Mesenchymal stem cell storage or transport preparation, and method for preparing and using the same.

Abstract

The present disclosure relates to a mesenchymal stem cell storage or transport formulation, its preparation, and methods of use. The present disclosure also includes a method for treating a subject with a disease, such as a wound, by locally administering mesenchymal stem cells stored or transported in the storage or transport formulation. The present disclosure also relates to a unit dosage of mesenchymal stem cells. In a preferred embodiment, the formulation comprises HypoThermosol®, human serum albumin, and Plasmalyte. TIFF2022552230000007.tif98170

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 62 / 912,368, filed on October 8, 2019, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] Array List This application contains an array list in computer - readable form, which is incorporated herein by reference.

[0003] Field of the Invention The present invention relates to a mesenchymal stem cell storage or transport formulation, a method for preparing a mesenchymal stem cell storage or transport formulation, and a method for using a mesenchymal stem cell storage or transport formulation. Such methods include a method for transporting mesenchymal stem cells in this storage or transport formulation, and a method for treating a subject having a disease, including the step of locally administering the mesenchymal stem cells stored or transported in this storage or transport formulation. It also relates to the unit dosage of mesenchymal stem cells.

Background Art

[0004] Background of the Invention Mesenchymal stem cells isolated from the amniotic membrane of the umbilical cord and their wound-healing properties were first reported in U.S. Patent Application 2006 / 0078993 (Patent Document 1) (leading to registered U.S. Patents 9,085,755 (Patent Document 2), 9,737,568 (Patent Document 3), and 9,844,571 (Patent Document 4)) and the corresponding International Patent Application WO2006 / 019357 (Patent Document 5). Since then, umbilical cord tissue has attracted attention as a source of pluripotent cells; stem cells isolated from the umbilical cord, and specifically from the amniotic membrane of the umbilical cord (also referred to as "cord lining stem cells"), are widely available and are therefore 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 (Non-Patent Document 1). On the other hand, such a population of mesenchymal stem cells from the amniotic membrane of the umbilical cord is described in U.S. Patent Application 2018 / 127721 (Patent Document 6) or the corresponding International Patent Application WO2018 / 067071 (Patent Document 7).

[0005] The mesenchymal stem cell population described in U.S. Patent Application 2018 / 127721 (Patent Document 6) or the corresponding International Patent Application WO2018 / 067071 (Patent Document 7) has the advantage that more than 99% of the stem cells in this population express three MSC markers CD73, CD90, and CD105, while lacking the expression of CD34, CD45, and HLA-DR. Therefore, such extremely homogeneous and clearly defined cell populations are, for example, described in 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 (Non-patent Literature 2), Sensebe et al, "Production of mesenchymal stromal / stem cells according to good manufacturing practices: a, review," Stem Cell Research & Therapy 2013, 4:66 (Non-patent Literature 3), Vonk et al., Stem Cell Research & Therapy (2015) 6:94 (Non-patent Literature 4), or Kundrotas Acta Medica Lituanica. 2012. Vol. 19. No. 2. P. This population of mesenchymal stem cells is an ideal candidate for clinical trials and cell-based therapies because it fully meets the generally accepted standards for human MSCs to be used in cell therapies, as defined in 75-79 (Non-Patent Document 5). As described in international patent application WO2018 / 067071 (Patent Document 7), this population of mesenchymal stem cells can be used in an undifferentiated state for wound healing purposes, such as in the treatment of burns.

[0006] However, stem cells, such as the mesenchymal stem cells mentioned above, are not typically applied / administered to patients at the site where they are generated. Often, a considerable amount of time elapses between cell harvesting and their further use. Therefore, there is a need for storage or transport formulations that maintain the cells viable and healthy over the period typically used for cell transport or storage.

[0007] Therefore, the object of the present invention is to provide a formulation suitable for the storage and / or transport of mesenchymal stem cells that satisfies this need. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] U.S. Patent Application 2006 / 0078993 [Patent Document 2] U.S. Patent No. 9,085,755 [Patent Document 3] U.S. Patent No. 9,737,568 [Patent Document 4] U.S. Patent No. 9,844,571 [Patent Document 5] WO2006 / 019357 [Patent Document 6] U.S. Patent Application 2018 / 127721 [Patent Document 7] WO2018 / 067071 [Non-patent literature]

[0009] [Non-Patent Document 1] Jeschke et al. Umbilical Cord Lining Membrane and Wharton's Jelly-Derived Mesenchymal Stem Cells: the Similarities and Differences; The Open Tissue Engineering and Regenerative Medicine Journal, 2011, 4, 21-27 [Non-Patent Document 2] 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 [Non-Patent Document 3] Sensebe et al,. “Production of mesenchymal stromal / stem cells according to good manufacturing practices: a, review”, Stem Cell Research & Therapy 2013, 4:66 [Non-Patent Document 4] Vonk et al., Stem Cell Research & Therapy (2015) 6:94 [Non-Patent Document 5] Kundrotas Acta Medica Lituanica. 2012. Vol. 19. No. 2. P. 75-79 [Overview of the project]

[0010] This objective is achieved by a method having the features of an independent claim, a mesenchymal stem cell storage or transport formulation, and a unit dose.

[0011] In the first phase, the present invention relates to a method for preparing a mesenchymal stem cell storage or transport formulation, The preparation contains approximately 500,000 to 10 million mesenchymal stem cells. a) Suspending mesenchymal stem cells in a predetermined volume of crystalloid solution containing approximately 0.5% or approximately 1% to approximately 5% (w / v) serum albumin, thereby obtaining a first cell suspension. b) A step of determining the concentration of mesenchymal stem cells in the first cell suspension and determining the volume of the first cell suspension required to prepare a formulation containing approximately 500,000 to 10,000,000 mesenchymal stem cells. c) The determined volume of the first cell suspension Approximately 0.5% or approximately 1% to approximately 5% (w / v) of serum albumin, as well as the following: i) Torolox, ii) Na + , iii) K + , iv) Ca 2+ , v) Mg 2+ , vi) Cl - , vii) H2PO4 - , viii) HEPES, ix) Lactobionate, x) Sucrose, xi) Mannitol, xii) Glucose, xiii) Dextran-40, xiv) Adenosine, and xv) Glutathione A liquid carrier of a certain volume containing The step of mixing with the mesenchymal stem cell storage or transport preparation containing approximately 500,000 to 10,000,000 mesenchymal stem cells. This provides a method that includes [something].

[0012] In the second aspect, the present invention provides a mesenchymal stem cell storage or transport formulation obtained by a method specified herein.

[0013] In a third aspect, the present invention provides a mesenchymal stem cell storage or transport formulation obtainable by a method specified herein.

[0014] In the fourth aspect, the present invention provides a method for transporting mesenchymal stem cells, comprising the step of transporting the mesenchymal stem cells in a mesenchymal stem cell storage or transport formulation as defined herein.

[0015] In the fifth aspect, the present invention provides a method for treating a subject having a disease, comprising the step of locally administering mesenchymal stem cells stored or transported in a mesenchymal stem cell storage or transport formulation as defined herein.

[0016] In the sixth aspect, the present invention provides a unit dose of mesenchymal stem cells that can be obtained by the method specified herein. [Brief explanation of the drawing]

[0017] The present invention will be better understood by referring to the detailed description, in conjunction with non-limiting embodiments and drawings.

[0018] [Figure 1-1] Figure 1 shows Lonza's technical information sheet for Dulbecco's Modified Eagle Medium, including the catalog number of the DMEM used to prepare an example of the culture medium (PTT-6) of the present invention in the experimental section. [Figure 1-2] See the explanation in Figure 1-1. [Figure 2] This shows Lonza's technical information sheet regarding Ham F12 culture medium. [Figure 3]The Lonza technical information sheet for DMEM:F12 (1:1) medium, including the catalog number of the DMEM:F12 (1:1) medium used to prepare an example of the culture medium (PTT-6) of the present invention in the Experiment section, is shown below. [Figure 4-1] Figure 4 shows the Life Technologies Corporation technical information sheet for M171 medium, including the catalog number of M171 medium used to prepare an example of the culture medium (PTT-6) of the present invention in the experimental section. [Figure 4-2] See the explanation in Figure 4-1. [Figure 5] The following is a list of the components used in the experiment to prepare medium PTT-6, including their commercial suppliers and catalog numbers. When medium PTT-6 is to be used in GMP manufacturing, it should not contain antibiotic reagents in accordance with the U.S. FDA's guidelines for the manufacture of biologics. [Figure 6A]Figure 6 shows the results of flow cytometry experiments analyzing the expression of mesenchymal stem cells isolated from umbilical cord tissue for the mesenchymal stem cell markers CD73, CD90, and CD105. For these experiments, mesenchymal stem cells were isolated from umbilical cord tissue by culturing the tissue in three different culture media, and then the mesenchymal stem cells were subcultured in each medium. In these experiments, the following three culture media were used: a) 90% (v / v / DMEM) supplemented with 10% FBS (v / v), b) culture medium PTT-4 as described in U.S. Patent Application 2006 / 0078993 and the corresponding International Patent Application WO2006 / 019357 (see paragraph

[0183] of WO2006 / 019357), consisting of 90% (v / v) CMRL1066 and 10% (v / v) FBS, and c) culture medium PTT-6 of the present invention, whose composition is described herein. In this flow cytometry analysis, two different samples of umbilical cord-lined mesenchymal stem cell (CLMC) populations were analyzed for each of the three culture media used. The results are shown in Figures 6a-6c. More specifically, Figure 6a shows the percentage of isolated mesenchymal umbilical cord-lining stem cells expressing the stem cell markers CD73, CD90, and CD105 after isolation and culture from umbilical cord tissue in DMEM / 10% FBS; Figure 6b shows the percentage of isolated mesenchymal umbilical cord-lining stem cells expressing the stem cell markers CD73, CD90, and CD105 after isolation and culture from umbilical cord tissue in PTT-4; and Figure 6c shows the percentage of isolated mesenchymal umbilical cord-lining stem cells expressing the stem cell markers CD73, CD90, and CD105 after isolation and culture from umbilical cord tissue in PTT-6. [Figure 6B] See the explanation in Figure 6A. [Figure 6C] See the explanation in Figure 6A. [Figure 7A]Figure 7 shows the results of flow cytometry experiments analyzing the expression of stem cell markers CD73, CD90, and CD105, CD34, CD45, and HLA-DR (human leukocyte antigen-antigen D-related), which are used to determine the suitability of multipotent human mesenchymal stem cells for cell therapy, in mesenchymal stem cells isolated from umbilical cord, and comparing them with the expression of these markers in bone marrow mesenchymal stem cells. For this experiment, mesenchymal stem cells from the amniotic membrane of the umbilical cord were isolated from umbilical cord tissue by culturing umbilical cord tissue in the culture medium PTT-6 of the present invention, while bone marrow mesenchymal stem cells were isolated from human bone marrow using a standard protocol. Figure 7a shows the percentage of isolated mesenchymal umbilical cord-lining stem cells that express the stem cell markers CD73, CD90, and CD105 and lack the expression of CD34, CD45, and HLA-DR after isolation and culture from umbilical cord tissue in PTT-6 medium. Figure 7b shows the percentage of isolated bone marrow mesenchymal stem cells that express CD73, CD90, and CD105 and lack the expression of CD34, CD45, and HLA-DR. [Figure 7B] See the explanation in Figure 7A. [Figure 8] The experimental setup for comparing different carriers is described below. First, the mesenchymal stem cell population described herein was grown in cell culture flasks. The amount of viable mesenchymal stem cells was counted, and then 2 million cells / vial were stored for varying periods in either PlasmaLyte-A or HypoThermosol®-FRS. After storage, cells were counted daily from day 1 to day 5 in ≤50 μl samples (total liquid volume 250 μl), and viability was examined by staining the cells with trypan blue. Furthermore, ≤80 μl samples were collected and analyzed on days 1, 3, and 5. After storage on days 1, 3, and 5, 100,000 MSCs at each time point were cultured in PTT-6 medium for 48 hours, and the supernatant obtained for cytokine assays of PDGF-AA, PDGF-BB, VEGF, IL-10, Ang-1, HGF, and TGFβ1 was measured using a FLEXMAP 3D system. [Figure 9]The survival data is summarized below. As can be seen from the graph on the left, after 7 days of storage in HypoThermosol®, 73% of the total number of cells at the start of storage (approximately 95%) were still viable. In contrast, after 7 days of storage in PlasmaLyte-A, only 42% of the total number of cells at the start of storage (approximately 94%) were still viable. All counts are based on paired readings within 10% of each other (according to SOP CR D2.600.1). Among the counts, cells stored in HypoThermosol® were significantly smaller, with smoother and more defined contours. In contrast, cells in Plasmalyte-A appeared in a variety of sizes. HypoThermosol® significantly supports membrane integrity and possibly survival over a 6-day period. Similar results are also shown in the graph on the right. [Figure 10] The results obtained when measuring the cell diameter of the cells are shown. The mesenchymal stem cell population described herein, when maintained in HypoThermosol®, exhibits a narrower diameter range compared to cells maintained in PlasmaLyteA. The comparison was made after 3 days of storage. [Figure 11] This graph shows the TGFβ1 concentration in the supernatant 48 hours after storage from the mesenchymal stem cell populations described herein, stored in HypoThermosol® or PlasmaLyte-A. As can be seen from the graph on the right, cells secrete approximately the same amount of TGFβ1 when stored in HypoThermosol® as when stored in PlasmaLyte-A. Generally, the amount of TGFβ1 secreted decreased over time (graph on the right). [Figure 12] A control experiment is shown. Here, PDGF-BB concentrations were measured in the supernatant after 48 hours from the mesenchymal stem cell populations described herein, stored in HypoThermosol® or PlasmaLyte-A. Since PDGF-BB is not normally secreted by the mesenchymal stem cell populations described herein, PDGF-BB was undetectable in any of the samples. [Figure 13]A control experiment is shown. Here, IL-10 concentrations were measured in the supernatant after 48 hours from the mesenchymal stem cell populations described herein, stored in HypoThermosol® or PlasmaLyte-A. Since IL-10 is not normally secreted by the mesenchymal stem cell populations described herein, IL-10 was undetectable in any of the samples. [Figure 14] This graph shows the VEGF concentrations in the supernatant after 48 hours from the mesenchymal stem cell populations described herein, stored in HypoThermosol® or PlasmaLyte-A. As can be seen from the graph on the right, on day 0, cells secrete approximately the same amount of VEGF when stored in HypoThermosol® or PlasmaLyte-A. On days 1 and 5, cells secreted more VEGF when stored in PlasmaLyte-A. Notably, after 3 days of storage, cells secreted more VEGF when stored in HypoThermosol® than when stored in PlasmaLyte-A. Therefore, up to day 3 of storage, HypoThermosol® is superior to PlasmaLyte-A. The more VEGF detected, the healthier the culture. Therefore, cells were healthier in HypoThermosol (trademark) than in PlasmaLyte-A after 3 days of storage, secreting more VEGF than those stored in PlasmaLyte-A. From day 5 onwards, storage in PlasmaLyte appears to be more advantageous, because at that point, cells stored in PlasmaLyte-A secreted more VEGF. Generally, the amount of secreted VEGF decreased over time (graph on the right). [Figure 15]This graph shows the PDGF-AA concentrations in the supernatant after 48 hours from the mesenchymal stem cell populations described herein, stored in HypoThermosol® or PlasmaLyte-A. As can be seen from the graph on the right, on day 0, cells stored in HypoThermosol® secrete approximately the same amount of PDGF-AA as those stored in PlasmaLyte-A. On days 1 and 5, cells stored in PlasmaLyte-A secreted more PDGF-AA. Notably, after 3 days of storage, cells stored in HypoThermosol® secreted more PDGF-AA than those stored in PlasmaLyte-A. Therefore, after 3 days of storage, cells stored in HypoThermosol® are healthier than cells stored in PlasmaLyte-A. After 5 days of storage, PlasmaLyte appears to become a more favorable carrier because, at that point, cells stored in PlasmaLyte-A secrete more PDGF-AA. Generally, the amount of secreted PDGF-AA decreased over time (graph on the right). [Figure 16]This graph shows the Ang-1 concentration in the supernatant of the mesenchymal stem cell populations described herein, stored in HypoThermosol® or PlasmaLyte-A, over a 48-hour period. As can be seen from the graph on the right, cells secrete approximately the same amount of Ang-1 on days 0 and 3 when stored in HypoThermosol® or PlasmaLyte-A. On day 5, cells secreted more Ang-1 when stored in PlasmaLyte-A. Notably, after 1 day of storage, cells secreted significantly more Ang-1 when stored in HypoThermosol® than when stored in PlasmaLyte-A. Therefore, cells stored in HypoThermosol® appear to be healthier than those stored in PlasmaLyte-A, at least for 48 hours up to 3 days of storage. From day 5 onwards, PlasmaLyte appears to become a more favorable carrier because, at this point, cells stored in PlasmaLyte-A secrete more Ang-1. Generally, the amount of Ang-1 secreted decreased over time (graph on the right). [Figure 17]This graph shows the HGF concentrations in the supernatant 48 hours after storage from the mesenchymal stem cell populations described herein, stored in HypoThermosol® or PlasmaLyte-A. As can be seen from the graph on the right, on day 0, cells stored in HypoThermosol® secrete approximately the same amount of HGF as those stored in PlasmaLyte-A. On days 3 and 5, cells secreted more HGF when stored in PlasmaLyte-A. Notably, after 1 day of storage, cells stored in HypoThermosol® secreted significantly more HGF than those stored in PlasmaLyte-A. Therefore, cells stored in HypoThermosol® appear to be healthier than cells stored in PlasmaLyte-A for at least 1 day (48 hours) up to 3 days of storage. From day 3 onward, PlasmaLyte-A appears to become a more favorable carrier because, at days 3 and 5, cells stored in PlasmaLyte-A secreted more HGF. Generally, the amount of HGF secreted decreased over time (graph on the right). [Figure 18-1]Figure 18 shows a photograph obtained from a preclinical trial using the mesenchymal stem cell population of the present invention in pigs. Pigs were induced with diabetes using 120 mg / kg streptozotocin, allowed to recover for 45 days, and then six 5 cm × 5 cm full-thickness wounds were created on their backs. Two pigs (n = 2) were treated twice a week for four weeks with 105 human mesenchymal stem cells per cm² as described herein. Two control pigs were treated with PBS. Wounds were photographed on postoperative day 0 (PO day 0) and every seven days until postoperative day 35. Wounds were analyzed for surface area size using ImageJ. By day 35, the addition of the mesenchymal stem cell population described herein resulted in closure of 10 out of 12 diabetic wounds (83%), compared to only 3 out of 12 (25%) in the PBS-treated control wounds. The wound healing rate was 0.8 cm² / day using the mesenchymal stem cell population described herein, compared to 0.6 cm² / day in control animals, representing a 33% improvement. [Figure 18-2] See the explanation in Figure 18-1. [Figure 18-3] See the explanation in Figure 18-1. [Figure 18-4] See the explanation in Figure 18-1. [Figure 18-5] See the explanation in Figure 18-1. [Figure 19-1] Figure 19 shows the Torolox datasheet available from Tocris. [Figure 19-2] See the explanation in Figure 19-1. [Figure 20-1] Figure 20 shows the datasheet for NaCl available from Sigma Aldrich. [Figure 20-2] See the explanation in Figure 20-1. [Figure 21] The datasheet for KH2PO4, available from Sigma Aldrich, is shown below. [Figure 22-1] Figure 22 shows the HEPES datasheet from Sigma Aldrich. [Figure 22-2] See the explanation in Figure 22-1. [Figure 23-1]Figure 23 shows the product sheet for sodium lactobionate from COMBI-BLOCKS. [Figure 23-2] See the explanation in Figure 23-1. [Figure 23-3] See the explanation in Figure 23-1. [Figure 24-1] Figure 24 shows the product sheet for sucrose from Sigma Aldrich. [Figure 24-2] See the explanation in Figure 24-1. [Figure 24-3] See the explanation in Figure 24-1. [Figure 24-4] See the explanation in Figure 24-1. [Figure 24-5] See the explanation in Figure 24-1. [Figure 25-1] Figure 25 shows the product sheet for mannitol from avantor. [Figure 25-2] See the explanation in Figure 25-1. [Figure 25-3] See the explanation in Figure 25-1. [Figure 25-4] See the explanation in Figure 25-1. [Figure 26] This shows the product sheet for glucose from Sigma Aldrich. [Figure 27-1] Figure 27 shows the product sheet for dextran-40 from Sigma Aldrich. [Figure 27-2] See the explanation in Figure 27-1. [Figure 27-3] See the explanation in Figure 27-1. [Figure 27-4] See the explanation in Figure 27-1. [Figure 28] This shows the product sheet for adenosine from Sigma Aldrich. [Figure 29] This shows the product sheet for glutathione from Sigma Aldrich. [Figure 30] This shows the product sheet for HypoThermosol®-FRS (HTS-FRS) from STEMCELL Technologies. [Figure 31-1] Figure 31 shows the product sheet for CaCl from Sigma Aldrich. [Figure 31-2] See the explanation in Figure 31-1. [Figure 31-3] See the explanation in Figure 31-1. [Figure 31-4] See the explanation in Figure 31-1. [Figure 31-5] See the explanation in Figure 31-1. [Figure 31-6] See the explanation in Figure 31-1. [Figure 31-7] See the explanation in Figure 31-1. [Figure 32] This shows the product sheet for MgCl from Sigma Aldrich. [Figure 33-1]Figure 33 shows the results of stability tests conducted over up to 3 days in a population of umbilical cord-lined mesenchymal stem cells seeded in the formulation of the present invention (Plasmalyte / HSA / HypoThermosol) as described herein. Figure 33a shows the results of MSC viability tests after storage in the formulation of the present invention. To mimic the transport and storage of the product before application to a wound, MSCs were stored at 2–8°C for 1–3 days. The results show that the cells did not show a significant decrease in viability up to 3 days under these conditions. Figure 33b shows the morphology of MSCs after storage at 2–8°C in the formulation of the present invention. MSCs were removed from an Aseptic Technologies (AT)-Closed Vial and cultured at 37°C for 24 hours before being photographed. As can be seen, cells obtained by 2 days under low-temperature storage were able to adhere to tissue culture plates and form a typical spindle shape. After storage at 2–8°C for 2.5 days, the cells gradually became spherical, suggesting that they were dying cells. Figure 33c shows the proliferation and metabolism of MSCs after storage in the formulation of the present invention. MSCs from the same culture analyzed in Figure 33a were assayed for lactate production as an indicator of metabolism and growth over 48 hours in culture at 37°C. Lactate is a product of glucose metabolism, and the inventors have verified that it is directly proportional to the rate of cell growth of MSCs. Cells stored at 2–8°C for 24 hours showed equivalent metabolism and growth to cells stored for 0 hours, and cells stored for 36 hours showed 86% of the control lactate production. Up to 72 hours at 2–8°C, cells showed only about 46% of the metabolism when subsequently cultured. Figure 33d shows lactate production by MSCs stored in the formulation of the present invention for 0, 1, 1.5, 2, 2.5, or 3 days, and then measured at 24 and 48 hours after culture. It can be seen that lactate production at 24 and 48 hours (day 1) by MSCs stored in the formulation of the present invention was the same as that of unstored MSCs (day 0). By day 3, lactate production had decreased by 40-45%. Figure 33e shows cytokine production measured at 24 hours at 37°C from the same culture analyzed in Figure 33c.Consistent with metabolic data, when cells were stored in the formulation of the present invention at 2-8°C for 24 hours, the ability of MSCs to produce angiopoietin-1 (Ang-1), transforming growth factor beta (TGF-β), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) was within 10-20% of that of the control (day 0). Figure 33f shows cytokine production measured from a different culture after 24 hours. The results indicate that when cells were stored in the formulation of the present invention at 2-8°C for 24 hours, the ability of MSCs to produce VEGF, angiopoietin-1, TGF-β, and HGF was maintained, but decreased by approximately 50% after storage for >2 days. [Figure 33-2] See the explanation in Figure 33-1. [Figure 33-3] See the explanation in Figure 33-1. [Figure 33-4] See the explanation in Figure 33-1. [Figure 33-5] See the explanation in Figure 33-1. [Figure 33-6] See the explanation in Figure 33-1. [Modes for carrying out the invention]

