culture medium may contain adenine at a final concentration of from about 0.01 to about 0.1 μg/ml of adenine, or from about 0.05 to about 0.1 μg/ml of adenine, hydrocortisone at a final concentration of from about 0, 1 to about 10 µg/ml hydrocortisone or about 1 to about 10 µg/ml hydrocortisone and/or 3,3',5-triiodo-L-thyronine (T3) sodium salt at a final concentration of about 0.5 to about 5 ng/ml/ml.

In embodiments of the cell culture medium, 500 ml of the cell culture medium of the present invention contains:

i. 250 ml DMEM

ii. 118 ml М171

iii. 118 ml DMEM/F12

iv. 12.5 ml fetal bovine serum (FBS) to obtain a final concentration of 2.5%

In other embodiments, the cell culture medium may further comprise

v. EGF at a final concentration of 10 ng/mL, and

vi. insulin at a final concentration of 5 mg/ml.

Both insulin and EGF can be added to the culture medium using a stock solution of choice so that the total culture medium volume does not exceed 500 ml.

In a specific example, the components of i.-vi. culture medium of the present invention are the components indicated in FIG. 5, that is, they are obtained from the respective manufacturers using the part number indicated in FIG. 5. Medium obtained by mixing components i. vi., as shown in Fig. 5 is also referred to here as "RTT-6". In this context, it should again be noted that in the manufacture of the medium of the present invention, components i.-vi., as well as any other ingredient, such as an antibiotic, from any other commercial supplier, can be used.

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

Finally, the invention also relates to a method of treating a patient with a disease comprising administering to the patient mesenchymal lining stem cells or a pharmaceutical composition containing stem cells as described herein. This disease may be any of the diseases described above. For treatment of a subject, the mesenchymal stem cell population of the invention may be administered by any suitable method, for example, including but not limited to topical administration, administration by implantation, or injection. Such a population of stem cells, for example, can be placed directly on a wound, such as a burn or diabetic wound (see international patent application WO 2007/046775). Alternatively, the stem cell population can also be implanted subcutaneously, for example directly under the skin, in fat deposits or in the peritoneum.

The invention will be further illustrated by the following non-limiting experimental examples.

Experimental examples

1. Cryopreservation of umbilical cord tissue prior to isolation of mesenchymal stem cells

Umbilical cord tissue (the umbilical cord was provided with the informed consent of the donor mother) was processed for subsequent isolation of mesenchymal stem cells from the umbilical cord amniotic membrane as follows.

1.1 Washing the umbilical cord tissue sample:

a. Remove the scalpels from the protective case.

b. The umbilical cord is held securely with forceps and a 10 cm long piece is cut out of it with a scalpel. Place the unused umbilical cord back into the original tissue cup.

c. Place a 10 cm long piece of umbilical cord in a new 150 mm culture dish. Instead of cups, a 150 mm culture dish can be used.

d. Use the lid of a 150 mm culture dish as a temporary storage space for the forceps and scalpel.

e. Withdraw 25 ml of Plasmalite A (Baxter, catalog # 2B2543Q) using a 30 ml syringe. Holding the syringe with one hand at an angle of 45°, Plasmalyte A is applied directly to the umbilical cord tissue.

f. While holding the culture dish at a slight angle, remove the Plasmalyte A using a 30 ml syringe and a blunt needle.

g. Collect used Plasmalyte A in a 300 ml transfer bag that serves as a waste container and dispose of it in a biohazard container.

h. Repeat the washing procedure, if necessary using a new culture dish for each wash. Ensure the removal of all blood clots on the surface. More Plasmalyte A can be used to clean tissues if necessary.

i. Place the tissue in a new labeled tissue culture dish to continue cutting the tissue. Place 20 ml of Plasmalite A in the cup so that the tissue does not dry out during cutting.

j. The umbilical cord is cut into equal approximately 1 cm segments, resulting in 10 segments.

k. Next, cut each 1 cm segment into smaller pieces of approximately 0.3 cm x 0.3 cm to 0.5 cm x 0.5 cm per segment.

l. Remove all Plasmalite A that was in the cup.

m. Withdraw 25 ml of Plasmalite A using a 30 ml syringe from the original Plasmalite A bag and dispense it directly onto the umbilical cord tissue.

n. Hold the culture dish at an angle to collect all of the Plasmalite A used for tissue washing on one side and remove it with a syringe and a blunt needle.

about. Repeat washing again. There should be no blood clots left.

NOTE. If the umbilical cord is not frozen immediately, the umbilical cord tissue is stored in Plasmalite A until ready to be frozen.

1.2 Cryopreservation of umbilical cord tissue:

a. Prepare cryopreservation solution:

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

ii. Label the 150 ml transfer bag "Tissue Freezing Solution" and connect the plasma transfer kit to the port using aseptic technique.

iii Remove 30 ml of Plasmalite A using a 30 ml syringe from the original Plasmalite A bag and transfer it to a transfer bag labeled "Tissue Freezing Solution" with the time and date of preparation.

iv. Withdraw 15 ml of 5% human serum albumin with a 20 ml syringe and transfer it to a labeled transfer bag.

v. Add 5 ml of DMSO to the transfer bag.

vi. Mix thoroughly and record the mixing of the freezing solution.

b. Remove Plasmalite A from the tissue before adding the freezing solution.

c. Using a 60 ml syringe, withdraw all 50 ml of freezing solution into the syringe and add approximately 30 ml of freezing solution to a 150 mm cell culture dish containing umbilical cord tissue. Install a blunt needle on the syringe so that it is sterile.

d. Shake the culture dish containing tissue and freezing solution once a minute for 10 minutes.