[0019] Detailed description of the invention As described above, in the first phase, the present invention relates to a method for preparing a mesenchymal stem cell storage or transport preparation, The preparation contains approximately 500,000 to 10 million mesenchymal stem cells. a) A step of suspending mesenchymal stem cells in a predetermined volume of crystalloid solution containing approximately 0.5 to 5% (w / v) serum albumin, thereby obtaining a first cell suspension. b) A step of determining the concentration of mesenchymal stem cells in the first cell suspension and determining the volume of the first cell suspension required to prepare a formulation containing approximately 500,000 to 10,000,000 mesenchymal stem cells. c) The determined volume of the first cell suspension Approximately 0.5 to 5% (w / v) of serum albumin, as well as the following: i) Torolox, ii) Na + 、 iii) K + 、 iv) Ca 2+ 、 v) Mg 2+ 、 vi) Cl - 、 vii) H2PO4 - 、 viii) HEPES、 ix) lactobionate, x) sucrose, xi) mannitol, xii) glucose, xiii) dextran - 40, xiv) adenosine, and xv) glutathione in a certain volume of liquid carrier and mixing them to obtain the mesenchymal stem cell storage or transport preparation containing about 500,000 to about 10 million mesenchymal stem cells A method comprising the above is targeted.

[0020] It has been surprisingly found in this application that using the mesenchymal stem cell storage or transport formulations described herein stabilizes the proliferation and metabolism of MSCs during storage / transport, improving MSC viability for up to 72 hours. For example, after storing mesenchymal stem cells in the mesenchymal stem cell storage or transport formulations of the present invention for 3 days, approximately 90% of the cells remained viable (see Figure 33a). Conversely, after storing in PlasmaLyte® for 3 days, only approximately 66% of the cells remained viable (see the example measured by hemocytometer and Figure 9). Thus, using the mesenchymal stem cell storage or transport formulations described herein makes it possible to transport / store stem cells for a period of time without substantially losing cell viability. In particular, as described in detail in the Experiments section, stem cells generally secreted more factors than after storage in PlasmaLyte-A, so storage in the mesenchymal stem cell storage or transport formulations of the present invention for shorter periods of 3 days or less is considered particularly beneficial. Furthermore, it has been surprisingly found that using the mesenchymal stem cell storage or transport formulations described herein makes it possible to recover more than 95% of the MSCs from the storage / transport container, thereby ensuring that the desired dose of cells can be administered to the patient.

[0021] As used herein, the terms “transport” or “transport” mean any transport. Such transport may be carried out by any vehicle, such as automobiles, trains, and airplanes, or by a person transporting / transporting a container containing stem cells in contact with a liquid carrier from one place to another. In one embodiment, transport is carried out from the place where the mesenchymal stem cells of interest (or, since both terms are used interchangeably herein, a population of mesenchymal stem cells) are produced to the place where the stem cells are administered (e.g., from a GMP facility where the stem cells or population of stem cells of interest are produced to the place where the stem cells or population of stem cells are administered, e.g., a clinic or medical office). However, the term “transport” may also refer to the storage of cells over a period of time in the same location. For example, stem cells may be stored after collection until they are applied to a subject at a certain location. The container on which stem cells can be stored or transported may be any container suitable for the method of the present invention.

[0022] The preparation of a mesenchymal stem cell storage or transport formulation includes the step of resuspending MSCs in a predetermined volume of crystalloid solution. In this invention, any volume of crystalloid solution suitable for adequately resuspending MSCs can be used as the predetermined volume. For example, the predetermined volume may be in the range of about 0.5 ml to about 15 ml. In one example, the predetermined volume may be in the range of about 1 ml to about 10 ml. In exemplary examples, the predetermined volume of crystalloid solution may be about 1 ml, about 2 ml, about 3 ml, about 4 ml, or about 5 ml. A first cell suspension is prepared by resuspending MSCs in a predetermined volume of crystalloid solution. Resuspension is usually performed after harvesting the mesenchymal stem cell / mesenchymal stem cell population after culturing for pharmaceutically effective administration.

[0023] After determining the concentration of MSCs in the first cell suspension and the volume of the first cell suspension required to prepare a formulation containing approximately 500,000 to 10 million mesenchymal stem cells, the first cell suspension is mixed with a certain volume of liquid carrier. The volume of the first cell suspension mixed with the liquid carrier may be approximately 0.5 ml to 10 ml. In the exemplary example, the total volume of the mesenchymal stem cell storage or transport formulation, i.e., the determined volume of the first cell suspension and the volume of the liquid carrier, is approximately 1 ml. Preferably, an amount of 500,000 to 10 million mesenchymal stem cells is selected to prepare a unit dose containing 500,000 to 10 million mesenchymal stem cells in a predetermined volume such as 1 ml, 2 ml, etc. In the present invention, the predetermined volume of crystalloid solution contains approximately 100,000 to 15 million viable MSCs. In one example, a predetermined volume of crystalloid solution contains approximately 500,000 to 10 million MSCs. In an exemplary example, a mesenchymal stem cell storage or transport preparation contains approximately 1 million, 2 million, 3 million, 4 million, 5 million, or 6 million MSCs. As used herein, the term “approximately” with respect to the number of mesenchymal stem cells may mean that the number may vary by a certain percentage. For example, “approximately” may mean a numerical variation / deviation of ±1% to approximately ±15%. Thus, “approximately” may also mean ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, or ±10%. In particular, such variations are apparent to those skilled in the art when mesenchymal stem cell storage or transport formulations are manually prepared for subsequent storage and / or transport to a site of administration, such as a wound healing clinic or medical practice (which is still the usual approach for preparing such live cell-based formulations).

[0024] In this invention, MSCs may have been recovered directly from a culture of MSC-containing tissue or from a culture of isolated MSCs or MSC populations before being resuspended in crystalloid solution. In either case, the MSCs may have been cultured in a cell culture vessel. Consequently, the MSCs used in this invention may have been recovered from a cell culture vessel before being resuspended in a predetermined volume of crystalloid solution.

[0025] Both the crystalloid solution and the liquid carrier of the present invention are supplemented with serum albumin. While we do not wish to be bound by theory, it is believed that serum albumin can improve the viability of mesenchymal stem cells / mesenchymal stem cell populations and also improve their recovery from the container in which the stem cells are stored for transport to the administration site. The concentration of serum albumin may be the same or different in the crystalloid solution and the liquid carrier. Preferably, the concentration of serum albumin is the same in both the crystalloid solution and the liquid carrier. In this regard, any concentration of serum albumin suitable for improving the viability of MSCs, for example, can be used. For example, the crystalloid solution and the liquid carrier may each contain serum albumin ranging from about 0.5% (w / v), about 0.6% (w / v), about 0.7% (w / v), about 0.8% (w / v), about 0.9% (w / v), or about 1.0% (w / v) to about 5% (w / v). In one such example, the crystalloid solution and liquid carrier may contain about 1% (w / v) to about 3% (w / v) of serum albumin. In the exemplary example, the crystalloid solution and liquid carrier each contain about 1% (w / v) of serum albumin. Any pharmaceutically appropriate serum albumin, e.g., bovine or human serum albumin, may be used herein. In the exemplary example, both the crystalloid solution and liquid carrier may contain human serum albumin (HSA). The serum albumin used herein is ideally obtained in pharmaceutically acceptable quality. An example of such pharmaceutical-grade serum albumin is a 25% solution (w / v) of human serum albumin, marketed under the trade name Plasbumin® by Grifols Therapeutics LLC, Clayton, North Carolina, USA.

[0026] The crystalloid solution may also contain one or more components suitable for supporting the growth and / or proliferation of MSCs. Such components may be minerals such as sodium, potassium, iron, magnesium, zinc, selenium, chloride, or a combination thereof. In one example, the crystalloid solution contains sodium, potassium, magnesium, and chloride. The crystalloid solution may be a commercially available solution containing further components suitable for supporting the growth and / or proliferation of MSCs. In one example, the crystalloid solution may be PlasmaLyte or Ringer's lactate solution. In the formulations of the present invention, the total amount of crystalloid solution may be limited to a specific percentage. For example, a mesenchymal stem cell storage or transport formulation may contain about 50% or less, about 40% or less, about 30% or less, about 20% or less, about 10% or less, or about 5% or less of crystalloid solution. In an exemplary example, a mesenchymal stem cell storage or transport formulation may contain about 30% or less, or about 20% or less, or about 10% or less of PlasmaLyte.

[0027] Transportation / storage can be carried out over any period of time. For example, transportation / storage can be carried out over approximately 7 days or less. It is also conceivable that transportation / storage could be carried out over approximately 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, or less. Therefore, transportation / storage can be carried out over approximately 48 hours, approximately 24 hours, or less.

[0028] It is also intended that transport / storage may take place at any temperature suitable for the method of the present invention. For example, transport / storage may take place at temperatures between approximately -5°C and approximately 15°C. Thus, it is also conceivable that transport / storage may take place at temperatures between approximately 2°C and approximately 8°C. Transport may also be carried out at temperatures above approximately -5°C, above approximately -10°C, above approximately -15°C, or above approximately -20°C. Furthermore, it is conceivable that transport / storage may take place at temperatures below 20°C, below 18°C, below 15°C, below 12°C, or below 10°C.

[0029] The methods of the present invention also assume that a stem cell population (or mesenchymal stem cells) is stored or transported at any appropriate concentration. As stated above, the terms “mesenchymal stem cells” and “mesenchymal stem cell population” may be used interchangeably herein. Where “mesenchymal stem cells” are referred to herein, these stem cells may also belong to the same mesenchymal stem cell population. For example, all mesenchymal stem cells may belong to a mesenchymal stem cell population in which about 97% or more, about 98% or more, or about 99% or more of their cells express CD73, CD90, and CD105, while lacking expression of CD34, CD45, and HLA-DR. Where the terms “carrier” or “liquid carrier” may be used in relation to a solution containing MSCs, PlasmaLyte, HSA, and Hyothermosol, it should be noted herein that the mesenchymal stem cell storage or transport formulation of the present invention may also mean the same. Therefore, when the solution contains MSCs, PlasmaLyte, HSA, and Hyothermosol, the terms “carrier” or “liquid carrier” and “stem cell storage or transport preparation” can also be used interchangeably. The stem cell populations used herein can be transported / stored at concentrations of, for example, approximately 70 million cells per ml of carrier, approximately 60 million cells per ml of carrier, approximately 50 million cells per ml of carrier, approximately 40 million cells per ml of carrier, approximately 30 million cells per ml of carrier, approximately 20 million cells per ml of carrier, approximately 10 million cells per ml of carrier, approximately 5 million cells per ml of carrier, approximately 4 million cells per ml of carrier, approximately 3 million cells per ml of carrier, approximately 2 million cells per ml of carrier, approximately 1 million cells per ml of carrier, approximately 500,000 cells per ml of carrier, approximately 100,000 cells per ml of carrier, or less than 100,000 cells per ml of carrier. Therefore, stem cell populations can be transported / stored at concentrations ranging from approximately 10 million cells per ml of carrier to approximately 1 million cells per ml of carrier.

[0030] The method of the present invention relates to the transport / storage of stem cells. In principle, any stem cells can be used in the method of the present invention. One characteristic feature of stem cells is their ability to self-replicate. "Self-renewal" is the ability to go through multiple cell cycles of cell division while maintaining an undifferentiated state. Methods for testing whether cells have the ability to self-replicate are known to those skilled in the art. For example, self-renewal can be tested by subculturing cells beyond 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more passages. Subculturing includes the step of dividing the cells before replating them as a single-cell suspension. A further characteristic of stem cells, as described elsewhere herein, is their diplopotency or pluripotency. In principle, diplopotency or pluripotency can be tested by differentiating the stem cells into different lineages.

[0031] In particular, the stem cell population used in the method of the present invention may be an embryonic stem cell population, an adult stem cell population, a mesenchymal stem cell population, or an induced pluripotent stem cell population.

[0032] As used herein, “embryonic stem cell population” means “pluripotent stem cell population.” As referred herein, pluripotent cells refer to a cell type that has the ability to self-replicate and differentiate into different cell types. Pluripotent stem cells can differentiate into virtually any cell, namely cells derived from any of the three primary germ layers: ectoderm, endoderm, and mesoderm. The term pluripotent stem cell also includes stem cells derived from the inner cell mass of the early embryo, known as the blastocyst. Notably, recent advances in embryonic stem cell research have made it possible to create new embryonic stem cell lines without destroying the embryo, for example, by using blastomere biopsy-based techniques that do not impair the embryo's developmental potential (Klimanskaya (2006) “Embryonic stem cells from blastomeres maintaining embryo viability.” Semin Reprod Med. 2013 Jan;31(1):49-55). Furthermore, numerous established embryonic stem cell lines are available in the art. Thus, it is possible to handle embryonic stem cells without requiring the destruction of the embryo. Pluripotent stem cells may be embryonic stem cells that were not obtained by destroying a human embryo. Therefore, pluripotent stem cells are embryonic stem cells obtained from an embryo without destroying the embryo.

[0033] As used herein, “adult stem cell population” refers to a population of pluripotent stem cells. A population of pluripotent stem cells can give rise to a limited number of cell types, and therefore their somatic cell fate is limited. For example, neural stem cells can give rise to both nerve cells and glial cells. Adult stem cells have the ability to self-replicate and can be obtained from any suitable source. For example, adult stem cells can be obtained from bone marrow, peripheral blood, brain, spinal cord, dental pulp, blood vessels, skeletal muscle, epithelium of the skin and digestive system, cornea, retina, liver, or pancreas.

[0034] The stem cell population used in the method of the present invention may also be a mesenchymal stem cell population. In this regard, it should be noted that the culture medium described herein (e.g., PTT-6) makes it possible to isolate a mesenchymal stem cell population (also referred to herein as "mesenchymal stem cells") from the amnion under conditions that allow for the proliferation of mesenchymal stem / progenitor cells without differentiation of the mesenchymal stem / progenitor cells. Thus, after isolating mesenchymal stem cells from the amnion as described herein, the isolated mesenchymal stem / progenitor cell population has the ability to differentiate into multiple cell types, as described in, for example, U.S. Patent Application 2006 / 0078993, U.S. Patent No. 9,085,755, International Patent Application WO2006 / 019357, U.S. Patent No. 8,287,854, or WO2007 / 046775. For example, as described in U.S. Patent Application 2006 / 0078993, mesenchymal stem cells from the amniotic membrane of the umbilical cord are spindle-shaped, express the genes POU5f1, Bmi-1, and leukemia suppressor (LIF), and secrete activin A and follistatin. Mesenchymal stem cells isolated in the present invention can be differentiated into any type of mesenchymal cell, including, but is not limited to, endocrine gland-derived cells such as adipocytes, cutaneous fibroblasts, chondrocytes, osteoblasts, tendinocytes, ligamentous fibroblasts, cardiomyocytes, smooth muscle cells, skeletal muscle cells, mucin-producing cells, insulin-producing cells (e.g., β-islet cells), or neuroectoderm cells. Stem cells isolated according to the methods described herein can be differentiated in vitro for later use as differentiated cells for medical purposes. An example of such an approach is the differentiation of mesenchymal stem cells into insulin-producing β-islet cells, which can then be administered, for example, by transplantation, to patients suffering from insulin deficiency, such as diabetes (see also WO2007 / 046775 in this regard). Alternatively, the mesenchymal stem cells described herein can be used in an undifferentiated state for cell-based therapies for wound healing purposes, such as the treatment of burns or chronic diabetic wounds.In these therapeutic applications, the mesenchymal stem cells of the present invention may help promote wound healing by interacting with the surrounding affected tissue, or they may differentiate into their respective skin cells (see, for example, WO2007 / 046775 again).

[0035] In this regard, it should be noted that MSCs may originate from any mammalian tissue or compartment / body part known to contain MSCs. In exemplary examples, MSCs may be umbilical cord MSCs, placental MSCs, umbilical cord-placental junction MSCs, umbilical cord blood MSCs, bone marrow MSCs, or adipose tissue-derived MSCs. Umbilical cord MSCs may be from (or originate from) any compartment of umbilical cord tissue containing MSCs, and may be mixed umbilical cord MSCs meaning not only amniotic membrane, perivascular MSCs, Wharton's gelatinous pulp MSCs, and amniotic membrane MSCs of the umbilical cord, but also MSCs containing two or more stem cells from these compartments. It should be noted that the mesenchymal stem cell populations described herein can be isolated and cultured from (i.e., derived from) any umbilical cord tissue, insofar as the umbilical cord tissue contains amniotic membrane (also referred to as “umbilical cord lining”). Thus, mesenchymal stem cell populations can be isolated from the entire umbilical cord (or a small piece thereof), as described in the Experiments section of this application. Therefore, this umbilical cord tissue may contain any other tissue / components of the umbilical cord in addition to the amniotic membrane. For example, as shown in Figure 16 of U.S. Patent Application 2006 / 0078993 or International Patent Application WO2006 / 019357, the amniotic membrane of the umbilical cord is the outermost part of the umbilical cord that covers it. In addition, the umbilical cord contains one vein (which carries oxygenated, nutrient-rich blood to the fetus) and two arteries (which carry deoxygenated, nutrient-depleted blood away from the fetus). For protection and mechanical support, these three blood vessels are embedded in Wharton's colloid, which is a gelatinous substance mainly composed of mucopolysaccharides. Thus, the umbilical cord tissue used herein may also contain this one vein, two arteries, and Wharton's colloid. The use of such a whole (intact) portion of the umbilical cord has the advantage that it does not need to separate the amniotic membrane from the other components of the umbilical cord. This reduces the isolation step, and consequently makes the methods described herein simpler, faster, less error-prone, and more economical—all crucial aspects of GMP production required for the therapeutic application of mesenchymal stem cells.Therefore, the isolation of mesenchymal stem cells can be initiated from an explant, and then, if a larger quantity of mesenchymal stem cells is desired for use, for example, in a clinical trial, the isolated mesenchymal stem cells can be subsequently subcultured. Alternatively, it is also possible to isolate mesenchymal umbilical cord-lining stem cells from the amnion by first separating the amnion from the other components of the umbilical cord and culturing the amnion in a culture medium, such as PTT-6. This culture can also be carried out on an explant, and optionally, the isolated mesenchymal stem cells are then subcultured. In this context, the terms “explant” or “explant method” are used in their usual sense in the art to refer to a method in which, once a tissue or tissue fragment has been recovered, it is placed in a cell culture dish containing a culture medium (growth medium), and stem cells migrate from the tissue to the surface of the dish over time. These primary stem cells can then be further augmented by micropropagation (subculture), as also described herein, and transferred to a new dish. In this regard, it should be noted that, from the perspective of generating cells for therapeutic purposes, a master cell bank of isolated mesenchymal stem cells can be obtained in the first step of isolating amniotic mesenchymal stem cells from the umbilical cord, and a working cell bank can be obtained in subsequent subculturing. In certain embodiments, the stem cell population is therefore a mesenchymal stem cell population. The mesenchymal stem cell population can be isolated from the amniotic membrane of the umbilical cord by a method that includes the step of culturing in a culture medium containing DMEM (Dulbeccoo's modified Eagle medium), F12 (Ham's F12 medium), M171 (Medium 171), and FBS (fetal bovine serum).By using such a culture medium, a population of mesenchymal stem cells was isolated from the amniotic membrane of the umbilical cord, and more than 90%, or even 99%, or more of these cells were positive for three mesenchymal stem cell markers: CD73, CD90, and CD105. At the same time, these stem cells lacked expression of CD34, CD45, and HLA-DR (see the Experiments section). This means that 99%, or even more, of the cells in this population expressed the stem cell markers CD73, CD90, and CD105, while not expressing the markers CD34, CD45, and HLA-DR.Such an extremely homogeneous and clearly defined cell population was first reported in concurrently pending U.S. Patent Application No. 15 / 725,913 (published as US2018 / 127721), filed on October 5, 2018, claiming priority to U.S. Provisional Patent Application No. 62 / 404,582, filed on October 5, 2017 (the contents of both of these are incorporated herein by reference as a whole), and in concurrently pending PCT Application PCT / SG2017 / 050500 (published as WO2018 / 067071), also filed on October 5, 2018, claiming priority to U.S. Provisional Patent Application No. 62 / 404,582, filed on October 5, 2017, and this stem cell population was first reported, for example, in Dominici et al., "Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement," Human mesenchymal stem cells are ideal candidates for clinical trials and cell-based therapies because they fully meet generally accepted standards for human mesenchymal stem cells to be used in cell therapy, as defined by 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. Furthermore, it is possible to obtain a large number of mesenchymal stem cells, such as 300-700 million per run, using bioreactors such as the Quantum cell proliferation system (see also the Experiments section).Therefore, the present invention makes it possible to transport and store the amount of stem cells necessary for therapeutic applications, such as use in wound healing, in a cost-effective manner. In addition, all components used to prepare the culture medium of the present invention are commercially available in GMP quality. Thus, the present invention opens a route for transporting and storing GMP-produced and highly homogeneous mesenchymal stem cell populations derived from umbilical cord amniotic membrane.

[0036] Therefore, in some embodiments, the mesenchymal stem cell population is an isolated population of mesenchymal stem cells from the amniotic membrane of the umbilical cord. It is further assumed that at least about 90% or more of the cells in the isolated stem cell population express each of the markers CD73, CD90, and CD105. For example, at least about 91% 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, and about 99% or more of the cells in the isolated mesenchymal stem cell population express each of the markers CD73, CD90, and CD105. In addition, or instead, at least about 90% or more, about 91% 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, and about 99% or more of the isolated mesenchymal stem cell population lacked expression of the following markers: CD34, CD45, and HLA-DR (human leukocyte antigen-antigen D related). In further examples, at least about 91% 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, and about 99% or more of MSCs may express CD73, CD90, and CD105, respectively, while at least about 90% or more, about 91% 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, and about 99% or more of MSCs may lack expression of CD34, CD45, and HLA-DR. In certain cases, approximately 97% or more, 98% or more, or 99% or more of MSCs express CD73, CD90, and CD105, while lacking expression of CD34, CD45, and HLA-DR.

[0037] The marker CD73 is known to those skilled in the art. In this regard, CD73 refers to surface antigen classification 73, also known as 5'-nucleotidase (5'-NT) or ecto-5'-nucleotidase. The sequence of the human CD73 protein may have the sequence with SEQ ID NO. 1. The marker CD90 is known to those skilled in the art. In this regard, CD90 refers to surface antigen classification 90, also known as thymocyte differentiation antigen 1 (Thy-1). The sequence of the human CD90 protein may have the sequence with SEQ ID NO: 2. The marker CD105 is known to those skilled in the art. CD105 is also known as endoglin (ENG). The sequence of the human CD105 protein may have the sequence with SEQ ID NO: 3.

[0038] When the mesenchymal stem cell population of the present invention (specifically, a population of mesenchymal stem cells in which at least about 98% or 99% express each of the markers CD73, CD90, and CD105, and lack the expression of each of the markers CD34, CD45, and HLA-DR) is used for clinical trials or as an approved therapy, the cell population of the working cell bank is typically used for this purpose. As described above, the mesenchymal stem cell population may lack the expression of the following markers: CD34, CD45, and HLA-DR. In this regard, it should be noted that the markers CD34, CD45, and HLA-DR are known to those skilled in the art. The human CD34 protein may have the sequence SEQ ID NO. 4. The human CD45 protein may have the sequence SEQ ID NO: 5. The human HLA-DR protein may have the sequence SEQ ID NO: 6.

[0039] Both the isolated stem cell population (which can constitute a master cell bank) and the subcultured stem cell population (which can constitute a working cell bank) can be stored, for example, in cryopreservation form.

[0040] As described above, this method for isolating mesenchymal stem cells from the amniotic membrane of the umbilical cord has the advantage that all components used in the culture medium of the present invention are available in GMP quality, and therefore, mesenchymal stem cells can be isolated under GMP conditions for subsequent therapeutic administration.

[0041] Therefore, the stem cell population may also be an induced pluripotent stem cell population. “Induced pluripotent stem cells,” as used herein, refer to adult somatic cells that have been genetically reprogrammed into an embryonic stem cell-like state by forcing the expression of genes and factors essential for maintaining the definitive characteristics of embryonic stem cells. Thus, induced pluripotent stem cells can be induced / created from non-pluripotent cells.

[0042] Induced pluripotent stem cells (iPSCs) represent a significant advance in stem cell research because they allow for the acquisition of pluripotent stem cells without the use of embryos. Mouse iPSCs were first reported in 2006 (Takahashi, K; Yamanaka, S (2006). "Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors." Cell 126 (4): 663-76), and human iPSCs (hiPSCs) were first reported in 2007 (Takahashi et al. (2007) "Induction of pluripotent stem cells from adult human fibroblasts by defined factors." Cell; 131(5):861-72). Mouse iPSCs exhibit key characteristics of pluripotent stem cells, including the expression of stem cell markers, the formation of tumors containing cells derived from all three germ layers, and the ability to contribute to many different tissues when injected into mouse embryos at very early developmental stages. Human iPSCs can also express stem cell markers and generate cells characteristic of all three germ layers. Such stem cell markers may include Oct3 / 4, Sox2, Nanog, alkaline phosphatase (ALP), and stem cell-specific antigens 3 and 4 (SSEA3 / 4). Furthermore, the chromatin methylation patterns of iPSCs are similar to those of embryonic stem cells (Tanabe, Takahashi, Yamanaka (2014) "Induction of pluripotency by defined factors." Proc. Jpn. Acad., 2014, Ser. B 90).

[0043] In addition, iPSCs can self-replicate in vitro and differentiate into all three germ layers. The pluripotency or potential for differentiation into different cell types of iPSCs can be tested, for example, by in vitro differentiation into nerve cells or glial cells, or by creating germline chimeric animals by blastocyst injection.

[0044] Methods for producing human induced pluripotent stem cells are known to those skilled in the art and are described, for example, in WO2009115295, WO2009144008, or EP2218778. Therefore, those skilled in the art can obtain iPSCs by any method. In principle, induced pluripotent cells can be obtained from any adult somatic cells (of the subject). Exemplary somatic cells include peripheral blood mononuclear cells (PBMCs) derived from blood, or fibroblasts obtained from skin tissue biopsy.

[0045] The present invention relates, in particular, to MSC storage or transport formulations obtained by the methods described herein, and to MSC storage or transport formulations obtainable by the methods described herein. Furthermore, the present invention relates to the transport of MSCs, including the step of transporting MSCs in the mesenchymal stem cell storage or transport formulation as defined herein. In this regard, the present invention includes contacting the stem cell population described herein with a liquid carrier. In the methods of the present invention, it is assumed that the stem cell population described herein is contacted with the carrier before transport / storage. In addition, or instead, the stem cell population is contacted with the carrier after its recovery. How recovery may be performed is described in detail elsewhere in this specification and in the Experiments section. For example, the stem cell population may be contacted with the carrier about 0 minutes, about 1 minute, about 5 minutes, about 10 minutes, about 30 minutes, about 45 minutes, about 60 minutes, or longer after its recovery.

[0046] Recovery may involve separating the stem cell population from the culture medium, for example, from PTT-6. Suitable techniques for such separation are known to those skilled in the art. For example, separation can be carried out by centrifuging the stem cells in the culture medium and decanting the culture medium.

[0047] The stem cell population is i) Torolox, ii) Na + , iii) K + , iv) Ca 2+ , v) Mg 2+ , vi) Cl - , vii) H2PO4 - , viii) HEPES, ix) Lactobionate, x) Sucrose, xi) Mannitol, xii) Glucose, xiii) Dextran-40, xiv) Adenosine, and xv) Glutathione It is brought into contact with a liquid carrier containing the liquid carrier.

[0048] "Trolox" refers to 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid with CAS number 53188-07-1. This is a water-soluble analogue of vitamin E and has been suggested to reduce oxidative stress or damage. Figure 19 shows the datasheet for Trolox available from Tocris. It is also commercially available from Sigma Aldrich (product number: 238813).

[0049] Na + and Cl - These are all well-known ions. Those skilled in the art know how to obtain them. For example, these ions may be added to a support as NaCl salts. GMP-grade NaCl is available from Sigma Aldrich. Figure 20 shows the datasheet for NaCl available from Sigma Aldrich.

[0050] Ca 2+ and Mg 2+ These are also well-known ions. Those skilled in the art know how to obtain them. These ions may be added to the support, for example, as CaCl2 or MgCl2 salts. Figure 31 shows the datasheet for CaCl2 available from Sigma Aldrich, and Figure 32 shows the datasheet for MgCl2 available from Sigma Aldrich.

[0051] K + and H2PO4 - (Dihydrogen phosphate) is also well known to those skilled in the art. This can be used, for example, as KH2PO4, available from Sigma Aldrich. Figure 21 shows the datasheet for KH2PO4 available from Sigma Aldrich.

[0052] HEPES, also known as 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (CAS number 7365-45-9), is commonly used as a zwitterionic organic chemical buffer. Those skilled in the art also know where to obtain HEPES, as it is commercially available. For example, it can be obtained from Sigma Aldrich; see the corresponding datasheet shown in Figure 22.

[0053] Lactobionate is the carboxylic acid anion of lactobionic acid. Lactobionic acid (4-O-β-galactopyranosyl-D-gluconic acid) is a sugar acid. Lactobionate can be used in different ways. When used as potassium lactobionate, it can, for example, provide osmotic support and prevent cell swelling, and when combined with sodium, it may have preservative properties. Alternatively, mineral salts of lactobionic acid can be used for mineral supplementation. For pharmaceutical applications, the antibiotic erythromycin can often be used, in particular, as erythromycin lactobionate. Those skilled in the art also know where to obtain lactobionate, for example, sodium lactobionate (CAS number: 27297-39-8), namely from COMBI-BLOCKS, for example; see the product sheet in Figure 23.

[0054] Sucrose, also known as D-Glc-(1→2)-β-D-Fru, α-D-glucopyranosylβ-D-fructofuranosyl, β-D-fructofuranosyl-α-D-glucopyranosyl, D(+)-saccharose, or sugar (CAS number 57-50-1), is commercially available like any other substance, and those skilled in the art will know where to purchase it. The corresponding product sheet for sucrose from Sigma Aldrich is shown in Figure 24.

[0055] Mannitol is a type of sugar alcohol (CAS Registry Number: 69-65-8). Those skilled in the art know how to obtain mannitol. For example, it can be obtained from Avantor. The respective product sheets are shown in Figure 25.

[0056] Glucose (CAS number: 50-99-7) is also well known to those skilled in the art and is commercially available. The respective product sheets from Sigma Aldrich are shown in Figure 26.

[0057] Dextran is a branched glucan composed of linear α(1→6)-linked glucose units and α(1→3)-linked initiation branches. The size of dextran ranges from 10,000 to 150,000 Kd. Dextran is used in many applications, including as a volume expander, stabilizer, matrix component, binding platform, lubricant, and physical structural component. Dextran 40 (CAS number: 9004-54-0), used in the carriers described herein, is typically used in the development of novel and improved preservation solutions for organ transplantation. Dextran 40 can be used to determine cell rigidity and flow parameters across cell layers. Dextran 40 can also be used as a colloidal plasma expander. Dextran-40 is commercially available and can be obtained, among other things, from Sigma Aldrich (product sheet shown in Figure 27).

[0058] Adenosine (CAS number 58-61-7) is a purine nucleoside composed of an adenine molecule linked to a ribose sugar molecule (ribofuranose) via a β-N9 glycosidic bond. Adenosine is commercially available, particularly from Sigma-Aldrich (the corresponding product sheet is shown in Figure 28).

[0059] Glutathione is also known as (2S)-2-amino-4-{[(1R)-1-[(carboxymethyl)carbamoyl]-2-sulfanylethyl]carbamoyl}butanoic acid. This component is commercially available, particularly from Sigma Aldrich (corresponding product sheet shown in Figure 29).

[0060] In principle, any liquid carrier containing the substances listed in i) to xv) above can be used in the method of the present invention. The carrier is a liquid carrier. Therefore, it is possible to dissolve the substances listed in i) to xv) in the liquid to form a solution / suspension. The liquid may be any suitable liquid. For example, the liquid may be a culture medium, water, buffer, or similar.

[0061] The carrier may additionally contain further pH buffers, energy substrates, free radical scavengers, and osmotic / colloid stabilizers—all of which are known to those skilled in the art. Furthermore, the liquid carrier may be serum-free and / or protein-free. The liquid carrier does not need to contain a bipolar aprotic solvent such as DMSO, for example. In particular, the liquid carrier may be a carrier described in WO2010 / 064054. The carrier may be HypoThermosol® or HypoThermosol®-FRS (HTS-FRS). HypoThermosol®-FRS (HTS-FRS) can be purchased from STEMCELL Technologies (according to the respective product sheets shown in Figure 30).

[0062] It is further assumed that the carrier is a transport / storage medium or an excipient. The transport / storage medium may be a natural medium, which consists only of naturally occurring biological fluids, additionally containing the substances listed in i) to xv) as described herein. The medium may also contain the substances listed in i) to xv) as described herein, as well as the addition of (further) nutrients (both organic and inorganic), vitamins, salts, O2 and CO2 gas phases, serum proteins, carbohydrates, and / or cofactors. In certain embodiments, the medium is serum-free and / or protein-free.

[0063] The carrier may also be an excipient. An "excipient" is a substance formulated together with the active ingredient of a pharmaceutical product. In this method, the active ingredient is a population of stem cells.

[0064] The carrier may further include a biocompatible scaffold or microcarrier. The scaffold or microcarrier may be, for example, a biodegradable polymer, most preferably poly(D,L-lactic acid-coglycolic acid) (PLGA). Alternatively, the scaffold or microcarrier may be a smooth, macroporous, or microporous structure containing a material including poly-L-lactide (PLLA), collagen, fibronectin, glycosaminoglycan (GAG), fibrin, starch, cellulose arabinogalactan (larch gum), alginic acid, agar, carrageenan, chitin, hyaluronic acid, dextran, gellan gum, pullulan, hydroxyapatite, polyhydroxyalkanoic acid (PHA), hydrogel, or other self-assembling materials such as peptide-based nanostructured fibrous scaffolds.

[0065] In principle, any amount of stem cells can be brought into contact with any amount of liquid carrier. In this regard, contact can be made by suspending the stem cell population at densities of approximately 70 million cells / ml, 60 million cells / ml, 50 million cells / ml, 40 million cells / ml, 30 million cells / ml, 20 million cells / ml, 10 million cells / ml, 5 million cells / ml, 4 million cells / ml, 3 million cells / ml, 2 million cells / ml, 1 million cells / ml, 500,000 cells / ml, 100,000 cells / ml, or at a density of less than 100,000 cells in 1 ml of carrier. In some embodiments, contact is made by suspending the stem cell population at a density of approximately 10 million cells / 1 ml of carrier.

[0066] After contacting a stem cell population with a mesenchymal stem cell storage or transport preparation, the stem cells that have been in contact with the mesenchymal stem cell storage or transport preparation can be dispensed into vials in volumes of approximately 50 ml, 20 ml, 10 ml, 5 ml, 4 ml, 3 ml, 2 ml, 1 ml, 0.5 ml, 0.25 ml, or less than 0.25 ml of the mesenchymal stem cell storage or transport preparation. For example, stem cells that have been in contact with a mesenchymal stem cell storage or transport preparation can be dispensed into a vial in a volume of approximately 1 ml.

[0067] It is further assumed that the method of the present invention does not involve a thawing or freezing step. This may include transporting / storing the stem cell population without the need to freeze and thaw the stem cell population after recovery.

[0068] The carrier used in the method of transporting / storing stem cell populations described herein is particularly suited to this purpose. One advantage of this carrier is that substantially all stem cells transported / stored therein remain viable. "Viable cells" means cells that are able to survive. Those skilled in the art know how to detect viable cells. One such method is to stain the cells with the dye trypan blue. Viable cells do not stain positively with trypan blue.

[0069] In this regard, the method of the present invention may result in the death of at most about 50%, about 40%, about 30%, about 20%, about 10%, or less than about 10% of the stem cells in a population during transport / storage, compared to the number / quantity of surviving stem cells before transport / storage.

[0070] The method of the present invention also intends that the stem cell population will have any cell diameter after transport / storage. Those skilled in the art know how to measure cell diameter. For example, cell size / diameter can be determined by taking a microscopic image and measuring the cell diameter using secondary software. The majority of stem cells in a stem cell population may therefore have a cell diameter of about 9 μm to about 20 μm after transport / storage. It is also assumed that the majority of stem cells in a stem cell population may have a cell diameter of about 12 μm to about 16 μm after transport.

[0071] Stem cells transported / stored in the carriers described herein secrete the same proteins / factors as living stem cells. For example, the method of the present invention is intended to enable a (mesenchymal) stem cell population to secrete approximately the same amount of TGF-beta-1 after transport / storage as it did before transport / storage. TGF-beta-1 (transforming growth factor beta, TGF-β1) is known to those skilled in the art and may include the sequence shown in SEQ ID NO. 7. In addition, or instead, after transport / storage, the (mesenchymal) stem cell population may secrete approximately the same amount of VEGF (vascular endothelial growth factor), PDGF-AA (platelet-derived growth factor subunit AA), Ang-1 (angiogenin-1), and / or HGF (hepatocyte growth factor) as it did before transport / storage. VEGF, PDGF-AA, Ang-1, and / or HGF are all known to those skilled in the art regarding their involvement in wound healing. In particular, VEGF may contain the sequence shown in SEQ ID NO. 8, PDGF-AA may have the sequence shown in SEQ ID NO. 9, Ang-1 may have the sequence shown in SEQ ID NO. 10, while HGF may have the sequence shown in SEQ ID NO. 11. In addition, or instead, PDGF-BB and / or IL-10 are essentially undetectable before and / or after transport. Both PDGF-BB (platelet-derived growth factor subunit BB) and / or IL-10 (interleukin-10) are also known to those skilled in the art. PDGF-BB may contain the sequence shown in SEQ ID NO. 12, while IL-10 may contain the sequence shown in SEQ ID NO. 13. The secretion of these factors can be determined by any suitable method, for example, by measuring the amount of proteins (e.g., PDGF-AA, PDGF-BB, VEGF, IL-10, Ang-1, HGF, or TGFβ1) secreted by stem cells into the carrier. Protein levels can be measured using commercially available antibody / immunoassay methods in an automated manner, for example, with systems such as the FLEXMAP 3D system (Luminex Corporation, Austin, Texas, USA).In this regard, it should be noted that the involvement of the proteins angiopoietin-1 (Ang-1), TGF-β1, VEGF, and HGF in the wound healing process is known to those skilled in the art. For the involvement of angiopoietin-1 in wound healing, see, for example, Li et al. Stem Cell Research & Therapy 2013, 4:113, "Mesenchymal stem cells modified with angiopoietin-1 gene promote wound healing." For information on the involvement of hepatocyte growth factor (HGF) in wound healing, specifically in the healing of chronic / non-healing wounds, see, for example, Yoshida et al., "Neutralization of Hepatocyte Growth Factor Leads to Retarded Cutaneous Wound Healing Associated with Decreased Neovascularization and Granulation Tissue Formation." J. Invest. Dermatol. 120:335-343, 2003; Li, Jin-Feng et al., "HGF Accelerates Wound Healing by Promoting the Dedifferentiation of Epidermal Cells through βl-Integrin / ILK Pathway." BioMed Research International 2013 (2013):470418; or Conway et al., "Hepatocyte growth factor regulation: An integral part of why wounds become chronic." Wound Rep Reg (2007) 15 683-692.For more information on the role of vascular endothelial growth factor (VEGF) in wound healing, specifically in the healing of chronic / non-healing wounds, see, for example, Froget et al., Eur. Cytokine Netw., Vol. 14, March 2003, 60-64, or Bao et al., "The Role of Vascular Endothelial Growth Factor in Wound Healing," J Surg Res. 2009 May 15; 153(2): 347-358.

[0072] For information on the involvement of transforming growth factor beta (including TGF-β1, TGF-β2, and TGF-β3) in wound healing, specifically in the healing of chronic / non-healing wounds, see, for example, Ramirez et al., "The Role of TGFb Signaling in Wound Epithelialization," Advances In Wound Care, Volume 3, Number 7, 2013, 482-491, or Pakyari et al., "Critical Role of Transforming Growth Factor Beta in Different Phases of Wound Healing," Advances In Wound Care, Volume 2, Number 5, 2012, 215-224.