e. Using forceps, select 8 randomly selected cut segments and place them in each of four 4 ml cryovials. Select 4 randomly selected segments and place them in one 1.8 ml cryovial. There should be no blood clots on these segments.

f. Fill each cryovial containing umbilical cord tissue with the remaining freezing solution to the 3.6 ml fill line for 4 ml tubes and the 1.8 ml line for the 1.8 ml Nunc vial.

g. Label one bottle of Bactec Lytic/10 - Anaerobic/F and one bottle of Bactec Plus Aerobic/F with a fabric identifier.

h. Remove 20 ml of the freezing solution from the culture dish using a syringe and a blunt needle, then wipe the Bactec vials with an alcohol swab, replace the blunt needle with an 18 g needle, and inoculate Bactec aerobic and anaerobic vials with 10 ml each.

i. Run the freezer at an adjustable speed.

j. After completion of freezing at a controlled rate, place the tubes in a liquid nitrogen freezer with continuous temperature control until further use.

2. Isolation of lining mesenchymal stem cells from umbilical cord tissue

2.1. Preparation of MSC processing medium from umbilical cord tissue:

a. To prepare 500 ml of PTT6 (culture/growth medium), add the following ingredients in this order:

i. DMEM, 250 ml

ii. M171, 118 ml

iii. DMEM F12, 118 ml

iv. PBS, 12.5 ml (final concentration 2.5%)

v. EGF, 1 ml (final concentration 10 ng/ml)

vi. Insulin, 0.175 ml (final concentration 5 µg/ml))

The above volumes of components i.-vi result in a total final volume of 499.675 ml of culture medium. If no additional components are added to the culture medium, the remaining 0.325 ml (to reach a total volume of 500 ml) can, for example, be any of components i-iv, i.e. DMEM, M171, DMEM/F12, or PBS. 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, an antibiotic stock solution such as penicillin-streptomycin-amphotericin can be added to give a final volume of 500 ml. It is also possible to add 0.325 ml of one or more of the following additives to the culture medium: adenine, hydrocortisone, 3,3',5-triiodo-L-thyronine (T3) sodium salt, thereby obtaining a total culture medium volume of 500 ml.

vii. Label the bottle "PTT6" with the date of preparation of the medium, the initial of the operator, the phrase "best before" and the date of expiration. The expiration date is the earliest expiration date for any component, or 1 month from the date of preparation, whichever comes first.

b. To prepare the wash medium (Hank's Buffered Salt Solution (HBSS) without calcium or magnesium and with 5% FBS), add 2.5 ml of FBS to 47.5 ml of HBSS in a 50 ml centrifuge tube. Label the tube "Washing Medium" with the initials of the operator and the date of preparation of the medium.

c. All media will be tested for sterility using Bactec Lytic/10 - Anaerobic/F (Becton Dickinson & Company) and Bactec Pluc+Aerobic/F (Becton Dickinson & Company) bottles. Inject 20 ml of prepared medium into each bottle.

2.2 Thawing cord tissue for MSC collection:

a. The defrosting starts when the operator is ready to process the sample in the clean room. It is not allowed to thaw more than 1 vial at a time, unless the vials come 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 wash medium consisting of 70-90% Plasmalite A in a clean room in a biological safety cabinet. Sterile filter the solution with a 0.2 µm syringe filter attached to a 10 ml syringe and store the solution in the refrigerator until use.

d. Place a processing label on a 50 ml conical tube.

e. Make sure that the temperature of the water bath was 36-38°C.

f. Take a tissue tube from liquid nitrogen storage and rapidly thaw in a water bath at 37° C. filled with 1 liter of sterile water. Freezer Vial Holder Mr. Frosty Nalgene Cryo 1°C floats with vials installed and can be used as a floating holder when thawing samples.

g. Remove the vial from the water bath and spray with a 70% isopropanol solution. It is recommended to remove the vial from the water bath when some floating ice is visible in the vial as confirmation that the internal temperature of the vial is less than 37°C.

h. Place the vial in the transfer window and alert the clean room technician.

2.3 Preparation for fabric processing:

a. Cord tissue processing should be done in an environmentally controlled (EM) clean room. At the end of each shift, a complete cleaning of the room and hood is carried out.

b. Prepare/clean the biosafety cabinet.

c. Perform a viable particle count while working in a biosafety cabinet.

d. Collect all necessary materials in the biosafety cabinet, checking each for damage to the packaging and expiration date. When handling syringes, serological pipettes, sterile forceps, scalpels, tissue plates and needles, care must be taken not to touch any surface that comes into contact with the sterile product. Only the outside of the syringe barrel, tubing, plunger tip and/or needle cap or cannula may be safely touched. The use of materials if the surface has been touched, or if they have touched a non-sterile surface, is not allowed.

e. Record lot numbers and expiration dates (if applicable) of all reagents and consumables used.

f. A thawed tube is obtained by cleaning it with a lint-free cloth moistened with 70% alcohol before transfer to the biological safety cabinet.

g. Using an aspiration needle with a syringe, remove as much liquid from the vial as possible. Avoid tissue suction.

h. Using sterile forceps, transfer the tissue to a sterile 100 mm Petri dish.

i. Add a 5 ml aliquot of wash medium to tissue fragments.

j. Shake the contents for 15-30 seconds, then remove the washing medium with a pipette or syringe using an aspiration needle. Repeat this washing process twice.

k. Add 2 ml of wash medium to the tissue to avoid drying of the tissue.

2.4. Initiation of MSC outgrowth from tissue:

a. Label the bottom of the 6-well plate "Overgrowth 1" with the MSC lot number or umbilical cord tissue identifier and the start date of growth. If a 60 mm tissue culture dish is used, divide the plate into 4 quadrants by drawing a grid on the bottom of the dish.