[0073] Turning to the culture medium used in the present invention, the culture medium may contain, for the isolation or culture of mesenchymal umbilical cord-lining stem cells, DMEM at a final concentration of approximately 55-65% (v / v), F12 at a final concentration of approximately 5-15% (v / v), M171 at a final concentration of approximately 15-30% (v / v), and FBS at a final concentration of approximately 1-8% (v / v). The value "% (v / v)" as used herein refers to the volume of each component relative to the final volume of the culture medium. This means that if DMEM is present in the culture medium at a final concentration of, for example, approximately 55-65% (v / v), then 1 liter of culture medium contains approximately 550-650 ml of DMEM.

[0074] In another embodiment, the culture medium may contain DMEM at a final concentration of approximately 57.5–62.5% (v / v), F12 at a final concentration of approximately 7.5–12.5% ​​(v / v), M171 at a final concentration of approximately 17.5–25.0% (v / v), and FBS at a final concentration of approximately 1.75–3.5% (v / v). In yet another embodiment, 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).

[0075] In addition to the above components, the culture medium may contain supplements that are advantageous for culturing mesenchymal umbilical cord-lining stem cells. The culture medium of the present invention may contain, for example, epidermal growth factor (EGF). If present, EGF may be present in the culture medium at a final concentration of about 1 ng / ml to about 20 ng / ml. In some of these embodiments, the culture medium may contain EGF at a final concentration of about 10 ng / ml.

[0076] The culture medium may also contain insulin. If present, insulin may be present at a final concentration of approximately 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.

[0077] The culture medium may further contain at least one of the following supplements: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). In such embodiments, the culture medium may contain all three: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). In these embodiments, the culture medium may contain adenine at a final concentration of about 0.05 to about 0.1 μg / ml, hydrocortisone at a final concentration of about 1 to about 10 μg / ml, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of about 0.5 to about 5 ng / ml.

[0078] In one embodiment, mesenchymal stem cells are cultured in PTT6 medium to obtain a highly purified population of mesenchymal stem cells described and used herein. In this regard, the PTT6 medium described herein is used to obtain a final volume of 500 ml of culture medium. i. DMEM 250 ml ii. M171 118 ml iii. DMEM / F12 118 ml iv. 12.5 ml of fetal bovine serum (FBS) to reach a final concentration of 2.5% (v / v) v. EGF with a final concentration of 10 ng / ml vi. Insulin with a final concentration of 5 μg / ml vii. Insulin 0.175 ml (final concentration 5 μg / ml) It should be noted that this is obtained by mixing the following.

[0079] "DMEM" refers to Dulbecco's Modified Eagle Medium, a modified version of the basic Eagle medium (BME), developed in 1969 (see Figure 1, which shows the datasheet for DMEM available from Lonza). The first DMEM formulation contained 1000 mg / L of glucose and was first reported for the culture of embryonic mouse cells. Since then, DMEM has become a standard medium for cell culture and is commercially available from various sources, including ThermoFisher Scientific (catalog no. 11965-084), Sigma Aldrich (catalog no. D5546), or Lonza, to name just a few suppliers. Therefore, any commercially available DMEM can be used in this invention. In a preferred embodiment, the DMEM used herein is DMEM medium available from Lonza under catalog no. 12-604F. This medium is DMEM supplemented with 4.5 g / L of glucose and L-glutamine. In another preferred embodiment, the DMEM used herein is Sigma Aldrich catalog number D5546, which contains 1000 mg / L glucose and sodium bicarbonate but does not contain L-glutamine.

[0080] "F12" medium refers to Ham F12 medium. This medium is also a standard cell culture medium and is a nutrient mixture originally designed to culture a wide variety of mammalian and hybridoma cells when used with serum in combination with hormones and transferrin (see Figure 2, which shows the datasheet for Ham F12 medium from Lonza). Any commercially available Ham F12 medium (for example, from just a few suppliers, ThermoFisher Scientific (catalog no. 11765-054), Sigma Aldrich (catalog no. N4888), or from Lonza) can be used in this invention. In a preferred embodiment, Ham F12 medium from Lonza is used.

[0081] "DMEM / F12" or "DMEM:F12" refers to a 1:1 mixture of DMEM and Ham F12 culture medium (see Figure 3, which shows the datasheet for DMEM:F12 (1:1) medium from Lonza). DMEM / F12 (1:1) medium is a widely used basic medium for supporting the growth of many different mammalian cells and is commercially available from various suppliers such as ThermoFisher Scientific (catalog no. 11330057), Sigma Aldrich (catalog no. D6421), or Lonza. Any commercially available DMEM:F12 medium can be used in this invention. In a preferred embodiment, the DMEM:F12 medium used herein is DMEM / F12 (1:1) medium available from Lonza under catalog no. 12-719F (DMEM:F12 with L-glutamine, 15 mM HEPES, and 3.151 g / L glucose).

[0082] "M171" refers to Culture Medium 171, developed as a basic medium for the culture and proliferation of normal human mammary epithelial cells (see Figure 4, which shows the datasheet for M171 medium from Life Technologies Corporation). This basic medium is widely used and commercially available from suppliers such as ThermoFisher Scientific or Life Technologies Corporation (catalog number M171500). Any commercially available M171 medium can be used in this invention. In a preferred embodiment, the M171 medium used herein is the M171 medium available from Life Technologies Corporation under catalog number M171500.

[0083] "FBS" refers to fetal bovine serum (also known as bovine fetal serum), that is, the blood fraction remaining after natural blood coagulation, followed by centrifugation to remove any remaining red blood cells. Fetal bovine serum is the most widely used serum supplement for in vitro cell culture of eukaryotic cells because it contains very low levels of antibodies, more growth factors, and allows for versatility in many different cell culture applications. It is preferable to obtain FBS from members of the International Serum Industry Association (ISIA), whose main focus is on the safety and safe use of serum and animal-derived products through proper origin tracing, truthfulness of labeling, and proper standardization and monitoring. ISIA member suppliers of FBS include, to name a few, Abattoir Basics Company, Animal Technologies Inc., Biomin Biotechnologia LTDA, GE Healthcare, Gibco by Thermo Fisher Scientific, and Life Science Production. In the currently preferred embodiment, FBS is obtained from GE Healthcare under catalog number A15-151.

[0084] As described above, the method for preparing a culture medium for isolating the mesenchymal stem cell population used in the present invention is to obtain a final volume of 500 ml of culture medium. i. DMEM 250 ml ii. M171 118 ml iii. DMEM / F12 118 ml iv. 12.5 ml of fetal bovine serum (FBS) to reach a final concentration of 2.5% (v / v) This includes the step of mixing the ingredients.

[0085] As explained above, DMEM / F12 medium is a 1:1 mixture of DMEM and Ham F12 medium. Therefore, 118 ml of DMEM / F12 medium contains 59 ml of DMEM and 59 ml of F12. Thus, when using this method to prepare the culture medium, the final concentration (v / v) in a total volume of 500 ml is as follows: -DMEM:250 ml + 59 ml = 309 ml, corresponding to 309 / 500 = 61.8% (v / v). -M171: 118 ml, equivalent to 118 / 500 = 23.6% (v / v). -F12: 59 ml, equivalent to 59 / 500 = 11.8% (v / v).

[0086] The embodiment of this method for preparing the culture medium is: v. 1 ml of EGF preservation solution (5 μg / ml) to achieve a final EGF concentration of 10 ng / ml, and vi. 0.175 ml of insulin storage solution (14.28 mg / ml) to achieve a final insulin concentration of 5 μg / ml. The process further includes the step of adding [a certain substance].

[0087] In these embodiments, it is noted herein that the above volumes of these components i-vi result in a final volume of 499.675 ml of culture medium. If no further components are added to the culture medium, the remaining 0.325 ml (to make a total volume of 500 ml) may be any of components i-iv, meaning, for example, DMEM, M171, DMEM / F12, or FBS. Alternatively, the concentration of the EGF or insulin storage solution can, of course, be adjusted so that the total volume of the culture medium is 500 ml. In addition, it is also noted that components i-iv do not necessarily have to be added in the order they are listed, and it is, of course, possible to mix these components in any order to reach the culture medium of the present invention. This means, for example, that M171 and DMEM / F12 can be mixed together and then combined with DMEM and FBS to obtain the final concentrations described herein, namely, a final concentration of approximately 55-65% (v / v) for DMEM, approximately 5-15% (v / v) for F12, approximately 15-30% (v / v) for M171, and approximately 1-8% (v / v) for FBS.

[0088] In another embodiment, the method further comprises the step of adding one or more of the supplements adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3) to DMEM in a volume of 0.325 ml, thereby making a culture medium with a total volume of 500 ml. In this embodiment, the final concentrations of these supplements in DMEM may be as follows: Adenine at approximately 0.05-0.1 μg / ml, for example, adenine at approximately 0.025 μg / ml, Approximately 1-10 μg / ml of hydrocortisone, Approximately 0.5 to 5 ng / ml of 3,3',5-triiodo-L-thyronine sodium salt (T3), for example, 1.36 ng / ml of 3,3',5-triiodo-L-thyronine sodium salt (T3).

[0089] In accordance with the above disclosure, the cell culture media used herein can be obtained or acquired by the methods for preparing the culture media described herein.

[0090] In addition, a method for isolating mesenchymal stem cells from the amniotic membrane of the umbilical cord, comprising the step of culturing amniotic tissue in a culture medium prepared by this method, is described herein.

[0091] Therefore, the present invention also, - DMEM with a final concentration of approximately 55-65% (v / v), - F12 with a final concentration of approximately 5-15% (v / v), - M171 with a final concentration of approximately 15-30% (v / v), and - FBS with a final concentration of approximately 1-8% (v / v) This applies to the use of cell culture media containing the following:

[0092] In certain embodiments of the culture medium described herein, the medium comprises DMEM at a final concentration of approximately 57.5–62.5% (v / v), F12 at a final concentration of approximately 7.5–12.5% ​​(v / v), M171 at a final concentration of approximately 17.5–25.0% (v / v), and FBS at a final concentration of approximately 1.75–3.5% (v / v). In other embodiments, the culture medium may comprise 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).

[0093] In addition, the culture medium may further contain epidermal growth factor (EGF) at a final concentration of approximately 1 ng / ml to approximately 20 ng / ml. In a particular embodiment, the culture medium contains EGF at a final concentration of approximately 10 ng / ml. The culture medium described herein may further contain insulin at a final concentration of approximately 1 μg / ml to 10 μg / ml. In such an embodiment, the culture medium may contain insulin at a final concentration of approximately 5 μg / ml.

[0094] The cell culture medium may further contain at least one of the following supplements: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). In certain embodiments, the culture medium contains all three: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). If present, the culture medium may contain adenine at a final concentration of approximately 0.01–0.1 μg / ml or approximately 0.05–0.1 μg / ml, hydrocortisone at a final concentration of approximately 0.1–10 μg / ml or approximately 1–10 μg / ml, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of approximately 0.5–5 ng / ml.

[0095] In the aspect of the cell culture medium, 500 ml of the cell culture medium of the present invention is i. DMEM 250 ml ii. M171 118 ml iii. DMEM / F12 118 ml iv. Fetal bovine serum (FBS) 12.5 ml (final concentration 2.5%) Includes. In a further embodiment, the cell culture medium is v. EGF at a final concentration of 10 ng / ml, and vi. Insulin with a final concentration of 5 μg / ml It may further include the following. Both insulin and EGF can be added to the culture medium using an optimal preservation solution, so that the total volume of the culture medium does not exceed 500 ml.

[0096] In certain cases, components i-vi of the culture medium used in the present invention are the components shown in Figure 5, meaning they are available from each manufacturer using the catalog numbers shown in Figure 5. The culture medium obtained by mixing components i-vi as shown in Figure 5 is also referred to herein as "PTT-6". In this regard, it should be noted again that components i-vi and any other components, such as antibiotics, from any other commercial supplier may be used to prepare the culture medium of the present invention.

[0097] In addition, the cell culture medium of the present invention may contain adenine at a final concentration of approximately 0.01 to approximately 0.1 μg / ml or approximately 0.05 to approximately 0.1 μg / ml, hydrocortisone at a final concentration of approximately 0.1 to 10 μg / ml, approximately 0.5 to approximately 10 μg / ml, or approximately 1 to approximately 10 μg / ml, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of approximately 0.1 to approximately 5 ng / ml or approximately 0.5 to approximately 5 ng / ml.

[0098] To obtain the mesenchymal stem cell population described herein, umbilical cord tissue can be cultured until an appropriate number of (primary) mesenchymal umbilical cord-lining stem cells proliferate from the tissue. In a typical embodiment, umbilical cord tissue is cultured until the cell proliferation of amniotic mesenchymal stem cells reaches a concentration density of approximately 70–80%. It should be noted herein that the terms “concentration density” or “cluster” are used in their usual sense in the art of cell culture and mean an estimate / indicator of the number of adherent cells in a culture dish or flask, referring to the percentage of the surface covered by cells. For example, 50 percent concentration means that approximately half of the surface is covered and there is still room for cell proliferation. 100 percent concentration means that the surface is completely covered by cells and there is no room left for cells to proliferate as a monolayer.

[0099] Once an appropriate number of primary cells (mesenchymal umbilical cord-lining stem cells) have been obtained from the umbilical cord-lining tissue using tissue explants, the mesenchymal stem cells are removed from the culture vessel used for cultivation. This allows for the creation of a master cell bank containing (primary) isolated mesenchymal stem cells from the amniotic membrane. Typically, since mesenchymal stem cells are adherent cells, removal is performed using standard enzymatic treatment. For example, the enzymatic treatment may include trypsin treatment, as described in U.S. Patent Application 2006 / 0078993, WO2006 / 019357, or WO2007 / 046775, which means that proliferating cells can be recovered by trypsin treatment (0.125% trypsin / 0.05% EDTA) for further growth. If the recovered mesenchymal stem cells are to be used, for example, to create a master cell bank, the cells may also be cryopreserved and stored for further use, as described below herein.

[0100] Once recovered, mesenchymal stem cells can be transferred to culture vessels for subculturing. Subculturing can also be started from frozen primary cells, i.e., from a master cell bank. For subculturing, any appropriate amount of cells can be seeded into a culture vessel such as a cell culture plate. Mesenchymal stem cells can be seeded for this purpose, for example, about 0.5 × 10⁶ 6 cells / ml ~ approx. 5.0×10 6 The cells can be suspended at a concentration of cells / ml in a suitable medium for subculturing (most conveniently, culture medium PTT-6). In one embodiment, the cells are subculturified in a medium of approximately 1.0 × 10⁶ cells / ml. 6Suspend at a concentration of cells / ml. Subculturing can be carried out by culturing in a simple culture flask, or by culturing in a multi-layer system such as CellStack (Corning, Corning, NY, USA) or Cellfactory (Nunc, part of Thermo Fisher Scientific Inc., Waltham, MA, USA), which can be stacked in an incubator. Alternatively, subculturing can also be carried out in a closed, self-contained system such as a bioreactor. Various designs of bioreactors are well known to those skilled in the art, and include, for example, 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," mentioned above. An example of a commercially available hollow fiber bioreactor is the Quantum® Cell Expansion System (Terumo BCT, Inc.), which is used for increasing bone marrow mesenchymal stem cells for clinical trials (see Hanley et al, Efficient Manufacturing of Therapeutic Mesenchymal Stromal Cells Using the Quantum Cell Expansion System, Cytotherapy. 2014 August; 16(8): 1048-1058). Another example of a commercially available bioreactor that can be used for subculturing the mesenchymal stem cell population of the present invention is the Xuri Cell Expansion System, available from GE Heathcare. Culturing mesenchymal stem cells in automated systems such as the Quantum® Cell Expansion System is particularly effective when a working cell bank for therapeutic application should be generated under GMP conditions and a large number of cells are required.

[0101] The subculture of mesenchymal umbilical cord-lining stem cells described herein is carried out in a culture medium described herein, such as PTT-6 medium. Thus, culture media such as PTT-6 can be used for both the isolation of mesenchymal stem cells from the amniotic membrane and the subsequent culture of the isolated primary cells by subculture. Similarly, in subculture, mesenchymal stem cells can be cultured until an appropriate number of cells have proliferated. In an illustrative embodiment, mesenchymal stem cells are subcultured until they reach a concentration density of about 70-80%.

[0102] The isolation and culture of mesenchymal umbilical cord-lining stem cell populations can be carried out under standard conditions for culturing mammalian cells. Typically, the method of the present invention for isolating mesenchymal umbilical cord-lining stem cell populations is carried out under conditions (temperature, atmosphere) commonly used for culturing cells of the species from which the cells originate. For example, human umbilical cord tissue and mesenchymal umbilical cord-lining stem cells are each typically cultured at 37°C in an air atmosphere containing 5% CO2. In this regard, it should be noted that the mesenchymal cells may originate from any mammalian species such as mouse, rat, guinea pig, rabbit, goat, horse, dog, cat, sheep, monkey, or human, and in one embodiment, human-derived mesenchymal stem cells are preferred.

[0103] Once the desired / appropriate number of mesenchymal umbilical cord-lining stem cells have been obtained from subculturing, they can be recovered by removing the mesenchymal stem cells from the culture vessels used for subculturing. Recovery of mesenchymal stem cells is typically carried out by enzymatic treatment, including trypsin treatment of the cells, as in this case as well. The isolated mesenchymal stem cells are then collected and either used immediately or stored for further use. Typically, storage is carried out by cryopreservation. The term "cryopreservation" is used herein in its usual sense to describe the process by which mesenchymal stem cells are preserved by cooling them to a sub-zero temperature, such as (typically) -80°C or -196°C (the boiling point of liquid nitrogen). Cryopreservation can be carried out as is known to those skilled in the art and may involve the use of cryoprotective agents such as dimethyl sulfoxide (DMSO) or glycerol, which slow the formation of ice crystals in the umbilical cord cells.

[0104] The isolated population of mesenchymal umbilical cord-lining stem cells obtained by the isolation methods described herein is highly distinct and highly homogeneous. In typical embodiments of the method, at least about 90% or more, about 91% 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, and about 99% or more of the isolated mesenchymal stem cells express the following markers: CD73, CD90, and CD105. In addition, in these embodiments, at least about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, and about 99% or more of the isolated mesenchymal stem cells may lack expression of the following markers: CD34, CD45, and HLA-DR. In certain embodiments, approximately 97% or more, approximately 98% or more, or approximately 99% or more of the isolated mesenchymal stem cell population expresses CD73, CD90, and CD105, while lacking expression of CD34, CD45, and HLA-DR.

[0105] Accordingly, consistent with the above disclosure, a population of mesenchymal stem cells isolated from the amniotic membrane of the umbilical cord, wherein at least about 90% or more of the cells in the stem cell population express each of the following markers: CD73, CD90, and CD105. In a preferred embodiment, at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, and about 99% or more of the cells in the isolated population of mesenchymal stem cells are CD73+, CD90+, and CD105+, meaning that this proportion of the isolated cell population expresses each of CD73, CD90, and CD105 (see the Experiments section of this application), which may be used herein. In addition, at least about 90% or more, about 91% 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, and about 99% or more of isolated mesenchymal stem cells may lack the expression of the following markers. In certain embodiments, about 97% or more, about 98% or more, or about 99% or more cells in an isolated mesenchymal stem cell population lack the expression of CD34, CD45, and HLA-DR, while expressing CD73, CD90, and CD105. Such a highly homogeneous population of mesenchymal stem cells derived from the amniotic membrane of the umbilical cord was first reported in U.S. Provisional Patent Application No. 62 / 404,582, filed October 5, 2016, and concurrently pending U.S. Patent Application No. 15 / 725,913, filed October 5, 2017, and concurrently pending PCT Application PCT / SG2017 / 050500, also filed October 5, 2017, and meets the criteria for mesenchymal stem cells to be used in cell therapy (see the Experiments section, and also see, for example, Sensebe et al. "Production of mesenchymal stromal / stem cells according to good manufacturing practices: a review," also mentioned above).In this regard, it should be noted that while this mesenchymal stem cell population can be obtained by the isolation method of the present invention, it can also be obtained by other methods such as cell sorting, if necessary.

[0106] The method for preparing a culture medium for isolating mesenchymal stem cells as described herein, to obtain a final volume of 500 ml of culture medium, i. DMEM 250 ml ii. M171 118 ml iii. DMEM / F12 118 ml iv. 12.5 ml of fetal bovine serum (FBS) to reach a final concentration of 2.5% (v / v) This includes the step of mixing the ingredients.

[0107] As explained above, DMEM / F12 medium is a 1:1 mixture of DMEM and Ham F12 culture medium.

[0108] Therefore, 118 ml of DMEM / F12 medium contains 59 ml of DMEM and 59 ml of F12. Thus, when this method is used to prepare the culture medium, the final concentration (v / v) in a total volume of 500 ml is as follows: DMEM:250 ml + 59 ml = 309 ml, corresponding to 309 / 500 = 61.8% (v / v). M171: 118 ml, equivalent to 118 / 500 = 23.6% (v / v). F12: 59 ml, equivalent to 59 / 500 = 11.8% (v / v).

[0109] The present invention also relates to a method for treating a subject having a disease, the method comprising the step of topically administering mesenchymal stem cells or a population described herein that have been stored or transported in a mesenchymal stem cell storage or transport solution, wherein the mesenchymal stem cells or stem cell population are administered within approximately 96 hours from the time the mesenchymal stem cell population is recovered. The method for treating the subject can be carried out as described in the international patent application WO2019 / 199229, “A Method Of Transporting Mesenchymal Stem Cells By Means Of A Transporting Solution And A Method Of Administering Stem Cells To Wounds,” which was published after the priority date of this PCT application and is incorporated herein by reference in its entirety for all purposes.

[0110] Similarly, the present invention also relates to a population of mesenchymal stem cells described herein for use in a method of treating a disease of interest, wherein the population of mesenchymal stem cells is administered topically within approximately 96 hours from the time the population of mesenchymal stem cells is harvested.

[0111] The subject to be treated may be any suitable subject. The subject may be a vertebrate, more preferably a mammal. Mammals include, but are not limited to, livestock, sports animals, pets, primates, dogs, horses, mice, and rats. Mammals may also be humans, dogs, cats, cattle, pigs, mice, rats, etc. Thus, in one embodiment, the subject is a vertebrate. The subject may also be a human subject. Thus, the subject may be a subject requiring treatment. As such, the subject may be suffering from a disease described elsewhere in this specification. In some embodiments, the subject is suffering from type 1 or type 2 diabetes mellitus with chronic foot ulcers. Preferably, the subject is negative for HLA antibodies against the mesenchymal stem cell population.

[0112] The mesenchymal stem cell population can be administered at any dose. The dose may be therapeutically effective. The "therapeutally effective dose" can vary depending on factors, including but not limited to, the activity of the cells used, the stability of the cells in the patient's body, the severity of the condition to be alleviated, the age and sensitivity of the patient being treated, adverse events, and similar factors, as will be apparent to those skilled in the art. The dose may be adjusted as various factors change over time.

[0113] The dosage to which mesenchymal stem cells are administered may also be a unit dose. For example, a mesenchymal stem cell population can be administered in unit doses of approximately 20 million cells, approximately 15 million cells, approximately 10 million cells, approximately 5 million cells, approximately 4 million cells, approximately 3 million cells, approximately 2 million cells, approximately 1 million cells, approximately 500,000 cells, approximately 250,000 cells, or less than 250,000 cells. In one example, mesenchymal stem cells may be administered in doses of approximately 3 million, approximately 5 million, or approximately 10 million cells. In a particular embodiment, a mesenchymal stem cell population is administered in unit doses of approximately 10 million cells.

[0114] Mesenchymal stem cells can be applied multiple times to the same subject. For example, stem cells can be applied once, twice, three times, or more times per week. In principle, any unit dose of mesenchymal stem cells can be applied as many times as is appropriate to cure or alleviate the disease. For example, a mesenchymal stem cell population can be applied once, twice, three times, or more times per week. A mesenchymal stem cell population can also be applied over a period of one, two, three, four, five, six, seven, eight, nine, ten, eleven weeks, or longer.

[0115] Therefore, a unit dose of approximately 20 million cells, approximately 15 million cells, approximately 10 million cells, approximately 5 million cells, approximately 4 million cells, approximately 3 million cells, approximately 2 million cells, approximately 1 million cells, approximately 500,000 cells, approximately 250,000 cells, or less than 250,000 cells is administered once or twice a week. A unit dose of approximately 20 million cells, approximately 15 million cells, approximately 10 million cells, approximately 5 million cells, approximately 4 million cells, approximately 3 million cells, approximately 2 million cells, approximately 1 million cells, approximately 500,000 cells, approximately 250,000 cells, or less than 250,000 cells may also be administered once or twice a week over a period of 3, 4, 5, 6, 7, 8, 10 weeks, or longer.

[0116] Mesenchymal stem cells or mesenchymal stem cell populations: approximately 1000 cells / cm² 2 ~About 5 million cells / cm 2 It is also intended by the method of treatment of the present invention that it may be applied in the dosage of cm. 2 This expression refers to the area of ​​the wound / skin to which stem cells are applied. The mesenchymal stem cell population is approximately 100,000 cells / cm². 2 300,000 cells / cm² 2 , or 500,000 cells / cm² 2 It is also conceivable that this dosage may be applied. The mesenchymal stem cell population also has approximately 100,000 cells / cm³ 2 , about 300,000 cells / cm 2 , or approximately 500,000 cells / cm² 2 This dosage can be administered twice a week for approximately 8 weeks.

[0117] The mesenchymal stem cell population is administered within approximately 96 hours of the time the mesenchymal stem cell population is harvested. How harvesting may be performed is described elsewhere in this specification. Mesenchymal stem cells or mesenchymal stem cell populations may also be applied within approximately 72 hours, approximately 48 hours, approximately 24 hours, approximately 12 hours, approximately 6 hours, or less, of the time the mesenchymal stem cell population is harvested. Between the time of harvesting and the time of application, the mesenchymal stem cell population may be transported or stored in the mesenchymal stem cell storage or transport formulation described in this invention. Therefore, the aspects described in this application regarding transport / storage in the mesenchymal stem cell storage or transport formulation are equally relevant to methods of treating a subject, including the step of administering MCS stored in the mesenchymal stem cell storage or transport formulation of this invention, with necessary modifications.

[0118] The method of treating a subject of the present invention helps to alleviate the disease affecting the subject. In principle, any disease that can be treated by the mesenchymal stem cell population described herein is meant herein. In particular, the disease may be a skin disease or a wound. The wound may result from any cause, for example, a burn, bite, trauma, surgery, or disease. The wound may also be caused by diabetes. Therefore, the wound may also be a diabetic wound. The wound may also be a diabetic foot ulcer. Notably, the mesenchymal stem cell population may be placed directly on the wound, for example, a burn or a diabetic wound (see International Patent Application WO2007 / 046775).

[0119] As described herein, between the collection of the mesenchymal stem cell populations described herein and their application to a subject, the cells may be transported / stored in a carrier as defined herein. Thus, the method of the present invention for treating a subject may also include the step of separating the mesenchymal stem cell population from the carrier before administering the mesenchymal stem cell population to the subject. Those skilled in the art will know how to separate cells from a carrier. For example, the separation of a mesenchymal stem cell population from a carrier may include centrifugation. In addition, or alternatively, the separation of a mesenchymal stem cell population from a carrier may include the step of removing the cell population from a vial by syringe.

[0120] After isolating stem cells from a mesenchymal stem cell storage or transport preparation, or after recovering mesenchymal stem cells, or after obtaining a mesenchymal stem cell population as described herein by any other method, these cells are applied topically to a subject. In principle, any method of topical administration is used herein. Administration of the mesenchymal stem cell population can be done by syringe. However, it is also possible to contact the mesenchymal stem cells in a cream, ointment, gel, suspension, or any other suitable substance before applying them to the subject. After application to the subject, the mesenchymal stem cell population may be held in place by a film or bandage. Examples of such films or bandages may be dressings such as Tegaderm® dressings and crepe bandages for covering the Tegaderm® dressings. For a more uniform distribution of cells, the application site may be gently massaged.

[0121] The present invention also relates to a unit dose of mesenchymal stem cells obtained or obtainable by the methods described herein. For example, a unit dose may contain, in a volume of 1 ml, about 20 million cells, about 15 million cells, about 10 million cells, about 5 million cells, about 4 million cells, about 3 million cells, about 2 million cells, about 1 million cells, about 500,000 cells, about 250,000 cells, or less than 250,000 cells of the mesenchymal stem cell population described herein.

[0122] It is also conceivable that the unit dose may contain approximately 10 million, 9 million, 8 million, 7 million, 6 million, 5 million, 4 million, 3 million, 2 million, 1 million, 500,000, 250,000, or 100,000 cells. In one example, the unit dose may contain approximately 1 million, 3 million, or 5 million cells. Preferably, the unit dose contains approximately 10 million cells. It is further conceivable that the unit dose contains approximately 1,000 to 5 million cells. The unit dose can be applied in doses of approximately 100,000, 300,000, or 500,000 cells. As described herein, the unit dose may be applied topically. For example, the unit dose may be 1 cm 2 It can be applied locally to the area.

[0123] A unit dose can be applied once, twice, three times, or more times per week. For example, a unit dose can be applied over one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks, eleven weeks, or more. A unit dose containing approximately 100,000 cells, approximately 300,000 cells, or approximately 500,000 cells is preferably 1 cm 2 This can be applied twice a week for eight weeks.

[0124] The unit dose can be contained in any suitable container. For example, the unit dose can be contained in a 1 ml vial. In such a case, for example, 0.1 ml of the vial is preferably 1 cm in diameter. 2 It can be applied per dose. The unit dose may be contained in the syringe.

[0125] In the unit dose of the present invention, cells may be in contact with a liquid carrier as defined herein. In this case, mesenchymal stem cells are separated from the carrier before administration. For example, cells can be centrifuged and isolated before administration to a subject. The carrier may include, or may be, any carrier described herein, such as HypoThermosol® or Hypothermosol®-FRS.

[0126] The unit dose of the present invention may include umbilical cord MSCs. As described above, the umbilical cord MSCs may be from (or derived from) any section of the umbilical cord tissue containing MSCs. Therefore, the unit dose may include amniotic membrane MSCs, perivascular MSCs, Wharton's gelatinous membrane MSCs, and umbilical cord amniotic membrane MSCs. The umbilical cord amniotic membrane MSCs may be highly distinct and highly homogeneous. Therefore, in one embodiment of the present invention, the unit dose may include and be used MSCs as described in international patent application WO2018 / 067071. Therefore, in a typical example of this method, a unit dose may include MSCs in which at least approximately 90% or more, approximately 91% or more, approximately 92% or more, approximately 93% or more, approximately 94% or more, approximately 95% or more, approximately 96% or more, approximately 97% or more, approximately 98% or more, and approximately 99% or more express the following markers: CD73, CD90, and CD105, respectively. Furthermore, a unit dose may include MSCs in which at least approximately 90% or more, approximately 91% or more, approximately 92% or more, approximately 93% or more, approximately 94% or more, approximately 95% or more, approximately 96% or more, approximately 97% or more, approximately 98% or more, and approximately 99% or more lack the expression of the following markers: CD34, CD45, and HLA-DR. In certain cases, the unit dose includes cases where approximately 97% or more, approximately 98% or more, or approximately 99% or more of MSCs express CD73, CD90, and CD105, while lacking expression of CD34, CD45, and HLA-DR.In further examples, at least about 91% 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, and about 99% or more of MSCs may express CD73, CD90, and CD105, respectively, while at least about 90% or more, about 91% 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, and about 99% or more of MSCs may lack expression of CD34, CD45, and HLA-DR. In certain cases, approximately 97% or more, 98% or more, or 99% or more of MSCs express CD73, CD90, and CD105, while lacking expression of CD34, CD45, and HLA-DR.

[0127] The treatment methods and unit doses of the present invention may include the use of viable cells. How viability can be tested is described elsewhere in this specification.

[0128] The present invention is further illustrated by the following non-limiting experimental examples.

[0129] The sequences used in this specification are shown in Table 1 below.

[0130] (Table 1) Sequences used in this specification TIFF0007861268000001.tif107170TIFF0007861268000002.tif219170TIFF0007861268000003.tif233170TIFF0007861268000004.tif219170 [Examples]

[0131] Experimental Examples 1. Cryopreservation of umbilical cord tissue before isolating mesenchymal stem cells. Umbilical cord tissue (the umbilical cord was donated with the mother's informed consent) was processed as follows to subsequently isolate mesenchymal stem cells from the amniotic membrane of the umbilical cord.

[0132] 1.1 Washing of umbilical cord tissue sample: a. Remove the surgical scalpel from its protective cover. b. Using forceps, firmly hold the umbilical cord and cut it into 10 cm long pieces using a surgical scalpel. Return the unused portion of the umbilical cord to the original tissue cup. c. Transfer the 10 cm long umbilical cord fragment to a new 150 mm culture dish. A 150 mm culture dish can also be used instead of a cup. d. Use the cover of the 150 mm culture dish as a place to put forceps and surgical scalpels. e. Dispense 25 ml of Plasmalyte A (Baxter, catalog # 2B2543Q) into a 30 ml syringe. Holding the syringe at a 45° angle with one hand, dispense the Plasmalyte A directly onto the umbilical cord tissue. f. While holding the culture dish at a slight angle, remove Plasmalyte A using a 30 ml syringe and blunt needle. g. Collect used Plasmalyte A in a 300 ml transfer bag that will serve as a waste container, and dispose of it in a biohazard waste bin. h. Repeat the washing procedure using a new culture dish for each wash, as needed. Ensure that all blood clots on the surface have been removed. If tissue cleansing is required, additional Plasmalyte A may be used. i. Place the tissue into a new, labeled tissue culture dish and continue cutting the tissue. Add 20 ml of Plasmalyte A to the dish to prevent the tissue from drying out during cutting. j. Cut the umbilical cord into sections of approximately 1 cm in size, for a total of 10 sections. k. Each 1 cm section is further cut into smaller pieces of approximately 0.3 cm × 0.3 cm to 0.5 cm × 0.5 cm each. l. Remove all Plasmalyte A from the dish. m. Withdraw 25 ml of Plasmalyte A from the original Plasmalyte A bag using a 30 ml syringe and dispense it directly onto the umbilical cord tissue sample. n. Hold the culture dish at an angle and collect all the Plasmalyte A used for tissue washing on one side, then remove it with a syringe and blunt needle. o. Repeat the washing process. No blood clots should remain.

[0133] Note: If the umbilical cord is not to be frozen immediately, the umbilical cord tissue should be kept in Plasmalyte A until just before freezing.

[0134] 1.2 Cryopreservation of umbilical cord tissue: a. Prepare the cryopreservation solution: i. Prepare 50 ml of a frozen solution consisting of 60% Plasmalyte A, 30% 5% human serum albumin, and 10% dimethyl sulfoxide (DMSO). ii. Label the 150 ml transfer bag with "tissue freezing solution" and attach the plasma transfer set to the port using sterile techniques. iii. Remove 30 ml of Plasmalyte A from the original Plasmalyte A bag using a 30 ml syringe and transfer it to a transfer bag labeled "Tissue Freezing Solution" along with the date and time the solution was prepared. iv. Take 15 ml of 5% human serum albumin using 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 frozen solution. b. Remove Plasmalyte A from the tissue before adding the freezing solution. c. Using a 60 ml syringe, draw out the entire 50 ml of frozen solution into the syringe and add approximately 30 ml of the frozen solution to a 150 mm cell culture dish containing umbilical cord tissue. Attach a blunt needle to the syringe and keep it sterile. d. Swirl the culture dish containing the tissue and frozen solution every minute for 10 minutes. e. Using forceps, select eight randomly chosen sections and place them into four 4 ml cryovials. Select four randomly chosen sections and place them into one 1.8 ml cryovial. These sections must not contain any blood clots. f. Fill each cryovial containing the umbilical cord tissue with the remaining frozen solution up to the 3.6 ml fill line for 4 ml tubes and up to the 1.8 ml line for 1.8 ml Nunc vials. g. Label one Bactec Lytic / 10 - Anaerobic / F bottle and one Bactec Pluc Aerobic / F bottle with tissue ID labels. h. Using a syringe and blunt needle, remove 20 ml of the frozen solution from the culture dish, wipe the Bactec vial with an alcohol swab, replace the blunt needle with an 18 g needle, and inoculate 10 ml each into the aerobic and anaerobic Bactec bottles. i. Activate the controlled speed freezer. j. After the controlled-speed freezing is complete, leave the unit in a liquid nitrogen freezer with continuous temperature monitoring until further use.

[0135] 2. Isolation of mesenchymal umbilical cord-lining stem cells from umbilical cord tissue 2.1. Preparation of culture medium for processing MSCs from umbilical cord tissue: a. To prepare 500 ml of PTT6 (culture medium / growth medium), add the following in the order listed: i. DMEM 250 ml ii. M171 118 ml iii. DMEM F12 118 ml iv. FBS 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).

[0136] The above volumes of components i-vi result in a final culture medium volume of 499.675 ml. If no further components are added to the culture medium, the remaining 0.325 ml (to make a total volume of 500 ml) may be any of components i-iv, meaning, for example, DMEM, M171, DMEM / F12, or FBS. Alternatively, the concentration of the EGF or insulin preservation solution can be adjusted so that the total volume of the culture medium is 500 ml. Alternatively, an antibiotic preservation solution such as penicillin-streptomycin-amphotericin can be added to make a final volume of 500 ml. It is also possible to add one or more of the supplements adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3) in a volume of 0.325 ml to the culture medium to make a total culture medium volume of 500 ml.

[0137] vii. Label the bottle with "PTT6" along with the date the culture medium was prepared, the operator's initials, and the word "Expiration Date" followed by the expiration date. The expiration date is the earlier of either the earliest expiration date of any of the components or one month after the preparation date.

[0138] b. To prepare rinse medium (Hanks buffer solution (HBSS) free of calcium and magnesium and containing 5% FBS), add 2.5 ml of FBS to 47.5 ml of HBSS in a 50 ml centrifuge tube. Label the tube with the operator's initials and the date the medium was prepared, along with the label "Rinse Medium". c. Test all media for sterility using Bactec Lytic / 10 - Anaerobic / F (Becton Dickinson & Company) and Bactec Plus + Aerobic / F (Becton Dickinson & Company). Pour 20 ml of the prepared media into each bottle.

[0139] 2.2 Thawing of umbilical cord tissue for MSC recovery: a. Thawing should begin when the operator is ready to process the sample in the cleanroom. Do not thaw more than one vial at a time, except when the vials originate from the same donor. b. Wipe the water bath with disinfectant and then with 70% isopropanol, and fill it with 1 L of sterile water. Heat the water bath to 36-38°C. c. Prepare 10 ml of rinse medium consisting of 70%–90% PlasmaLyte A under a biosafety cabinet in a cleanroom. Sterile filter this solution using a 0.2-μm syringe filter attached to a 10 ml syringe, and keep the solution refrigerated until use. d. Attach a processing label to the 50 ml conical tube. e. Ensure the water bath temperature is between 36 and 38°C. f. Remove the tissue vials from liquid nitrogen storage and rapidly thaw them in a 37°C water bath filled with 1 L of sterile water. The vial holder of the Mr. Frosty Nalgene Cryo 1°C freezing container can be used as a floating rack to hold the vials in place and thaw the samples. g. Remove the vials from the water bath and spray them with a 70% isopropanol solution. The appropriate time to remove the vials from the water bath is when you can see small ice crystals floating inside the vials—this suggests that the internal temperature of the vials is below 37°C. h. Place the vial in the pass-through and inform the cleanroom technician.

[0140] 2.3 Preparation for tissue processing: a. Umbilical cord tissue processing must be performed in an environmental monitoring (EM) cleanroom. The room and hood must be thoroughly cleaned at the end of each shift. b. Prepare / clean the biosafety cabinet. c. Perform biological particle counting while working inside the biosafety cabinet. d. Gather all necessary items into the biosafety cabinet, checking for packaging damage and expiration dates. When handling syringes, serum pipettes, sterile forceps, surgical scalpels, tissue plates, and needles, never touch any surfaces that may come into contact with sterile products. Only the outside of syringes, tubes, plunger tips, and / or needle caps or cases may be handled safely. Discard any items if any surfaces are touched or if any surface comes into contact with a non-sterile surface. e. Record the lot number and expiration date (if applicable) of all reagents and supplies used. f. Receive the thawed vials by cleaning them with a lint-free wipe moistened with 70% alcohol and then moving them into a biosafety cabinet. g. Using an aspiration needle attached to a syringe, remove as much liquid as possible from the vial. Avoid aspirating tissue. h. Using sterile forceps, transfer the tissue to a sterile 100 mm Petri dish. i. Add a fixed fraction of 5 ml of rinse medium to the tissue fragment. j. Swirl the contents for 15-30 seconds, then remove the rinse medium using a pipette or syringe with a suction needle. Repeat this rinsing process twice. k. Add 2 mL of rinse medium to the tissue to prevent it from drying out.

[0141] 2.4. Initiation of MSC proliferation from tissue: a. Label the bottom of the 6-well plate with the MSC lot number or umbilical cord tissue ID and the start date of proliferation, along with the label "Proliferation 1". If using a 60 mm tissue culture dish, draw a grid on the bottom of the dish to divide the plate into four sections. b. Using sterile disposable forceps, place one 3×3 mm to 5×5 mm tissue sample into each well. If using a 60 mm tissue culture dish, place the tissues in the center of each section, keeping them separated (more than 1 cm apart). c. Fill each well with 3 ml of PTT6. d. Using a suction needle attached to a 30 ml syringe, remove just enough culture medium to barely cover the tissue. Do not tilt the plate. Do not touch the bottom of the wells with the suction needle. e. Observe cell proliferation daily (24 ± 6 hours) using an inverted optical microscope. A real-time cell culture imaging system may be used instead of an optical microscope. f. Replace the culture medium daily. Always allow the medium to equilibrate to room temperature before use. i. Remove the culture medium by aspirating it. ii. Add 3 ml of PTT6. iii. Aspirate until the tissue is barely immersed in the culture medium. g. Once cell proliferation is observed in the tissue, transfer the tissue to a new 6-well plate using the same procedure as in 4.a-4.e above, except that the plate is labeled "Proliferation 2". Maintain cell proliferation in the "Proliferation 1" plate by adding 2 ml of PTT6 to each well. Observe the concentration density daily. Replace the culture medium every 2-3 days (always equilibrate the medium to room temperature before use). h. Repeat steps 4a-4e, except that when cell proliferation is observed in the "Proliferation 2" plate, label the plate "Proliferation 3". Maintain cell proliferation in the "Proliferation 2" plate by adding 2 ml of PTT6 to each well. Observe the concentration density daily. Replace the culture medium every 2-3 days (always equilibrate the medium to room temperature before use). i. Discard the tissue when proliferation is observed in the "Proliferation 3" plate. If the tissue is very small and does not hinder cell proliferation, discard the tissue during subculturing. j. Once the cells reach a density of 40-50%, monitor them daily to prevent excessive growth. k. When the cells reach a density of 70-80%, subculture the cells. Do not allow the cells to grow beyond a density of 80%.