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

c. Fill each well with 3 ml PTT6.

d. Using an aspiration needle attached to a 30 ml syringe, withdraw enough medium to barely cover the tissue. Do not tilt your tablet. Do not touch the bottom of the well with the aspiration needle.

e. Cell growth is observed daily using an inverted light microscope (24±6 hours). Instead of a light microscope, a real-time cell culture imaging system can be used.

f. Change environment every day. Before use, the medium must be equilibrated to room temperature.

i. Aspirate the medium.

ii. Add 3 ml PTT6.

iii. Aspirate until the tissue is barely immersed in the medium.

g. When cell growth is observed from the tissue, transplant the tissue into a new 6-well plate using the same procedure as in steps 4.a-4.e above, except that the plate is labeled "Overgrowth 2". Support cell growth in the "Growth 1" plate by adding 2 ml of PTT6 to each well. Monitor confluency every day. Replace the medium every 2-3 days (be sure to equilibrate the medium to room temperature before use).

h. When cell growth is observed on the "Overgrowth 2" plate, repeat steps 4.a-4.e, except that the plate is labeled "Overgrowth 3.". Support cell growth on the Sprawl 2 plate by adding 2 ml of PTT6 to each well. Monitor confluency every day. Replace the medium every 2-3 days (be sure to equilibrate the medium to room temperature before use).

i. When growth is observed on the "Overgrowth 3" plate, the tissue is discarded. If the tissue pieces are very small and do not appear to interfere with cell growth, the tissue is discarded by subculture.

j. When the cells reach 40-50% confluency, monitor the cells every day to prevent over-proliferation.

k. When cells reach 70-80% confluence, cells are subcultured. Do not allow cells to expand beyond 80% confluence.

When tissue explants are approximately 1-3 mm in size, and tissue/cell culture explantation is performed in a 175 mm culture dish, the average number of mesenchymal stem cells harvested from the explant is typically approximately 4000-6000 cells/explant. Accordingly, when mesenchymal stem cells are grown simultaneously from 48 explants, approximately 300,000 cells can be harvested. These 300,000 mesenchymal stem cells collected from explants can then be used for subculture by seeding a 175 cm ² cell culture flask with these 300,000 cells as described in the following example 2.5 (this can be called "Passage 1!"). Mesenchymal stem cells obtained from this passage 1 can then be used to re-seed the 175 cm ² flasks (Passage 2) and expand the cells as described in the following example 2.5 Cells obtained in both Passage 1 and bank" by cryopreservation, it being considered that the mesenchymal stem cells obtained after Passage 2 represent the Master cell bank, which will be used for further expansion of mesenchymal stem cells, for example, in a bioreactor, as described below in example 2.7.

2.5. Subculture of MSC in cell culture dishes

a. Obtain viable particles by working in a biosafety cabinet. All media are equilibrated to room temperature before use.

b. When cell proliferation reaches approximately 70-80% confluence, the cells are subcultured.

i. Remove PTT6 from the Petri dish.

ii. Wash with HBSS without calcium or magnesium.

iii. Add 0.2 ml IX TrypLE-EDTA and mix for 1-2 minutes.

iv. Tilt the dish 30-45° to allow the cells to move under the force of gravity. Light tapping on the side of the tablet accelerates the release.

v. Add 1 ml PTT6. Carefully pipet up and down, then transfer the cells to a 15 ml centrifuge tube. Use a clean pipette tip for each well. Cells from all 6 wells can be pooled into one 15 ml tube.

vi. Centrifuge for 10 minutes at 1200 rpm.

vii. Remove the supernatant and resuspend the cells with 5 ml of PTT6.

c. Subculture MSC

i. Aliquot 50 μl of cell suspension and analyze for TNC and viability by trypan blue displacement.

ii. Cells are counted using a hemocytometer. Expected result is 20-100 cells/sq. If this number is greater than 100, dilute the original sample 1:5 and repeat the trypan blue method using a hemocytometer.

iii. Count the number of viable cells/ml and the total number of viable cells:

1. Number of viable cells/ml = number of viable cells × dilution factor × 10 ⁴

2. Total Viable Cells = Viable Cells × Dilution Factor × Total Volume × 10 ⁴

iv. Calculate % 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 ;

3. "X" = 10 ⁷ /10 ⁶ cells/ml or 10 ml, so you can bring the total cell volume to 10 ml by adding 5 ml to the cell suspension (ie 5 ml).

vi. If the cell suspension is less than 10 ⁶ /ml, determine the volume required to inoculate 2×10 ⁶ cells for each 150 mm Petri dish or 175 cm ² flask.

1. Volume for 2×10 ⁶ cells=2×10 ⁶ cells ÷ number of viable cells/ml

2. For example, if the number of viable cells/ml is 8×10 ⁵ cells/ml, 2×10 ⁶ cells ÷ 8×10 ⁵ cells/ml or 2.5 ml are needed.

vii. Set aside 0.5 ml for analysis of MSC markers.

viii. 2×10 ⁶ cells are seeded in each 150 mm Petri dish or 175 cm ² flask with 30 ml PTT6.

ix. Cells are monitored for adherence, colony formation and confluence every three days. When the cells reach 40-50% confluence, observe the cells every one to two days to prevent overexpansion. Do NOT allow cells to expand beyond 80% confluence. Instead of a light microscope, a real-time cell culture monitoring system can be used.

x. Change medium every 2-3 days.