[0142] When tissue explants are approximately 1–3 mm in size and tissue explant / cell culture is performed in a 175 mm square culture dish, the average number of mesenchymal stem cells recovered from the explants is typically around 4,000–6,000 cells per explant. Therefore, when mesenchymal stem cells are simultaneously grown from 48 explants, approximately 300,000 cells can be obtained at harvest. These 300,000 mesenchymal stem cells collected from the explants are then cultured in a 175 cm square culture dish as described in Example 2.5 below. 2 By seeding 300,000 such cells into a cell culture flask, they can be used for subculturing (this may be referred to as the first subculturing). Then, using the mesenchymal stem cells obtained from this first subculturing, they can be cultured again at 175 cm as described in Example 2.5 below. 2 The cells can be seeded in a flask (second passage) and increased in size. Cells obtained from both the first and second passages can be "banked" by cryopreservation, and mesenchymal stem cells obtained after the second passage are considered to represent a master cell bank, which can be used to further increase the mesenchymal stem cells, for example, in a bioreactor, as described in Example 2.7 below.

[0143] 2.5. Subculture of MSCs in a cell culture dish a. Perform biological particle counting while working inside the biosafety cabinet. Equilibrium all culture media to room temperature before use. b. When the cell proliferation reaches a concentration of approximately 70-80%, the cells are subcultured. i. Remove the PTT6 from the Petri dish. ii. Rinse with HBSS without calcium and magnesium. iii. Add 0.2 ml of 1× TrypLE-EDTA and swirl for 1 - 2 minutes. iv. Tilt the dish at 30 - 45° so that the cells can move downward by gravity flow. Gently tap the side of the plate to promote detachment. v. Add 1 ml of PTT6. Gently pipette up and down, then transfer the cells to a 15 ml centrifuge tube. Use a clean pipette tip for each well. Pool the cells from all 6 wells into a single 15 ml tube. vi. Centrifuge at 1200 rpm for 10 minutes. vii. Remove the supernatant and resuspend the cells in 5 ml of PTT6. c. Passage the MSCs. i. Aliquot 50 μl of the cell suspension and assay for TNC and viability by trypan blue exclusion assay. ii. Count the cells using a hemocytometer. Predict to count 20 - 100 cells / section. If the number is more than 100, dilute the original sample 1:5 and repeat the trypan blue method using a hemocytometer. iii. Count viable cells / ml and total viable cells: 1. Viable cells / ml = number of viable cells × dilution factor × 10 4 2. Total viable cells = number of viable cells × dilution factor × total volume × 10 4 iv. Count % viability: 1. % viability = number of viable cells × 100 / (number of viable cells + number of dead cells) v. Dilute the cell suspension to 1.0×10 6 cells / ml: 1. "X" volume = total viable cells / 10 6 cells / ml 2. For example, if the total number of viable cells is 1.0×10 7 cells; 3. "X" = 10 7 / 10 6cells / ml, i.e., 10 ml, and thus, by adding 5 ml to the cell suspension (which is 5 ml), the total cell volume is made 10 ml. vi. If the cell suspension is less than 10 6 cells / ml, determine the volume required to seed 2×10 2 cells into each 150 mm Petri dish or 175 cm 6 flask. 1. Volume for 2×10 6 cells = 2×10 6 cells ÷ viable cells / ml 2. For example, if the viable cells / ml is 8×10 5 cells / ml, then 2×10 6 cells ÷ 8×10 5 cells / ml, i.e., 2.5 ml, is required. vii. Aliquot 0.5 ml for MSC marker analysis. viii. Seed 2×10 6 cells into each 150 mm Petri dish or 175 cm 2 flask with 30 ml of PTT6. ix. Observe every 3 days for attachment, colony formation, and confluency. When the cells reach 40 - 50% confluency, observe the cells daily to every 2 days to prevent overgrowth. Do not allow the cells to grow beyond 80% confluency. Instead of an optical microscope, a real-time cell culture monitoring system can be used. x. Replace the medium every 2 - 3 days.

[0144] 2.6 Cryopreservation of MSC cells a. Perform bioparticle counting during work in the biosafety cabinet. b. When the cells reach 70 - 80% confluency, detach the cells from each 150 mm Petri dish or 175 cm 2 flask using 2 ml of 1×TrypLE-EDTA. i. Remove PTT6 from the Petri dish. ii. Wash with 5 ml of HBSS or PBS that does not contain calcium and magnesium. iii. Add 2 ml of 1×TrypLE-EDTA and swirl for 1-2 minutes. iv. Tilt the dish 30-45° to allow the cells to move downwards due to gravity. Gently tap the sides of the petri dish to help facilitate detachment. Add 10 ml of v. PTT6 to inactivate TrypLE. Mix thoroughly to dissociate the cell clumps. vi. Using a Pasteur pipette, transfer the cells to a 15 ml centrifuge tube. vii. Centrifuge at 1200 rpm for 10 minutes. viii. Aspirate the culture medium and resuspend it in 10 ml of PTT6. ix. Dispense 50 μl and determine the total number of viable cells and the percentage of viability as described above. Cell counting should be performed within 15 minutes, as cells may begin to aggregate. c. Prepare cells for cryopreservation. i. Prepare cell suspension media and cryopreservation media, and freeze the cells.

[0145] 2.7. Subculturing (enlargement) of MSCs in a Quantum Bioreactor (Terumo BTC, Inc.) It is also possible to increase the number of MSCs using a Quantum bioreactor. The starting cell count for growth in a Quantum bioreactor should be 20 to 30 million cells per run. A typical yield per run is 300 to 700 million MSCs at harvest. The bioreactor is operated according to the manufacturer's protocol. The mesenchymal stem cells thus obtained are typically cryopreserved (see below) and become a working cell bank.

[0146] Materials / Reagents: 1. Quantum Enlargement Set 2. Quantum waste liquid bag 3. Quantum culture medium bag 4. Quantum Inlet Bag 5. PTT6 6. PBS 7. Fibronectin 8. TrypLE 9. 3 ml Syringe 10. Glucose Test Strip 11. Lactate Test Strip 12. 60 ml Cell Culture Plate or Equivalent 13. Medical Grade 5% CO2 Gas Mixture 14. 50 ml Combichip

[0147] Equipment: 1. Biosafety Cabinet 2. Glucose Meter (Bayer Healthcare / Ascensia Contour Blood Glucose Meter) 3. Lactate Plus (Nova Biomedical) 4. Peristaltic Pump with Head 5. Centrifuge, Eppendorf 5810 6. Sterile Tube Connector 7. M4 Continuous Pipettor 8. RF Sealer

[0148] Procedure: 1. Preparation of Quantum Bioreactor a) Pre - preparation of Quantum Bioreactor b) Coating of Bioreactor: 1) Prepare fibronectin solution in a biosafety cabinet. 1) Equilibrate lyophilized fibronectin to room temperature (≥15 minutes at room temperature). 2) Add 5 ml of sterile distilled water; do not swirl or stir. 3) Make fibronectin into solution over 30 minutes. 4) Transfer the fibronectin solution to the cell inlet bag containing 95 ml of PBS using a 10 ml syringe with an 18g needle. 2) Connect the bag to the "reagent" line. 3) Check for air bubbles (air bubbles can be removed by using "IC air removal" or "EC air removal" and by using "cleaning" as the inlet supply source). 4) Open or set the bioreactor coating program (Figure 1, steps 3-5). 5) Run the program. 6) While the program is running and coating the bioreactor, prepare a 4 L medium bag of PTT6 medium. 7) Connect the culture medium bag to the IC culture medium line using a sterile tube connector. 8) Once the bioreactor coating stage is complete, remove the cell inlet bags used with the fibronectin solution using an RF sealer. c) Washing away excess fibronectin d) Acclimatization of the bioreactor with culture medium 2. Cell culture in a Quantum bioreactor a) Cell loading and adhesion using a homogeneous suspension: b) Nutritional support and culture of cells 1) Select the culture medium flow rate to supply nutrients to the cells. 2) Sample lactate and glucose daily. 3) Adjust the flow rate of the culture medium as the lactate level rises. The actual maximum acceptable lactate concentration is determined by the flask culture from which the cells originated. Ensure that there is sufficient PTT6 medium in the medium bag. Replace the PTT6 medium bag with a new one if necessary. 4) Once the flow rate reaches the desired value, measure the lactate level every 8-12 hours. If the lactate level does not decrease, or if the lactate level continues to rise, collect the cells. 3. Cell recovery from the Quantum bioreactor a) Once lactate concentration has not decreased, collect the cells after the final sampling for lactate and glucose. b) Cell retrieval: 1) Using a sterile tube connector, connect the cell inlet bag filled with TrypLE 100 ml to the "reagent" line. 2) Ensure there is enough PBS in the PBS bag. If not, use a sterile tube connector to connect a new bag containing at least 1.7 liters of PBS to the "wash" line. 3) Execute the recovery program. 4. Cryopreservation of cells 1) Once the cells have been collected, transfer them to a 50 ml centrifuge tube to pellet them. 2) Resuspend the cells in 25 ml of cold cell suspension solution. Count the cells using a Sysmex or Biorad cell counter. Attach the cell count report to each Quantum processing batch record. 3) Cell concentration 2 × 10 7 Adjust to pieces / ml. 4) Add an equal volume of cryopreservation solution and mix thoroughly (do not shake or vortex). 5) Using a serial pipette, add 1 ml of the cell suspension in the cryopreservation agent to each 1.8 ml vial. Cryopreserve using a controlled-speed freezer with the CRF program as described in SOP D6.100 CB cryopreservation. 6) Store the vials in the designated liquid nitrogen storage space. 7) Attach the CRF execution report to the form for each MSC P3-Quantum processing batch.

[0149] 3. Analysis of stem cell marker expression in mesenchymal umbilical cord-lining stem cell populations isolated from umbilical cord tissue using different culture media. Flow cytometry experiments were performed to analyze the expression of mesenchymal stem cells isolated from the umbilical cord for the mesenchymal stem cell markers CD73, CD90, and CD105.

[0150] For these experiments, mesenchymal stem cells were isolated from umbilical cord tissue by culturing the tissue in three different culture media, as described in Example 2, and then the mesenchymal stem cells were subcultured in each medium.

[0151] In these experiments, the following three culture media were used: a) 90% (v / v / DMEM) supplemented with 10% FBS (v / v), b) culture medium PTT-4 as described in U.S. Patent Application US2008 / 0248005 and the corresponding International Patent Application WO2007 / 046775 (see paragraph

[0183] of WO2007 / 046775), consisting of 90% (v / v) CMRL1066 and 10% (v / v) FBS, and c) culture medium PTT-6 of the present invention, whose composition is described herein. In this flow cytometry analysis, two different samples of umbilical cord-lined mesenchymal stem cell (CLMC) populations were analyzed for each of the three culture media used.

[0152] The following protocol was used for flow cytometry analysis.

[0153] material and method TIFF0007861268000005.tif230150

[0154] procedure a) Isolation and culture of cells from the umbilical cord lining membrane 1. As described in Example 2, the explant tissue samples were incubated in cell culture plates, immersed in each culture medium, and then maintained in a CO2 incubator at 37°C. 2. The culture medium was changed every 3 days. 3. Cell proliferation from tissue culture explants was monitored under a light microscope. 4. At approximately 70% compaction, the cells were separated from the dish by trypsin treatment (0.0125% trypsin / 0.05% EDTA) and used for flow cytometry experiments. b) Trypsin treatment of experimental cells 1. Remove the culture medium from the cell culture plate. 2. Since FBS interferes with the enzymatic action of trypsin, gently rinse with sterile 1x PBS to remove any trace amounts of FBS. 3. Add 1X trypsin to the cell culture plate and incubate at 37°C for 3-5 minutes. 4. Observe the cells under a microscope to ensure they have been removed. Neutralize the trypsin by adding complete medium containing FBS (DMEM containing 10% FBS). 5. Using a pipette, break up cell clumps by pipetting the cells against the plate wall in the culture medium. Collect the cell suspension and transfer it to a 50 ml centrifuge tube. 6. Add sterile 1×PBS to the plate, rinse it, and collect the cell suspension in the same centrifuge tube. 7. Centrifuge this at 1800 rpm for 10 minutes. 8. Discard the supernatant and resuspend the cell pellet in PBA medium. c) Cell counting 1. Preferably, the hemocytometer and its coverslip should be washed with 70% ethanol, dried, and then wiped with Kimwipes (lint-free paper) to ensure they are clean and dry. 2. Transfer a small amount of the suspended cells into a microcentrifuge tube and remove it from the BSC hood. 3. Stain the suspended cells with an equal volume of trypan blue. For example, add 500 μl of trypan blue to 500 μl of suspension (dilution factor = 2X, resulting in a 0.2% trypan blue solution). 4. Trypan blue is toxic and can lead to an increase in non-viable cells and the production of pseudocell counts; therefore, cells should not be exposed to trypan blue for longer than 30 minutes. 5. Add 20 μl of the cell suspension mixture to each chamber of the hemocytometer and observe under a light microscope. a. For a total of eight compartments in the upper and lower chambers, count the number of viable cells (bright cells; non-viable cells readily absorb trypan blue and are therefore darker in color) in each compartment of the hemocytometer. The total cell count is (average cell count / compartment) × 10 4 It is given as cells / ml. d) Cell staining i. Preparation before staining cells Each cell suspension, containing 50,000 cells, is divided into three tubes (CD73, CD90, CD105) in pairs and two tubes (negative control). ii. Staining with primary antibody (Ab) Add 1 μl [0.5 mg / ml Ab] of primary antibody to 100 μl of cell suspension and incubate at 4°C for 45 minutes. • Adjust to 1 ml with PBA. Centrifuge at 8000 rpm for 5 minutes at 4°C. Remove the supernatant. Add 1 ml of PBA and resuspend the pellet. Centrifuge at 8000 rpm for 5 minutes at 4°C. Remove the supernatant. Resuspend in 100 µl of PBA. iii. Staining with secondary Ab - Under the dark Add 1 µl [0.5 mg / ml ab] of secondary antibody to 100 µl of cell suspension and incubate at 4°C for 30 minutes. • Adjust to 1 ml with PBA. Centrifuge at 8000 rpm for 5 minutes at 4°C. Remove the supernatant. Add 1 ml of PBA and resuspend the pellet. Centrifuge at 8000 rpm for 5 minutes at 4°C. Remove the supernatant. • For flow cytometry analysis, resuspend in 200-300 µl of PBA. Transfer the cells to a FACS tube for reading using BD FACS CANDO flow cytometry.

[0155] The results of flow cytometry analysis are shown in Figures 6a to 6c. Figure 6a shows the percentage of isolated mesenchymal umbilical cord-lining stem cells expressing the stem cell markers CD73, CD90, and CD105 after isolation and culture from umbilical cord tissue in DMEM / 10% FBS; Figure 6b shows the percentage of isolated mesenchymal umbilical cord-lining stem cells expressing the stem cell markers CD73, CD90, and CD105 after isolation and culture from umbilical cord tissue in PTT-4; and Figure 6c shows the percentage of isolated mesenchymal umbilical cord-lining stem cells expressing the stem cell markers CD73, CD90, and CD105 after isolation and culture from umbilical cord tissue in PTT6-. As can be seen from Figure 6a, the population isolated using DMEM / 10% FBS as the culture medium had approximately 75% CD73+ cells, 78% CD90+ cells, and 80% CD105+ cells (average of two experiments), whereas the number of CD73-positive, CD90-positive, and CD105-positive mesenchymal stem cells after isolating / culturing umbilical cord tissue using PTT-4 culture medium (see Figure 6b) was approximately 87% (CD73+ cells), 93% (CD90+ cells), and 86% (CD105+ cells) on average of two experiments. The purity of the mesenchymal stem cell population obtained by culturing in the PTT-6 medium of the present invention was at least 99.0% for all three markers (CD73, CD90, CD105), which means that the purity of this cell population is significantly higher than that of culture using PTT-4 medium or DMEM / 10% FBS. Furthermore, and more importantly, the mesenchymal stem cell population obtained by culturing in PTT-6 is essentially a 100% pure and distinct stem cell population. This makes the stem cell population of the present invention an ideal candidate for stem cell-based therapies. Thus, this population of mesenchymal umbilical cord-lining stem cells can serve as the optimal standard for such stem cell-based therapeutic approaches.

[0156] The findings shown in Figure 6 are further supported by the results of flow cytometry analysis shown in Figures 7a and 7b. Figure 7a shows the percentage of isolated mesenchymal umbilical cord-lining stem cells (mesenchymal stem cells of the amniotic membrane of the umbilical cord) that expressed the stem cell markers CD73, CD90, and CD105, and lacked the expression of CD34, CD45, and HLA-DR, after isolation and culture from umbilical cord tissue in PTT-6 medium. As shown in Figure 7a, the mesenchymal stem cell population contained 97.5% viable cells, 100% of which expressed CD73, CD90, and CD105 respectively (see the "CD73+CD90+" and "CD73+CD105+" columns), while 99.2% of the stem cell population did not express CD45, and 100% of the stem cell population did not express CD34 and HLA-DR (see the "CD34-CD45-" and "CD34-HLA-DR-" columns). Therefore, the mesenchymal stem cell population obtained by culturing in PTT-6 medium is essentially a 100% pure and distinct stem cell population that meets the criteria for enabling mesenchymal stem cells to be used in cell therapy (95% or more of the stem cell population expresses CD73, CD90, and CD105, while 98% or more of the stem cell population lacks expression of 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 is noted herein that the amniotic mesenchymal stem cells of the present invention are adherent to plastic under standard culture conditions, differentiate into osteoblasts, adipocytes, and chondrocytes in vitro, and meet generally accepted standards for the use of mesenchymal stem cells in cell therapy, see U.S. Patent No. 9,085,755, U.S. Patent No. 8,287,854, or WO2007 / 046775.

[0157] Figure 7b shows the proportion of isolated bone marrow mesenchymal stem cells expressing CD73, CD90, and CD105, and lacking expression of CD34, CD45, and HLA-DR. As shown in Figure 7b, the bone marrow mesenchymal stem cell population contained 94.3% viable cells, of which 100% expressed CD73, CD90, and CD105 respectively (see the "CD73+CD90+" and "CD73+CD105+" columns), while only 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 the "CD34-CD45-" and "CD34-HLA-DR-" columns). Therefore, bone marrow mesenchymal stem cells, considered to be the optimal standard for mesenchymal stem cells, exhibit far less uniformity / purity with respect to stem cell markers than the mesenchymal stem cell population (from the umbilical cord amniotic membrane) of this application. This finding also indicates that the stem cell population of the present invention could be an ideal candidate for stem cell-based therapies and could serve as the optimal standard for stem cell-based therapeutic approaches.

[0158] 4. Experiments demonstrating that the mesenchymal stem cell population of the present invention can be transported / stored in HypoThermosol®: To analyze the health and viability of the mesenchymal stem cells described herein in different storage or transport carriers, two different carriers were compared to each other. Specifically, carrier HypoThermosol®-FRS was compared to carrier PlasmaLyte-A. Both are commercially available. The product sheet for HypoThermosol®-FRS is shown in Figure 30, and its composition is described elsewhere herein. Each 100 mL of PlasmaLyte contains 526 mg of sodium chloride, USP (NaCl); 502 mg of sodium gluconate (C6H) 11 It contains 368 mg of sodium acetate trihydrate (USP (C2H3NaO2·3H2O)), 37 mg of potassium chloride (USP (KCl)), and 30 mg of magnesium chloride (USP (MgCl2·6H2O)). PlasmaLyte does not contain antibacterial agents. The pH of PlasmaLyte is adjusted to 7.4 (6.5-8.0) with sodium hydroxide.

[0159] The experimental setup for comparison is shown in Figure 8. First, the mesenchymal stem cell population described herein was grown in cell culture flasks. The number of viable mesenchymal stem cells was counted, and then 2 million cells / vial were stored for various periods in either PlasmaLyte-A or HypoThermosol®-FRS. After storage, cells were counted daily from days 1 to 5 using ≤50 μl of sample (total liquid volume 250 μl), and viability was examined by staining the cells with trypan blue. Furthermore, ≤80 μl of sample was collected and analyzed on days 1, 3, and 5. In addition, the supernatant was obtained and frozen. Subsequently, PDGF-AA, PDGF-BB, VEGF, IL-10, Ang-1, HGF, and TGFβ1 were measured using the FLEXMAP 3D system.

[0160] Figure 9 summarizes the survival data. As can be seen from the graph on the left, after 7 days of storage in HypoThermosol®, 73% of all cells (approximately 95%) that were stored at the start of storage were still viable. In contrast, after 7 days of storage in PlasmaLyte-A, only 42% of all cells (approximately 94%) that were stored at the start of storage were still viable. All counts are based on paired readings within 10% of each other (according to SOP CR D2.600.1). Among the counts, cells stored in HypoThermosol® were significantly smaller, with smoother and more defined contours. In contrast, cells in Plasmalyte-A appeared in a variety of sizes. HypoThermosol® significantly supports membrane integrity and possibly survival over a period of one week (6 days). Similar results are also shown in the graph on the right.

[0161] Figure 10 shows the results obtained when measuring the cell diameter of the cells. The mesenchymal stem cell population described herein, when maintained in HypoThermosol®, exhibits a narrower diameter range compared to cells maintained in PlasmaLyteA. The comparison was made after 3 days of storage.

[0162] Figure 11 shows the TGFβ1 concentration in the supernatant 48 hours after storage from the mesenchymal stem cell populations described herein, stored in HypoThermosol® or PlasmaLyte-A. As can be seen from the graph on the right, cells secrete approximately the same amount of TGFβ1 when stored in HypoThermosol® and when stored in PlasmaLyte-A. Generally, the amount of TGFβ1 secreted decreased over time (graph on the right).

[0163] Figures 12 and 13 show the control experiment. Here, PDGF-BB and IL-10 concentrations were measured in the supernatant of the mesenchymal stem cell population described herein, stored in HypoThermosol® or PlasmaLyte-A, after 48 hours. Since neither PDGF-BB nor IL-10 are normally secreted by the mesenchymal stem cell population described herein, neither PDGF-BB nor IL-10 were detectable in any of the samples.

[0164] Figure 14 shows the VEGF concentrations in the supernatant after 48 hours from the mesenchymal stem cell populations described herein, stored in HypoThermosol® or PlasmaLyte-A. As can be seen from the graph on the right, on day 0, cells secrete approximately the same amount of VEGF when stored in HypoThermosol® or PlasmaLyte-A. On days 1 and 5, cells secreted more VEGF when stored in PlasmaLyte-A. Notably, after 3 days of storage, cells secreted more VEGF when stored in HypoThermosol® than when stored in PlasmaLyte-A. Therefore, after 3 days of storage, HypoThermosol® is superior to PlasmaLyte-A. The more VEGF detected, the healthier the culture. Therefore, cells are healthier in HypoThermosol (trademark) than in PlasmaLyte-A after 3 days of storage, by secreting more VEGF than those stored in PlasmaLyte-A. After 5 days of storage, PlasmaLyte appears to become a more favorable carrier, because at 5 days, cells stored in PlasmaLyte-A secreted more VEGF. Generally, the amount of secreted VEGF decreased over time (graph on the right).

[0165] Figure 15 shows the PDGF-AA concentrations in the supernatant after 48 hours from the mesenchymal stem cell populations described herein, stored in HypoThermosol® or PlasmaLyte-A. As can be seen from the graph on the right, on day 0, cells stored in HypoThermosol® secreted approximately the same amount of PDGF-AA compared to those stored in PlasmaLyte-A. On days 1 and 5, cells secreted more PDGF-AA when stored in PlasmaLyte-A. Notably, after 3 days of storage, cells stored in HypoThermosol® secreted more PDGF-AA than those stored in PlasmaLyte-A. Therefore, after 3 days of storage, cells stored in HypoThermosol® are healthier than cells stored in PlasmaLyte-A. After 5 days of storage, PlasmaLyte appears to become a more favorable carrier because, at 5 days, cells stored in PlasmaLyte-A secreted more PDGF-AA. Generally, the amount of secreted PDGF-AA decreased over time (graph on the right).

[0166] Figure 16 shows the Ang-1 concentration in the supernatant of the mesenchymal stem cell populations described herein, stored in HypoThermosol® or PlasmaLyte-A, over 48 hours. As can be seen from the graph on the right, cells secrete approximately the same amount of Ang-1 on days 0 and 3 when stored in HypoThermosol® or PlasmaLyte-A. On day 5, cells secreted more Ang-1 when stored in PlasmaLyte-A. Notably, after 1 day of storage, cells secreted significantly more Ang-1 when stored in HypoThermosol® than when stored in PlasmaLyte-A. Therefore, cells stored in HypoThermosol® appear to be healthier than those stored in PlasmaLyte-A, at least for 48 hours up to 3 days of storage. After 5 days of storage, PlasmaLyte appears to become a more favorable carrier because, at this point, cells stored in PlasmaLyte-A secrete more Ang-1. Generally, the amount of Ang-1 secreted decreased over time (graph on the right).

[0167] Figure 17 shows the HGF concentrations in the supernatant 48 hours after storage from the mesenchymal stem cell populations described herein, stored in HypoThermosol® or PlasmaLyte-A. As can be seen from the graph on the right, on day 0, cells stored in HypoThermosol® secreted approximately the same amount of HGF as those stored in PlasmaLyte-A. On days 3 and 5, cells secreted more HGF when stored in PlasmaLyte-A. Notably, after 1 day of storage, cells stored in HypoThermosol® secreted significantly more HGF than those stored in PlasmaLyte-A. Therefore, cells stored in HypoThermosol® appear to be healthier than cells stored in PlasmaLyte-A for at least 1 day (48 hours) up to 3 days of storage. From day 3 onward, PlasmaLyte-A appears to become a more favorable carrier because, at days 3 and 5, cells stored in PlasmaLyte-A secreted more HGF. Generally, the amount of HGF secreted decreased over time (graph on the right).

[0168] In summary, based on the data above, we can conclude that storage of the mesenchymal stem cell population of the present invention in HypoThermosol® is superior to storage in PlasmaLyte-A, particularly with respect to storage during the first three days.

[0169] 5. Experiments demonstrating that the mesenchymal stem cell population of the present invention possesses wound-healing properties through local treatment of pigs: Preclinical trials were also conducted using 10-week-old female Yorkshire Landrace pigs (50 kg). The procedure was performed at the SingHealth Experimental Medicine Centre in Singapore. The pigs were induced with diabetes using 120 mg / kg streptozotocin, allowed to recover for 45 days, and then six 5 cm × 5 cm full-thickness wounds were created on their backs (see Figure 18). Two pigs (n = 2) were subjected to a 1 cm wound. 2 10 hits 5Two control pigs were treated with the human mesenchymal stem cell population described herein, twice a week for four weeks. Two control pigs were treated with PBS. Wounds were photographed on postoperative day 0 (PO day 0) and every seven days until postoperative day 35. Wounds were analyzed for surface area size using ImageJ. By day 35, the addition of the mesenchymal stem cell population described herein resulted in closure of 10 out of 12 diabetic wounds (83%) compared to only 3 out of 12 (25%) in the PBS-treated control wounds. The rate of wound healing was 0.6 cm in the control animals. 2 Compared to / day, using the mesenchymal stem cell population described herein, 0.8 cm 2 The improvement was 33% per day. The results of this study are summarized in Figure 18.

[0170] Although the pig model is not naturally occurring, its skin structure is the most similar to that of humans. This data suggests that the umbilical cord-lined mesenchymal stem cell population of the present invention improves wound healing without the risk of serious adverse side effects. Therefore, these data strongly support the hypothesis that the human umbilical cord-lined mesenchymal stem cell population described herein can promote the healing of chronic wounds by suppressing inflammation and promoting angiogenesis. Furthermore, no signs of inflammation were clearly present when heterogeneous mesenchymal stem cells were used in either mice or pigs, and therefore, the likelihood of allogeneic mesenchymal stem cells causing any serious adverse effects in humans is very low.

[0171] 6. Experiments demonstrating the efficacy of the mesenchymal stem cells described herein in topical treatments for humans: Experiments demonstrating the efficacy of the mesenchymal stem cells described herein in topical treatments for humans are described in WO2007 / 046775. In particular, as described in Examples 23-26 of WO2007 / 046775, mesenchymal stem cells (UCMCs) from the amniotic membrane of the umbilical cord were able to alleviate full-thickness burns (Example 23), partial-thickness wounds (Example 24), non-healing radiation wounds (Example 25), and non-healing diabetic wounds and non-healing diabetic foot wounds (Example 26). Notably, according to Example 2 of WO2007 / 046775, the mesenchymal stem cells were resuspended in PTT-4 medium.

[0172] Notably, as shown in Figures 6b and 6c, the stem cell population obtained by culturing using PTT6 (as used herein) medium is significantly more homogeneous than the cell population obtained by using PTT4 medium (as used in WO2007 / 046775). Since PTT-4 was used as the medium for mesenchymal stem cells in Examples 23-26 of WO2007 / 046775, it is clear that the even more homogeneous mesenchymal stem cell population isolated after culturing in PTT-6 (as used herein) has similar beneficial effects in wound healing applications such as full-thickness burns, partial-thickness wounds, non-healing radiation wounds, and non-healing diabetic wounds and non-healing diabetic foot wounds.

[0173] 7. Experiments demonstrating the efficacy of the mesenchymal stem cells described herein in topical treatments for humans: This is a planned dose-escalation study of the mesenchymal stem cell population obtained as described herein, conducted at the University of Colorado Anschutz Medical Campus in Aurora, Colorado. The objective of this study is to determine a safe dose of the mesenchymal stem cell population (human umbilical cord-lined mesenchymal stem cells) described herein. This is a single-center dose-escalation study involving a total of 15 subjects, with 5 subjects enrolled in each of the three dose levels. Group 1, consisting of 5 patients, received an MSC count of 100,000 cells / cm³. 2(Skin / wound area) treatment is administered twice a week for 8 weeks. Group 2, consisting of 5 patients, had a MSC count of 300,000 cells / cm². 2 The treatment is administered twice a week for eight weeks. Group 3, consisting of five patients, had a MSC count of 500,000 cells / cm². 2 The drug is administered twice a week for eight weeks. This schedule is continued until the highest dose is reached, or until at least two subjects at a dose level experience an allergic reaction of ≥Grade 2 suspected to be related to the mesenchymal stem cell population obtained herein, or until two or more subjects at a dose level experience an unexpected, treatment-related serious adverse event or dose-limiting toxicity within 14 days after the initial dose of the mesenchymal stem cell population obtained as described herein. All patients will be evaluated for anti-HLA antibody production and wound closure 30 days after treatment. At present, HLA antibody production is not considered an absolute contraindication at any particular dose, but it will be a factor in the overall safety assessment. This is an open-label study, and all subjects will take the study drug, and all investigators will know the dose each subject receives. The secondary endpoint of this study is significant improvement in wound condition. These endpoints are based on the rate of wound closure, the percentage of wound area that closed successfully, and the percentage of wounds that closed completely, as measured using the Silhouette Wound Measurement and Documentation System. This device is FDA approved for this purpose.

[0174] Subject population Patients with type 1 or type 2 diabetes who have chronic foot ulcers that have not healed after at least 30 days of conventional treatment and who are negative for HLA antibodies against the mesenchymal stem cell population described herein. Patients will continue conventional wound care for the first two weeks starting at the time of enrollment, at which point they will have already been screened for diabetic foot ulcers that have not healed for 30 days. At this point, photographic recording and measurement of wound parameters will begin. Conventional dressings will be changed twice a week for the first two weeks, and thereafter the mesenchymal stem cell population described herein will be applied to the wound at the specified concentration twice a week. Wounds treated with the mesenchymal stem cell population described herein will also be covered with Tegaderm® and crepe dressings.

[0175] Dose level The purpose of this study is to determine a safe dose of human umbilical cord-lined mesenchymal stem cells as described herein for further testing. Patients will be given three doses: 100,000 cells / cm². 2 Skin / wound area, 300,000 cells / cm² 2 , or 500,000 cells / cm² 2 One of these treatments is administered twice a week for eight weeks. Each dose of 100,000 cells represents 0.1 ml of the mesenchymal stem cell population described herein, from a vial containing 1 million cells / ml in HypoThermosol.

[0176] Dosage plan This is a safety and tolerability study of the escalating doses of mesenchymal stem cells described herein. The objective of this study is to determine a safe dose of human umbilical cord-lined mesenchymal stem cells described herein for further study. Five subjects will be enrolled in each of the three dose levels. Group 1, consisting of five patients, will receive 100,000 MSCs / cm². 2 The skin / wound site receives treatment twice a week for 8 weeks. Group 2, consisting of 5 patients, had MSC levels of 300,000 cells / cm². 2 The treatment is administered twice a week for eight weeks. Group 3, consisting of five patients, had a MSC count of 500,000 cells / cm².2 The drug is administered twice a week for eight weeks. This schedule is continued until the highest dose is reached, or until at least two subjects at a dose level experience an allergic reaction of ≥Grade 2 suspected to be related to the mesenchymal stem cells described herein, or until two or more subjects at a dose level experience an unexpected, treatment-related serious adverse event or dose-limiting toxicity within 30 days after the initial dose of the mesenchymal stem cell population described herein. All patients will be evaluated 30 days after treatment for anti-HLA antibody production and the degree of wound closure. At present, HLA antibody production is not considered an absolute contraindication at any particular dose, but it will be a factor in the overall safety assessment. This is an open-label study, and all subjects will take the study drug, and all investigators will know the dose each subject receives.

[0177] Route of administration The mesenchymal stem cell population described herein is applied topically to a cleaned diabetic foot ulcer and held in place with a Tegaderm® bandage.

[0178] Administration Procedure After appropriate wound cleaning as needed, position the patient prone with the affected leg bent at a 90° angle. Gently swirl this vial of the mesenchymal stem cell population described herein to ensure even distribution of cells. Then, using a sterile syringe, dispense 1 cm of the vial. 2 The elevated foot is treated by extracting 100,000 (0.1 ml) to 500,000 (0.5 ml) cells from each foot and placing them in the center of the wound. The wound is then sealed with a Tegaderm® membrane and gently massaged to distribute the cells evenly. The foot is kept elevated for 5 minutes to allow the cells to settle and adhere. The foot is then wrapped with a crepe bandage to cover the Tegaderm® dressing.

[0179] 8. Preparation of mesenchymal stem cell storage or transport formulations (including stem cell populations in which more than 99% of cells express CD73, CD90, and CD105 respectively, and lack expression of CD34 and HLA-DR) Preparation for processing after the fourth cell passage (Stage 4 processing): Stage 4 processing is typically performed in an environmental monitoring (EM) cleanroom. The necessary solutions and equipment must be prepared in advance for use.

[0180] Transfer the cells from the cryovial to a labeled 50 ml centrifuge tube.

[0181] Use complete PTT6 medium that has been removed from the refrigerator within 5 minutes (record the time the PTT6 medium is removed from the refrigerator). Slowly add 9 ml of complete PTT6 medium to the cells while gently swirling to promote mixing.

[0182] Centrifuge at 1200 rpm at room temperature (15-25°C) for 5 minutes to pellet the cells. Record the use of the centrifuge on the Centrifuge Periodic Preventive Maintenance Record Sheet - CR, and verify its performance according to the SOP Centrifuge Preventive Maintenance.

[0183] Remove the supernatant, resuspend the cells in sufficient complete PTT6 medium, and count them.

[0184] Counting: Cell counting is performed in TC20 (with or without trypan blue) to determine cell concentration. TC20 is suitable for a wide range of cell concentrations (5 × 10⁶). 4 ~1 × 10 7 Since it corresponds to individual cells / ml, sample dilution is usually not necessary.

[0185] To determine the survival rate, count cells in a hemocytometer according to the same SOP. Ensure that a total of at least 200 cells are counted.

[0186] If both viability and cell concentration are desired via a hemocytometer, the suspension may need to be diluted to correspond to the hemocytometer's range (20-100 cells per outer square). If an estimated 10 million thawed cells are resuspended, a volume of 6 ml should provide that range.

[0187] Therapeutic culture (P4): Seed 300,000 live cells per 175 cm² flask in 30 ml of complete PTT6 medium and incubated at 35–39°C and 4–6% CO2. Ensure the incubator preventive maintenance is up-to-date according to the SOP Incubator Preventive Maintenance and General Use guidelines. Label the flasks with P1–P4 MSC treatment labels.

[0188] Once most of the cells have adhered (preferably overnight), perform a rough inspection of the indicator flask under an inverted Nikon microscope located in a cleanroom to determine if there are any areas in the flask containing significantly high cell density. If so, use those areas for continuous monitoring with CytoSmart. If seeding is done in multiple flasks, a single flask may be used as a representative "indicator" flask. As an option, set CytoSmart email alert notifications to 60%, 70%, and 80% density relative to the "indicator" flask.

[0189] Every 2-3 days, replace the culture medium with 30 ml of fresh, pre-warmed complete PTT6 per flask and continue the incubation.

[0190] When the cell proliferation reaches a density of 80% ± 10%, the MSCs are harvested as follows: Rinse each flask with 10 ml of Ca2+ and Mg2+-free HBSS.

[0191] Add 5 ml of 1×TrypLE to each flask. Tilt the flask to coat the entire surface, then tilt the flask again and immediately aspirate and remove most of the TrypLE using a sterile serum pipette, leaving only enough TrypLE to cover the surface. Discard the aspirated TrypLE.

[0192] Desorb the cells (10-20 minutes at 15-25°C). Tilt the flask 30-45° to allow the cells to move downwards due to gravity. Gently tap the sides of the flask to promote desorption. Monitor the flask under an inverted microscope to confirm that all cells have desorbed.

[0193] Ca 2+ and Mg 2+ Add 5 ml of HBSS-free solution to the first flask. Gently pipette up and down, then transfer the cell suspension to the next flask. Repeat this process until cells have been collected from all flasks, then transfer to a 50 ml centrifuge tube labeled with a processing label.

[0194] New 5ml Ca 2+ and Mg 2+ Repeat this process with HBSS-free solution and mix with the suspension.

[0195] Under a microscope, confirm that all cells have been removed, and if necessary, repeat the process a third time to collect all cells from the flask.

[0196] Centrifuge the mixed cell suspension at 1200 rpm and 15-25°C for 5 minutes. Record the use of the centrifuge on the Centrifuge Periodic Preventive Maintenance Record Sheet - CR and verify its performance.

[0197] Prepare the recovered cell suspension: Remove the supernatant without disturbing the pellet, and resuspend the cells in 1.0 ml of complete PTT6 medium per flask using an appropriately sized serum pipette. The medium does not need to be preheated.

[0198] Resuspend the cells in complete PPT6 medium and centrifuge at 1200 rpm at room temperature for 5 minutes.

[0199] Remove the complete PTT6 supernatant without disturbing the pellet, and gently resuspend the pellet in 1.0 ml of "1% HSA in Plasmalyte" per flask using an appropriately sized serum pipette. This is the recovered cell suspension. From this point onward, keep the recovered cell suspension in a cooling block.

[0200] Count the recovered cell suspension: Prior to each sample for counting from the recovered cell suspension, ensure that the cells are thoroughly mixed.

[0201] To determine cell concentration (with or without trypan blue), count cells in a TC20 according to the SOP cell counting and viability assay. Since the TC20 accommodates a wide range of cell concentrations (5 × 10⁴ to 1 × 10⁷ cells / ml), sample dilution is usually not necessary.

[0202] To determine the survival rate, count cells in a hemocytometer according to the same SOP. Ensure that a total of at least 200 cells are counted.

[0203] Prepare the vial-filled suspension (place it in a 50ml conical flask and cool it in a cooling block): Based on the previous count of the recovered cell suspension, determine the volume of recovered cell suspension and "1% HSA in HypoThermosol" required to prepare the desired patient dose. Label the conical tube appropriately. The conical tubes containing the vial-filled suspension are kept in their own pre-cooled cooling blocks.

[0204] HypoThermosol and the prepared "1% HSA in HypoThermosol" should be stored and used within the refrigerated temperature range (2-8°C), and therefore the vial-filled suspension should be kept in a cooling block.

[0205] Record the volume of each component (HSA, Plasmalyte-A, and HypoThermosol-FRS) used to prepare this final suspension. Based on these volumes, also record the volumes of HSA, Plasmalyte, and HypoThermosol present in each AT-Closed Vial.

[0206] Count the vial-filled suspension: Prior to each sample for counting from the vial-filled suspension, ensure that the cells are thoroughly mixed.

[0207] To determine cell concentration (with or without trypan blue), count the cells in a TC20 according to the SOP cell counting and viability assay.

[0208] To determine survival rate, count cells in a hemocytometer according to the same SOP. Ensure that a total of at least 200 cells are counted. The survival rate test can only be performed once in the VLS.

[0209] Fill the AT-Closed Vial as follows: Take the pre-packaged syringe and needle out of the refrigerator and place them in the biosafety cabinet (BSC).

[0210] Remove the pre-filled AT-Closed Vial from the refrigerator during the CoolRackSV10 / XT Cooling Core assembly and place the device inside the BSC cabinet. Start the timer to ensure that filling is completed within 30 minutes.

[0211] Wipe the injection port with an alcohol swab.

[0212] Before filling the vial, insert a sterile 22G needle near the center of the stopper to puncture the vial (this is to avoid pressurizing the vial during filling).

[0213] Swirl the vial-loaded suspension to mix, then slowly draw it into the syringe without introducing air bubbles. Make a hole in the center of the stopper with the syringe and inject 1.0 ml into each AT-Closed Vial, taking care not to introduce air bubbles (read from meniscus to meniscus on the syringe).

[0214] Remove the filling syringe, then remove the pressure release needle.

[0215] Cover the vial port with the included cap and press firmly. Return to the CoolRackSV10, store at 2-8°C, and transport to destination.

[0216] Sample collection for cytokine evaluation after P4 (cytokine assays should be performed at least once for each lot number, i.e., for the same CBU # and donor tissue lot #): Based on the concentration of the vial-filled suspension described above, dispense a sufficient volume of the vial-filled suspension into at least one well of a 6-well plate so that a total of 100,000 (live + dead) cells are dispensed per well. Add the vial-filled suspension directly to the sufficient amount of complete PTT6 medium already added to each well so that the total volume in each well is 2 ml. Note the incubation start time.

[0217] Incubate for 48 hours ± 1 hour. Perform the following at the end of incubation.

[0218] Take one representative CytoSmart image, randomly positioned near the center of each well.

[0219] Measure lactate from each well and report the results on the lactate test results sheet.

[0220] Collect the culture medium from each well and centrifuge at 1200 rpm at room temperature for 5 minutes. Record the use of the centrifuge on the Centrifuge Periodic Preventive Maintenance Record Sheet - CR and verify its performance.

[0221] Dispense the culture medium supernatant into cryotubes and freeze within one hour of collection. Record the storage location in the batch record.

[0222] 9. Stability testing of MSC growth and metabolism during storage / transport. Cells from umbilical cord tissue and early passages have been stored at -195°C and tested for stability.