2.6 Cryopreservation of MSC cells

a. Obtain viable particles by working in a biosafety cabinet.

b. When the cells reach 70-80% confluency, separate the cells using 2 ml of 1X TrypLE-EDTA for every 150 mm Petri dish or 175 cm ² flask.

i. Remove PTT6 from the Petri dish.

ii. Wash with 5 ml HBSS or PBS without calcium or magnesium.

iii. Add 2 ml of 1X TrypLE-EDTA and shake for 1-2 minutes.

iv. Tilt the dish 30-45° to allow the cells to move under the force of gravity. Light tapping on the side of the tablet accelerates the release.

v. Add 10 ml PTT6 to inactivate TrypLE. Mix well to separate cell clumps.

vi. Transfer the cells to a 15 ml centrifuge tube using a Pasteur pipette.

vii. Centrifuge for 10 minutes at 1200 rpm.

viii. Aspirate the medium and resuspend with 10 ml of PTT6.

ix. Aliquot 50 µl and determine the total number of viable cells and % viability as above. Cell counts must be completed within 15 minutes, as cells may begin to clot.

With. Preparation of cells for cryopreservation

i. Prepare cell suspension medium and cryopreservation medium and freeze cells

2.7. Subculture (propagation) of MSC in a Quantum bioreactor (Terumo VTS, Inc.)

The Quantum bioreactor can also be used to propagate MSCs. The initial number of cells for expansion in the Quantum Bioreactor should be between 20 and 30 million cells per run. Typical yield per cycle is 300 to 700 million MSC at harvest. The bioreactor is operated according to the manufacturer's protocol. Mesenchymal stem cells thus obtained are usually cryopreserved (see below) and used as a working cell bank.

MATERIALS/REAGENTS:

1. Quantum Breeding Kit

2. Quantum waste bag

3. Package for Quantum environment

4. Quantum introduction package

5. PTT6

6. FSB

7. Fibronectin

8. TrypLE

9. 3 ml syringe

10. Glucose test strips

11. Lactate test strips

12. 60 ml cell culture plate or equivalent

13. Gas mixture 5% CO ₂ medical grade

14. Combination tip 50ml

EQUIPMENT:

1. Biosafety box

2. Glucometer (Bayer Healthcare/Ascensia Contour Blood Glucose Meter)

3. Lactate Plus (Nova Biomedical)

4. Peristaltic pump with outlet

5. Centrifuge, Eppendorf 5810

6. Sterile connector

7. M4 pipette dispenser

8. RF tablet sealer

PROCEDURE:

1. Preparing the Quantum bioreactor

a) Charging the Quantum bioreactor

b) Bioreactor coating:

1) Prepare the fibronectin solution in the biosafety cabinet.

1) Allow lyophilized fibronectin to acclimate to room temperature (≥15 min at room temperature)

2) Add 5 ml of sterile distilled water; without shaking or stirring

3) Allow fibronectin to go into solution for 30 minutes.

4) Using a 10 ml syringe with an 18g needle attached, transfer the fibronectin solution to a Ccell management bag containing 95 ml of PBS.

2) Attach the bag to the "reagent" line

3) Check for bubbles (bubbles can be removed using the "Air IC" or "Air EC" modes and using "Purge" as the inlet source.

4) Open or set up a program to coat the bioreactor (FIG. 1, steps 3-5)).

5) Run the program

6) While the bioreactor coating program is running, prepare a bag of 4 L of PTT6 medium.

7) Connect the media bag to the IC Media line using a sterile connecting tube.

8) When the bioreactor coating steps are completed, detach the cell injection bag used for the fibronectin solution with the RF sealing device.

c) Flushing out excess fibronectin

d) Conditioning the bioreactor with the medium

2. Cultivation of cells in a Quantum bioreactor

a) Loading and attaching cells with a standardized suspension:

b) Nutrition and cell culture

1) Choose the flow rate of the medium to feed the cells.

2) Take samples for lactate and glucose every day.

3) Adjust the flow rate of the medium as the level of lactate increases. The actual maximum tolerable concentration of lactate will be determined by the culture in the flask from which the cells originate. Determine if there is enough PTT6 medium in the bag. If necessary, replace the package for the PTT6 environment with a new package for the PTT6 environment.

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 the lactate level continues to increase, the cells are harvested.

3. Collection of cells from the Quantum bioreactor

a) When the lactate concentration does not decrease, collect the cells after sampling for lactate and glucose for the last time.

b) Collection of cells:

1) Connect the cell injection bag filled with 100 ml TrypLE to the "Reagent" line using a sterile connecting tube.

2) Make sure that there is enough FSB in the FSB package. If not, connect a new bag with at least 1.7 liters of PBS to the "Flush" line using a sterile connecting tube.

3) Run the collection program

4. Cryopreservation of cells

1) After collection, the cells are transferred to a 50 ml centrifuge tube to pellet the cells.

2) Resuspend using 25 ml of cold cell suspension solution. Count the number of cells using a Sysmex or Biorad cell counter. Attach the cell count report to the appropriate Quantum batch processing protocol.

3) Bring the concentration of cells to 2×10 ⁷ /ml

4) Add equal volume of cryopreservation solution and mix well (do not shake or vortex)

5) Using a microdoser, add 1 ml of cell suspension in cryopreservation to each 1.8 ml vial. Cryopreserve using a CRF program as described in SOP D6.100 CB "Cryopreservation Using Rate Controlled Freezers"

6) Store the vials in a designated liquid nitrogen storage facility.

7) Attach the CRF run report to the form corresponding to the P3-Quantum MSC processing batch record.

3. Analysis of expression of stem cell markers in populations of lining mesenchymal stem cells isolated from umbilical cord tissue using various nutrient media

Flow cytometry experiments were performed to analyze umbilical cord-derived mesenchymal stem cells for expression of mesenchymal stem cell markers CD73, CD90, and CD 105.

For these experiments, mesenchymal stem cells were isolated from umbilical cord tissue by culturing the umbilical cord tissue in three different culture media followed by subculturing the mesenchymal stem cells in the appropriate medium as described in Example 2.

The following three culture media were used in these experiments: a) 90% (v/v) DMEM supplemented with 10% FBS (v/v), b) PTT-4 culture medium described in US Patent Application 2006/ 0078993 and the corresponding international patent application WO 2006/019357, which consists of 90% (v/v) CMRL1066 and 10% (v/v) PBS (see paragraph [0183] WO 2006/019357 and c) culture medium according to of the present invention PPT-6, the composition of which is described here. In this flow cytometry analysis, two different samples of the umbilical cord mesenchymal stem cell (CLMC) population were analyzed for each of the three culture media used.

The following protocol was used for flow cytometry analysis.

Materials and methods

Procedure

a) Isolation and culture of cells from the umbilical cord membrane

1. Explant tissue samples were incubated in a cell culture plate and immersed in the appropriate medium, then stored in a CO ₂ incubator at 37°C as described in Example 2.

2. The medium was changed every 3 days.

3. Cell growth from tissue culture explants was monitored by light microscopy.

4. At approximately 70% confluency, cells were detached from the dish by trypsinization (0.0125% trypsin/0.05% EDTA) and used for flow cytometry experiments.

b) Trypsinization of cells for experiments

1. Remove media from cell culture plate

2. Gently wash with sterile 1X PBS to remove traces of PBS as PBS will interfere with trypsin enzymatic action.

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

4. Examine the cells under a microscope to make sure they are moved. Neutralize trypsin by adding complete medium containing FBS (DMEM with 10% FBS).

5. Use a pipette to break up cell clumps by pipetting the cells in the medium against the wall of the plate. Collect and transfer the cell suspension to 50 ml centrifuge tubes.

6. Add sterile 1X PBS to the plate and wash it. Collect the cell suspension in the same centrifuge tube.

7. Centrifuge at 1800 rpm for 10 minutes.

8. Remove the supernatant and resuspend the cell pellet with PBA medium.

c) Cell count

1. Ensure that the hemocytometer and its coverslip are clean and dry, preferably by rinsing them with 70% ethanol and drying them before wiping them with Kim wipes (lint-free paper).

2. Aliquot a small amount of cells in suspension into a microcentrifuge tube and remove from the biosafety cabinet.

3. Stain cells in suspension with an equal volume of trypan blue, eg 500 µl of trypan blue is added to 500 µl of suspension (dilution factor = 2X, resulting in a 0.2% trypan blue solution).

4. Avoid exposing cells to trypan blue for more than 30 minutes because trypan blue is toxic and will increase the number of non-viable cells, resulting in an incorrect cell count.

5. Add 20 µl of the cell suspension mix to each chamber of the hemocytometer and view under a light microscope.

Count the number of viable cells (clear cells; non-viable cells readily absorb trypan blue and therefore darken) in each quadrant of the hemocytometer to give a total of 8 quadrants in the upper and lower chambers.

The total number of cells is presented as (Average number of cells/quadrant) × 10 ⁴ cells/ml.

d) Cell staining

i. Preparation before cell staining

• The cell suspension is aliquoted into 3 tubes (CD73, CD90, CD 105) in duplicate and 2 tubes (negative control) each containing 50,000 cells.

ii. Staining with primary antibody (Ab)

• Add 1 µl [0.5 mg/ml Ab] of primary antibody to 100 µl of cell suspension. Incubate at 4°C for 45 min.

• Adjust the volume of PBA to 1 ml.

• Centrifuge at 8000 rpm at 4°C for 5 minutes.

• Remove the supernatant.

• Add 1 ml PBA and resuspend pellet

• Centrifuge at 8000 rpm at 4°C for 5 minutes.

• Remove the supernatant.

• Resuspend in 100 µl PBA.

iii. Secondary Ab staining - in the dark

• Add 1 µl [0.5 mg/ml antibody] secondary antibody to 100 µl cell suspension. Incubate at 4°C for 30 min.

• Adjust the volume of PBA to 1 ml.

• Centrifuge at 8000 rpm at 4°C for 5 minutes.

• Remove the supernatant.

• Add 1 ml PBA and resuspend pellet

• Centrifuge at 8000 rpm at 4°C for 5 minutes.

• Remove the supernatant

• Resuspend in 200-300 µl PBA for flow cytometry analysis

• Transfer the cells to a FACS tube for reading the flow cytometry result on a BD FACS CANDO cytometer.

The results of the analysis by flow cytometry are shown in FIG. 6a-6c. On FIG. 6a shows the percentage of isolated lining mesenchymal stem cells expressing CD73, CD90 and CD105 stem cell markers after isolation from umbilical cord tissue and cultured in DMEM/10% PBS, FIG. 6b shows the percentage of isolated lining mesenchymal stem cells expressing CD73, CD90 and CD105 stem cell markers after isolation from umbilical cord tissue and cultured in PTT-4, and FIG. 6c shows the percentage of isolated lining mesenchymal stem cells expressing CD73, CD90 and CD105 stem cell markers after isolation from umbilical cord tissue and cultured in PPT-6. As seen from FIG. 6a, the population isolated using DMEM/10% FBS as culture medium contains approximately 75% CD73 ⁺ cells, 78% CD 90 ⁺ cells and 80% CD105 ⁺ cells (average of two experiments), while after isolation/culturing cord tissue using PPT-4 culture medium (see Fig. 6b), the number of mesenchymal stem cells that are CD73-positive, CD90-positive and CD105-positive is approximately 87% (CD73 ⁺ cells), 93%/CD90 ⁺ cells) and 86% (CD105 ⁺ cells) on average from two experiments. The purity of the population of mesenchymal stem cells obtained by cultivation in the PTT-6 medium according to the present invention is at least 99.0% with respect to all three markers (CD73, CD90, CD105), which means the purity of this cell population is much higher, than when cultured using PPT-4 medium or DMEM/10% FBS. In addition, and more importantly, the population of mesenchymal stem cells obtained by culturing in PTT-6 is essentially a 100% pure and defined population of stem cells. This makes the stem cell population of the present invention an ideal candidate for stem cell therapy. Thus, this population of lining mesenchymal stem cells may become the gold standard for such stem cell-based therapeutic approaches.

The results shown in FIG. 6 is further supported by the flow cytometric analysis results shown in FIG. 7a and Fig. 7b. On FIG. 7a shows the percentage of isolated lining mesenchymal stem cells (umbilical cord amniotic membrane mesenchymal stem cells) that express CD73, CD90 and CD 105 stem cell markers and do not express CD34, CD45 and HLA-DR after isolation from umbilical cord tissue and dilution in PTT-6 medium. . As shown in FIG. 7a, the mesenchymal stem cell population contained 97.5% viable cells, of which 100% expressed each of CD73, CD90, and CD105 (see rows "CD73+CD90+" and "CD73+CD105+"), while 99.2% of the population stem cells did not express CD45, and 100% of the stem cell population did not express CD34 and HLA-DR (see rows "CD34-CD45-" and "CD34-HLA-DR-"). Thus, the population of mesenchymal stem cells obtained by culturing in PTT-6 medium is essentially a 100% pure and defined stem cell population that meets the criteria that mesenchymal stem cells must meet for use in cell therapy (95% or more stem cell population expresses CD73, CD90, and CD105, while 98% or more of the stem cell population does not express CD34, CD45, and HLA-DR, see Sensebe et al., "Production of mesenchymal stromal/stem cells according to good manufacturing practices: a review", see above). It should be noted that true amniotic membrane mesenchymal stem cells adhere to plastic under standard culture conditions and differentiate in vitro into osteoblasts, adipocytes and chondroblasts, see US Pat. No. 9,085,755, US Pat. accepted criteria for the use of mesenchymal stem cells in cell therapy.

On FIG. 7b shows the percentage of isolated bone marrow mesenchymal stem cells that express CD73, CD90 and CD105 and do not express CD34, CD45 and HLA-DR. As shown in FIG. 7b, the bone marrow mesenchymal stem cell population contained 94.3% viable cells, of which 100% expressed each of CD73, CD90, and CD105 (see rows "CD73+CD90+" and "CD73+CD105+"), while after 62.8% of the bone marrow stem cell population lacked CD45 expression, and 99.9% of the stem cell population lacked CD34 and HLA-DR expression (see rows "CD34-CD45-" and "CD34-HLA-DR-"). Thus, bone marrow mesenchymal stem cells, which are considered the gold standard of mesenchymal stem cells, are much less homogeneous/pure in terms of stem cell markers than the population of mesenchymal stem cells (umniotic cord membrane) of the present application. This finding also shows that the population stem cells of the present invention may be an ideal candidate for stem cell based treatment and may become the gold standard for stem cell based therapeutic approaches.

It will be apparent to a person skilled in the art that various substitutions and modifications to the invention disclosed in this application can be made without departing from the scope and spirit of the invention.

All patents and publications mentioned in the description of the invention are indicative at the level of the average person in the field of technology to which the present invention pertains. All patents and publications are incorporated herein by reference to the same extent as if each individual publication were specifically and individually indicated for inclusion by reference.

The invention illustratively described in this application can be appropriately practiced in the absence of any element or elements, any limitation or limitations not specifically disclosed in this application. Thus, for example, the terms "comprising", "including", etc. should be read broadly and without limitation. In addition, the terms and expressions used herein have been used as terms of description and not limitation, and the use of such terms and expressions is not intended to exclude any equivalents to the features or parts shown and described, but it is recognized that various modifications are possible within the scope the claimed invention. Thus, it should be understood that although the present invention has been specifically disclosed by way of preferred embodiments and optional features, modifications and variations of the inventions embodied therein disclosed in this application may be used by those skilled in the art, and that such modifications and variations fall within the scope of the present invention. The invention has been described here broadly and in general terms. Each of the narrower species and subgenus groups falling within the general disclosure also forms part of the invention. This includes a general description of the invention, with a condition or negative limitation removing any subject from the genus, whether or not the excluded material has been specifically cited here. Furthermore, if features or aspects of the invention are described in terms of Markush groups, those skilled in the art will appreciate that the invention is also thus described in terms of any individual member or subgroup of members of the Markush group. Other embodiments of the invention will become apparent from the following claims.

Claims (79)

1. A method for isolating a population of mesenchymal stem cells from the amniotic membrane of the umbilical cord, including culturing umbilical cord tissue in a culture medium containing DMEM (Eagle's medium modified according to Dulbecco), F12 (Ham's F-12 medium), M171 (171 medium) and FBS (fetal bovine serum), said culture medium containing DMEM at a final concentration of approximately 55 to 65% (v/v), F12 at a final concentration of approximately 5 to 15% (v/v), M171 at a final concentration of approximately 15 to 30% (v/v) and FBS at a final concentration of approximately 1 to 8% (v/v). 2. The method according to claim 1, characterized in that said culture medium contains DMEM at a final concentration of approximately 57.5 to 62.5% (v/v), F12 at a final concentration of approximately 7.5 to 12, 5% (v/v), M171 at a final concentration of approximately 17.5 to 25.0% (v/v) and PBS at a final concentration of approximately 1.75 to 3.5% (v/v). about.). 3. The method according to claim 2, characterized in that said culture medium contains DMEM at a final concentration of approximately 61.8% (v/v), F12 at a final concentration of approximately 11.8% (v/v), M171 at a final concentration of approximately 23.6% (v/v) and PBS at a final concentration of approximately 2.5% (v/v). 4. The method according to any one of paragraphs. 1-3, characterized in that said culture medium additionally contains epidermal growth factor (EGF) at a final concentration of from about 1 ng/ml to about 20 ng/ml. 5. The method according to any one of paragraphs. 1-4, characterized in that said culture medium contains EGF at a final concentration of approximately 10 ng/ml. 6. The method according to any one of paragraphs. 1-5, characterized in that said culture medium contains insulin at a final concentration of approximately 1 μg/ml to 10 μg/ml. 7. The method according to any one of paragraphs. 1-6, characterized in that said culture medium contains insulin at a final concentration of approximately 5 μg/ml. 8. A method according to any one of the preceding claims, characterized in that said culture medium further comprises at least one of the following additives: adenine, hydrocortisone and 3,3',5-triiodo-L-thyronine (T3) sodium salt. 9. A method according to any one of the preceding claims, characterized in that said culture medium contains all three of adenine, hydrocortisone and 3,3',5-triiodo-L-thyronine (T3) sodium salt. 10. The method according to p. 9, characterized in that the specified culture medium contains adenine at a final concentration of from about 0.01 to about 0.1 µg/ml of adenine, hydrocortisone at a final concentration of from about 0.1 to about 10 µg/ml hydrocortisone and/or 3,3',5-triiodo-L-thyronine (T3) sodium salt at a final concentration of about 0.5 to about 5 ng/ml. 11. The method of any one of the preceding claims, comprising culturing umbilical cord tissue until the amniotic membrane mesenchymal stem cell outgrowth reaches approximately 70-80% confluence. 12. The method of claim 11, comprising recovering the mesenchymal stem cells from the culture container used for culture. 13. The method according to claim 12, characterized in that the extraction of mesenchymal stem cells from the culture container is carried out by enzymatic treatment. 14. The method according to p. 13, characterized in that the enzymatic treatment includes trypsinization. 15. The method according to any one of paragraphs. 12-14, characterized in that the mesenchymal stem cells are transferred for subculturing into a culture container for subculturing. 16. The method according to claim 15, characterized in that the mesenchymal cells are suspended for subculturing at a concentration of 1.0×10 ⁶ cells/ml. 17. The method according to p. 16, characterized in that the mesenchymal stem cells are subcultivated in a culture medium as defined in any of paragraphs. 1-9. 18. The method of claim 17, wherein the mesenchymal stem cells are subcultured until the mesenchymal stem cells reach approximately 70-80% confluence. 19. The method according to any one of paragraphs. 15-18, characterized in that subculturing is carried out in an autonomous bioreactor. 20. The method according to claim 19, characterized in that the bioreactor is selected from the group consisting of a parallel plate bioreactor, a hollow fiber bioreactor, and a microfluidic bioreactor. 21. The method according to any one of the preceding claims, characterized in that the umbilical cord tissue is a piece of the entire umbilical cord or umbilical cord amniotic membrane. 22. The method according to any one of the preceding claims, characterized in that the cultivation is carried out in a cell culture incubator with CO ₂ at a temperature of 37°C. 23. The method of claim 22, comprising recovering mesenchymal stem cells from a culture container used for subculturing. 24. The method according to p. 23, characterized in that the extraction of mesenchymal stem cells from the culture container is carried out by enzymatic treatment. 25. The method according to p. 24, characterized in that the enzymatic treatment includes trypsinization. 26. The method of claim 25, further comprising collecting isolated mesenchymal stem cells. 27. The method of any 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 isolated mesenchymal stem cells express the following markers: CD73, CD90 and CD105. 28. 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 isolated mesenchymal stem cells do not express the following markers: CD34, CD45 and HLA-DR (leukocyte antigen human - D-related antigen). 29. The method according to any one of paragraphs. 27 or 28, characterized in that about 97% or more, about 98% or more, about 99% or more of the isolated mesenchymal stem cells express CD73, CD90 and CD105 and do not express CD34, CD45 and HLA-DR. 30. A method according to any one of the preceding claims, further comprising preserving the isolated stem/progenitor cells for further use. 31. The method according to p. 30, characterized in that the preservation is carried out by cryopreservation. 32. An isolated umbilical cord amniotic membrane mesenchymal stem cell population for cell therapy, wherein at least about 97% or more of the cells in the isolated stem cell population express each of the following markers: CD73, CD90, and CD105 and do not express each of CD34, CD45, and HLA -DR. 33. The population of mesenchymal stem cells according to claim 32, characterized in that at least about 98% or more, about 99% or more of the cells of the isolated population of mesenchymal stem cells express each of CD73, CD90 and CD105 and do not express each of CD34, CD45 and HLA-DR. 34. The population of mesenchymal stem cells according to any one of paragraphs. 32, 33, characterized in that the population can be obtained by the method defined in any of paragraphs. 1-29. 35. The population of mesenchymal stem cells according to any one of paragraphs. 32, 33, characterized in that the population obtained by the method defined in any of paragraphs. 1-29. 36. Pharmaceutical composition for cell therapy, containing an isolated population of mesenchymal stem cells of the amniotic membrane of the umbilical cord, characterized in that at least 97% or more cells of the stem cell population express each of the following markers: CD73, CD90 and CD105 and do not express each of the following markers: CD34, CD45 and HLA-DR. 37. Pharmaceutical composition according to claim 36, characterized in that the pharmaceutical composition is adapted for systemic or topical use. 38. A pharmaceutical composition according to claim 36 or 37, further comprising a pharmaceutically acceptable excipient. 39. A method for obtaining a culture medium suitable for isolating a population of mesenchymal stem cells from the amniotic membrane of the umbilical cord, including mixing the following components: i. 250 ml DMEM, ii. 118 ml M171, iii. 118 ml DMEM/F12, iv. 12.5 ml of fetal bovine serum (FBS) (final concentration 2.5%) to obtain a volume of 498.5 ml of culture medium. 40. The method of claim 39, further comprising adding v. 1 ml EGF stock solution (5 µg/ml) to achieve a final concentration of 10 ng/ml), vi. 0.175 ml stock insulin solution (14.28 mg/ml) to a final concentration of 5 µg/ml. 41. The method of claim 40, further comprising adding one or more of the following additives to DMEM: adenine, hydrocortisone, 3,3',5-triiodo-L-thyronine (T3) sodium salt to obtain a total volume of 500 ml of culture medium. 42. The method according to p. 41, characterized in that the final concentration of additives in DMEM is as follows: about 0.05 to 0.1 µg/ml adenine, e.g. about 0.025 µg/ml adenine, approximately 1 to 10 mcg/ml hydrocortisone, approximately 0.5 to 5 ng/ml 3,3',5-triiodo-L-thyronine sodium (T3), e.g. 1.36 ng/ml 3,3',5-triiodo-L-thyronine sodium ( T3). 43. Cell culture medium, which can be obtained by the method according to any one of paragraphs. 39-42. 44. A method for isolating mesenchymal stem cells from the amniotic membrane of the umbilical cord, including culturing tissue of the amniotic membrane in a culture medium prepared by the method defined in any of paragraphs. 39-42. 45. Cell culture medium containing: - DMEM at a final concentration of approximately 55 to 65% (v/v), - F12 at a final concentration of approximately 5 to 15% (v/v), - M171 at a final concentration of approximately 15 to 30% (v/v) and - PBS at a final concentration of approximately 1 to 8% (v/v). 46. Cell culture medium according to claim 45, characterized in that said culture medium contains DMEM at a final concentration of approximately 57.5 to 62.5% (v/v), F12 at a final concentration of approximately 7.5 to 12.5% (v/v), M171 at a final concentration of approximately 17.5 to 25.0% (v/v), and PBS at a final concentration of approximately 1.75 to 3.5% (v/v). ./about.). 47. Cell culture medium according to claim 46, characterized in that said culture medium contains DMEM at a final concentration of approximately 61.8% (v/v), F12 at a final concentration of approximately 11.8% (v/v). ), M171 at a final concentration of approximately 23.6% (v/v) and PBS at a final concentration of approximately 2.5% (v/v). 48. Cell culture medium according to any one of paragraphs. 45-47, characterized in that said culture medium additionally contains epidermal growth factor (EGF) at a final concentration of from about 1 ng/ml to about 20 ng/ml. 49. Cell culture medium according to any one of paragraphs. 45-48, characterized in that said culture medium contains EGF at a final concentration of approximately 10 ng/ml. 50. Cell culture medium according to any one of paragraphs. 45-49, characterized in that said culture medium contains insulin at a final concentration of approximately 1 μg/ml to 10 μg/ml. 51. A cell culture medium according to claim 50, characterized in that said culture medium contains insulin at a final concentration of approximately 5 μg/ml. 52. Cell culture medium according to any one of paragraphs. 45-51, characterized in that said culture medium additionally contains at least one of the following additives: adenine, hydrocortisone and sodium salt of 3,3',5-triiodo-L-thyronine (T3). 53. A cell culture medium according to claim 52, characterized in that said culture medium includes all three of the following additives: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine (T3) sodium salt. 54. Cell culture medium according to claim 52 or 53, characterized in that said culture medium contains adenine at a final concentration of from about 0.05 to about 0.1 µg/ml of adenine, hydrocortisone at a final concentration of from about 1 to about 10 µg /ml of hydrocortisone and/or sodium salt of 3,3',5-triiodo-L-thyronine (T3) at a final concentration of from about 0.5 to about 5 ng/ml. 55. Cell culture medium according to any one of paragraphs. 45-54, characterized in that 500 ml of this cell culture medium contains: i. 250 ml of DMEM, ii. 118 ml M171, iii. 118 ml DMEM/F12, iv. 12.5 ml fetal bovine serum (FBS) (final concentration 2.5%). 56. Cell culture medium according to claim 55, additionally containing v. EGF at a final concentration of 10 ng / ml, vi. insulin at a final concentration of 5 μg / ml, vii. insulin 0.175 ml (final concentration 5 µg/ml). 57. Cell culture medium according to claim 55 or 56, additionally containing adenine at a final concentration of from about 0.05 to about 0.1 μg / ml of adenine, hydrocortisone at a final concentration of from about 1 to about 10 μg / ml of hydrocortisone and / or 3,3',5-triiodo-L-thyronine (T3) sodium salt at a final concentration of about 0.5 to about 5 ng/mL. 58. The use of cell culture medium according to any one of paragraphs. 45-57 for the isolation of mesenchymal stem cells from the amniotic membrane of the umbilical cord. 59. The use of cell culture medium according to any one of paragraphs. 45-57 for culturing mesenchymal stem cells from the amniotic membrane of the umbilical cord.

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