[0223] Initial stability testing of the mesenchymal umbilical cord-lining stem cells (MSCs) described herein (see Figure 7 for this regard), which have a purity of over 99% with respect to positive and negative markers, was performed on the final product consisting of viable MSCs in HypoThermosol® alone. Adhesion of the mesenchymal stem cells was observed during the actual manufacturing process as a result of the inherent properties of the mesenchymal stem cells and the viscosity of HypoThermosol®.

[0224] To reduce cell loss when MSCs are placed in HypoThermosol alone during distribution, to mitigate MSC adhesion to plastic at various stages of Stage 4 processing, and to maximize the recovery of the drug product from vials used during distribution, it has been found that the addition of two pharmaceutically inactive components, Plasmalyte and human serum albumin (HSA), can optimize the quality of the final drug product.

[0225] To confirm that the addition of these two pharmaceutically inactive ingredients does not adversely affect the stability of the final drug product, new stability tests were conducted. The results are shown in Figure 33.

[0226] Survival analysis Mesenchymal stem cells were seeded in AT-Closed Vials (registered trademark) at a rate of 106 cells per vial in 1 mL of Plasmalyte / HSA / HypoThermosol (registered trademark). Individual vials were sampled at various time points, and viability was manually assessed using trypan blue (hemocytometer). The total cell count was then tallied using an automated system (TC20).

[0227] To mimic the transport and storage of the product before application to wounds, MSCs were stored at 2–8°C for 1–3 days. As shown in Figure 33a, the cells did not show a significant decrease in viability up to 3 days under these conditions.

[0228] Visual Analysis MSCs were removed from AT-Closed Vials and cultured at 37°C for 24 hours before imaging. As can be seen below, cells obtained within 2 days under low-temperature storage adhered to tissue culture plates and formed typical spindle-shaped structures. After storage at 2–8°C for 2.5 days, the cells gradually took on a spherical shape, suggesting they were dying cells. The results are shown in Figure 33b.

[0229] Analysis of proliferation and metabolism MSCs from the same culture shown in Figure 33a were assayed for metabolic and growth indicators, specifically lactate production, over 48 hours of culture at 37°C. Cells stored at 2–8°C for 24 hours showed similar metabolism and growth to cells stored for 0 hours, while cells stored for 36 hours showed 86% of the control lactate production. Up to 72 hours at 2–8°C, subsequent culture showed only about 46% of the metabolic rate. The results are shown in Figure 33c.

[0230] Each vial was further tested on days 0, 1, 1.5, 2, 2.5, and 3, based on an established 3-day cell viability threshold. Trypan blue viability testing was performed immediately after cell removal from the sealed vials, and no significant decrease in viability was observed over 2.5 days (ranging from 92–98%). Cells were also placed in standard PTT6 medium at a concentration of 10⁵ cells / cm³. 2Cells were plated, and lactate production was measured at 24 and 48 hours. Lactate is a product of glucose metabolism, and the inventors have verified that it is directly proportional to the rate of cell growth in MSCs. Figure 33d shows lactate production by MSCs stored in Plasmalyte / HSA / HypoThermosol® for 0, 1, 1.5, 2, 2.5, or 3 days, and then measured at 24 and 48 hours of culture. Lactate production at 24 and 48 hours (day 1) by MSCs stored in Plasmalyte / HSA / HypoThermosol® for 24 hours was the same as that of unstored MSCs (day 0). By day 3, lactate production had decreased by 40-45%.

[0231] Analysis of cytokine production Cytokine production was measured from the same culture after 24 hours at 37°C. Consistent with metabolic data, when cells were stored at 2–8°C for 24 hours, the ability of MSCs to produce Ang-1, TGFβ, VEGF, and HGF was within 10–20% of the control (day 0). Results shown in Figure 33e indicate that when cells were stored in Plasmalyte / HSA / HypoThermosol® at 2–8°C for 24 hours, the ability of MSCs to produce VEGF, angiopoietin-1, TGF-β, and HGF was maintained. However, the ability of MSCs to produce VEGF and angiopoietin-1 decreased by approximately 50% after >2 days of storage. HGF results were similarly maintained for 24 hours but decreased by >70% after >2 days of storage. The TGF-β results indicate that when stored in Plasmalyte / HSA / HypoThermosol (registered trademark) at 2-8°C for >2 days, the ability of MSCs to produce TGF-β is maintained at approximately 75%.

[0232] The results obtained from the initial cytokine analysis were validated by further analysis of cytokine production in MSCs stored in Plasmalyte / HSA / HypoThermosol® for 0, 1, 1.5, 2, 2.5, or 3 days (Figure 33e). The results indicate that when cells were stored in Plasmalyte / HSA / HypoThermosol® at 2–8°C for 24 hours, the ability of MSCs to produce VEGF, angiopoietin-1, and TGF-β was maintained. Furthermore, after storage for >2 days, the secretion levels of VEGF and angiopoietin-1 decreased by approximately 50%, and the secretion level of TGF-β decreased by approximately 25%.

[0233] In summary, based on the viability, appearance, metabolism, and cytokine production demonstrated by the cells during these tests, an expiration date of 72 hours from the closure of the product vial can be set. Thus, since it can be reached by air transport to virtually any location in the world (developed countries) within 72 hours, the storage and transport formulation of the present invention essentially makes it possible to transport viable MSCs from an MSC production facility to virtually any location in the world where the MSCs can be administered to the target. Therefore, the storage and / or transport formulation of the present invention significantly reduces the complexity of GMP manufacturing and supply chains for pharmaceutically appropriate mesenchymal stem cell / stem cell populations, thereby making mesenchymal stem cell-based therapies more readily available to the general public.

[0234] The present invention is further characterized by the following: 1. A method for preparing a mesenchymal stem cell storage or transport preparation, The preparation contains approximately 500,000 to 10 million mesenchymal stem cells. a) A step of suspending mesenchymal stem cells in a predetermined volume of crystalloid solution containing approximately 0.5% to approximately 5% (w / v) serum albumin, thereby obtaining a first cell suspension. b) A step of determining the concentration of mesenchymal stem cells in the first cell suspension and determining the volume of the first cell suspension required to prepare a formulation containing approximately 500,000 to 10,000,000 mesenchymal stem cells. c) The determined volume of the first cell suspension Approximately 0.5% to 5% (w / v) of serum albumin, as well as the following: i) Torolox, ii) Na+, iii) K+, iv) Ca2+, v) Mg2+, vi) Cl-, vii) H2PO4-, viii) HEPES, ix) Lactobionate, x) Sucrose, xi) Mannitol, xii) Glucose, xiii) Dextran-40, xiv) Adenosine, and xv) Glutathione A liquid carrier of a certain volume containing The step of mixing with the mesenchymal stem cell storage or transport preparation containing approximately 500,000 to 10,000,000 mesenchymal stem cells. Methods that include... 2. The method of item 1, wherein the predetermined volume of the crystalloid solution used to suspend the mesenchymal stem cells is approximately 1 ml to approximately 10 ml. 3. The method of item 1 or 2, wherein the determined volume of the first cell suspension is mixed with the volume of the liquid carrier, and the total volume of the mesenchymal stem cell storage or transport preparation is approximately 1 ml. 4. The method described in item 2, wherein the formulation contains approximately 500,000 to 10 million living mesenchymal stem cells. 5. One of the methods described in items 1-4, wherein the formulation contains approximately 1 million, 3 million, or 5 million mesenchymal stem cells. 6. Any of the methods described above, in which "approximately" with respect to the number of mesenchymal stem cells means ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, or ±10%. 7. Any of the above methods, wherein the mesenchymal stem cells are recovered from the cell culture vessel before being resuspended in a predetermined volume of crystalloid solution. 8. Any of the above methods, wherein both the crystalloid solution and the liquid carrier contain serum albumin at the same concentration. 9. The method of item 8, wherein both the crystalloid solution and the liquid carrier contain approximately 0.5% to approximately 5% (w / v) of serum albumin. 10. The method of item 8 or 9, wherein both the crystalloid solution and the liquid carrier contain approximately 1% to approximately 5% (w / v) serum albumin. 11. Any method from items 8 to 10, wherein both the crystalloid solution and the liquid carrier contain approximately 1% to approximately 3% (w / v) serum albumin. 12. Any method from items 8 to 11, wherein both the crystalloid solution and the liquid carrier contain approximately 1% (w / v) serum albumin. 13. Any of the methods described above, wherein the serum albumin is human serum albumin. 14. Any of the above methods, wherein the crystalloid solution contains sodium, potassium, magnesium, and chloride. 15. Any of the methods described above, wherein the crystalloid solution is PlasmaLyte or Ringer's lactate solution. 16. The method of item 15, wherein the mesenchymal stem cell storage or transport preparation contains 20% or less PlasmaLyte. 17. Any of the methods described above, wherein the mesenchymal stem cells are mesenchymal stem cells selected from the group consisting of umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, umbilical cord-placental junction mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, bone marrow mesenchymal stem cells, and adipose tissue-derived mesenchymal stem cells. 18. The method of item 17, wherein umbilical cord mesenchymal stem cells are selected from a group consisting of amniotic mesenchymal stem cells, perivascular mesenchymal stem cells, Wharton's gelatinous mesenchymal stem cells, and amniotic mesenchymal stem cells of the umbilical cord. 19. The method of item 17 or 18, wherein the mesenchymal stem cells of the amniotic membrane of the umbilical cord constitute a mesenchymal stem cell population, and at least about 90% or more of the cells in the mesenchymal stem cell population express the following markers: CD73, CD90, and CD105, respectively. 20. At least approximately 90% or more of the cells in the mesenchymal stem cell population lack the expression of the following markers: CD34, CD45, and HLA-DR, according to the method of item 19. 21. The method of item 19 or 20, wherein at least about 91% 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, or about 99% or more of the cells in the mesenchymal stem cell population express CD73, CD90, and CD105 respectively, and lack expression of CD34, CD45, and HLA-DR (human leukocyte antigen-antigen D related) respectively. 22. Mesenchymal stem cell storage or transport preparations obtained by any of the methods specified in items 1 to 21. 23. Mesenchymal stem cell storage or transport preparations obtainable by any of the methods specified in items 1 to 21. 24. A method for transporting mesenchymal stem cells, The step of transporting mesenchymal stem cells in a mesenchymal stem cell storage or transport formulation as defined in item 22 or 23. Methods that include... 25. A method of transport described in item 24, where the transport takes place over approximately 7 days or less. 26. The method of item 24 or 25, where the transport takes place over approximately 6 days, approximately 5 days, approximately 4 days, approximately 3 days, approximately 2 days, approximately 1 day, or less than approximately 1 day. 27. Any method described in items 24-26, where the transport takes approximately 48 hours, approximately 24 hours, or less. 28. Transportation takes place at a temperature of approximately -5°C to approximately 15°C, using one of the methods described in items 24 to 27. 29. Transportation takes place at a temperature of approximately 2°C to 8°C using one of the methods described in items 24 to 28. 30. Transportation is carried out at temperatures above approximately -5°C, above approximately -10°C, above approximately -15°C, or above approximately -20°C, using any of the methods described in items 24-29. 31. A method for treating a subject with a disease, The step of locally administering mesenchymal stem cells stored or transported in a mesenchymal stem cell storage or transport formulation as defined in item 22 or 23. Methods that include... 32. The method of item 31, wherein mesenchymal stem cells are isolated from a mesenchymal stem cell storage or transport preparation, and the mesenchymal stem cells are administered to a subject. 33. The method of item 32, comprising centrifugation, for the isolation of mesenchymal stem cells from a mesenchymal stem cell storage or transport preparation. 34. The methods of items 32 and 33 for isolating mesenchymal stem cells from a mesenchymal stem cell storage or transport preparation, comprising removing a cell population from a vial by syringe. 35. Any method described in items 31-34, including the step of administering mesenchymal stem cells by syringe. 36. Any of the methods described in items 31-35, in which mesenchymal stem cells are administered in doses of approximately 3 million, 5 million, or 10 million cells. 37. A method of items 31-36 applied to a mesenchymal stem cell population within approximately 72 hours, approximately 48 hours, approximately 24 hours, approximately 12 hours, approximately 6 hours, or less from the time the mesenchymal stem cell population is harvested. 38. The method of item 37, wherein the mesenchymal stem cells are applied within approximately 72 hours, approximately 48 hours, approximately 24 hours, approximately 12 hours, approximately 6 hours, or less from the time the mesenchymal stem cells are collected. 39. The disease is a skin disease or wound, by any of the methods described in items 31-38. 40. A wound resulting from a burn, bite, trauma, surgery, or disease, as described in item 39. 41. The method of item 40, wherein the wound is caused by diabetes, and preferably the wound is a diabetic wound. 42. The wound is a diabetic foot ulcer, method of item 41. 43. A dose of approximately 10 million cells, approximately 5 million cells, approximately 4 million cells, approximately 3 million cells, approximately 2 million cells, approximately 1 million cells, approximately 500,000 cells, approximately 250,000 cells, or less than 250,000 cells, administered once or twice a week, using one of the methods described in items 31-42. 44. The method of item 43, wherein a dose of approximately 10 million cells, approximately 5 million cells, approximately 4 million cells, approximately 3 million cells, approximately 2 million cells, approximately 1 million cells, approximately 500,000 cells, approximately 250,000 cells, or less than 250,000 cells is administered once or twice per week over a period of 3, 4, 5, 6, 7, 8, 10 weeks, or longer. 45. Any method of items 31-44, wherein mesenchymal stem cells are applied locally and covered with a film or bandage. 46. ​​One of the methods described in items 31-45, in which mesenchymal stem cells are administered at a dose of approximately 1,000 cells / cm2 to approximately 5 million cells / cm2. 47. Mesenchymal stem cells are administered at a dose of approximately 100,000 cells / cm², approximately 300,000 cells / cm², or approximately 500,000 cells / cm², using any of the methods described in items 31-46. 48. Mesenchymal stem cells are administered once, twice, or more times per week, using one of the methods described in items 31-47. 49. Any method described in items 31-48, wherein mesenchymal stem cells are applied for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or longer. 50. Mesenchymal stem cells are administered twice a week for approximately 8 weeks at doses of approximately 100,000 cells / cm², approximately 300,000 cells / cm², or approximately 500,000 cells / cm², using one of the methods described in items 31-49. 51. A unit dose of mesenchymal stem cells obtained by any of the methods specified in items 1 to 21. 52. A unit dose of mesenchymal stem cells obtainable by any of the methods specified in items 1 to 21. 53. A unit dose of item 51 or 52 containing approximately 500,000 to 10,000,000 mesenchymal stem cells in a volume of 1 ml. 54. A unit dose of item 53 containing approximately 1 million, 3 million, or 5 million cells. 55. A unit dose of any of items 52-54, in which umbilical cord mesenchymal stem cells are selected from the group consisting of amniotic mesenchymal stem cells, perivascular mesenchymal stem cells, Wharton's colloid mesenchymal stem cells, and umbilical cord amniotic mesenchymal stem cells. 56. The mesenchymal stem cells of the amniotic membrane of the umbilical cord constitute a mesenchymal stem cell population, wherein at least about 90% or more of the cells in the mesenchymal stem cell population express the following markers: CD73, CD90, and CD105, respectively, in the unit dose of item 55. 57. The unit dose of item 56 in which at least approximately 90% or more of the cells in the mesenchymal stem cell population lack expression of the following markers: CD34, CD45, and HLA-DR. 58. Unit doses of item 56 or 57 in which at least approximately 91% or more, approximately 92% or more, approximately 93% or more, approximately 94% or more, approximately 95% or more, approximately 96% or more, approximately 97% or more, approximately 98% or more, or approximately 99% or more cells of the mesenchymal stem cell population express CD73, CD90, and CD105 respectively, and lack expression of CD34, CD45, and HLA-DR (human leukocyte antigen-antigen D-related), respectively.

[0235] It will be readily apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0236] All patents and publications referenced herein represent the level of skill of those skilled in the art in which the present invention relates. All patents and publications are incorporated herein by reference to the same extent that each individual publication is incorporated by reference specifically and individually.

[0237] The inventions described exemplary herein can be adequately carried out in the absence of any one or more elements or limitations not specifically disclosed herein. Therefore, terms such as “comprising,” “including,” and “containing” should be interpreted comprehensively and non-restrictively. Furthermore, the terms and expressions used herein are descriptive rather than restrictive, and the use of such terms and expressions is not intended to exclude any equivalent of the exhibited and described features or any part thereof, and it should be recognized that various modifications are possible within the scope of the claimed invention. Thus, while the invention has been specifically disclosed by preferred embodiments and optional features, it should be understood that modifications and alterations of the inventions embodied herein are left to those skilled in the art, and that such modifications and alterations are considered to be within the scope of the invention. The invention is described broadly and generically herein. Each of the narrower species and subgenera groups that fall within the scope of the generic disclosure also forms part of the invention. This includes generic descriptions of the invention using conditional or negative limitations that exclude any subject matter from the group, regardless of whether the excluded subject matter is specifically mentioned herein. In addition, where a feature or aspect of the invention is described in terms of a Markush group, those skilled in the art will recognize that the invention is also described in terms of any individual member or subgroup of any member of that Markush group. Further aspects of the invention will become apparent from the appended claims.

[0238] As used herein, the term “about” is understood to mean that there may be variations in each value or range (pH, concentration, percentage, molar concentration, number of amino acids, time, etc.) of up to 5%, 10%, 15%, or even up to 20% of a given value. For example, if a formulation contains about 5 mg / ml of the compound, this is understood to mean that the formulation may have 4–6 mg / ml, preferably 4.25–5.75 mg / ml, more preferably 4.5–5.5 mg / ml, and even more preferably 4.75–5.25 mg / ml, and most preferably 5 mg / ml. As used herein, an interval defined as “X–Y” is considered equivalent to an interval defined as “between X and Y.” Both intervals clearly include an upper and lower limit. This means that, for example, the interval "5 mg / ml to 10 mg / ml" or "between 5 mg / ml and 10 mg / ml" includes concentrations of 5, 6, 7, 8, 9, and 10 mg / ml, as well as any given intermediate value.

Claims

1. A method for preparing a mesenchymal stem cell storage or transport preparation, Mesenchymal stem cells are mesenchymal stem cells from the amniotic membrane of the umbilical cord, and this preparation contains 500,000 to 10,000,000 mesenchymal stem cells. a) Suspending mesenchymal stem cells in a crystalline liquid of a pre-specified volume to obtain a first cell suspension, wherein the crystalline liquid contains 526 mg of sodium chloride, 502 mg of sodium gluconate (C 6 H 11 NaO 7 ), 368 mg of sodium acetate trihydrate (C 2 H 3 NaO 2 ・3H 2 O), 37 mg of potassium chloride (KCl), and 30 mg of magnesium chloride (MgCl 2 ・6H 2 O) per 100 mL, does not contain an antibacterial agent, and has a pH adjusted to 7.4 (6.5 - 8.0) with sodium hydroxide, and the PlasmaLyte (registered trademark) further contains 0.5% - 3% (w / v) of serum albumin; b) A step of determining the concentration of mesenchymal stem cells in the first cell suspension and determining the volume of the first cell suspension required to prepare a formulation containing 500,000 to 10,000,000 mesenchymal stem cells. c) A step of mixing the determined volume of the first cell suspension with a certain volume of liquid carrier to obtain the mesenchymal stem cell storage or transport preparation containing 500,000 to 10,000,000 mesenchymal stem cells, wherein after mixing the mesenchymal stem cell storage or transport preparation contains 20% or less of PlasmaLyte®, and the liquid carrier is 0.5% to 3% (w / v) serum albumin and the following: i) Torolox, keen on + 、 iii) K + 、 iv) As 2+ 、 v) Mg 2+ 、 vi) Cl - 、 vii) H 2 AFTER 4 - 、 viii) HEPES, ix) Lactobionate, x) Sucrose, xi) Mannitol, xii) Glucose, xiii) Dextran-40, xiv) Adenosine, and xv) Glutathione stages Methods that include...

2. The method according to claim 1, wherein the predetermined volume of the crystalloid solution used to suspend the mesenchymal stem cells is 1 ml to 10 ml.

3. The method according to claim 1 or 2, wherein the determined volume of the first cell suspension is mixed with the volume of the liquid carrier, and the total volume of the mesenchymal stem cell storage or transport preparation is 1 ml.

4. The method according to claim 2, wherein the formulation contains 500,000 to 10,000,000 living mesenchymal stem cells.

5. The method according to any one of claims 1 to 4, wherein the formulation comprises 1 million, 3 million, or 5 million mesenchymal stem cells.

6. The method according to any one of claims 1 to 5, wherein the mesenchymal stem cells are recovered from the cell culture vessel before being resuspended in a predetermined volume of crystalloid solution.

7. The method according to any one of claims 1 to 6, wherein both the crystalloid solution and the liquid carrier contain the same concentration of serum albumin.

8. The method according to claim 7, wherein both the crystalloid solution and the liquid carrier contain 1% to 3% (w / v) serum albumin.

9. The method according to claim 7 or 8, wherein both the crystalloid solution and the liquid carrier contain 1% (w / v) serum albumin.

10. The method according to any one of claims 1 to 9, wherein the serum albumin is human serum albumin.

11. The method according to any one of claims 1 to 10, wherein the mesenchymal stem cells of the amniotic membrane of the umbilical cord constitute a mesenchymal stem cell population, and at least 90% or more of the cells of the mesenchymal stem cell population express the following markers: CD73, CD90, and CD105, respectively.

12. The method according to claim 11, wherein at least 90% or more of the cells in the mesenchymal stem cell population lack the expression of the following markers: CD34, CD45, and HLA-DR.

13. The method according to claim 11 or 12, wherein at least 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, and 99% or more of the cells in the mesenchymal stem cell population express CD73, CD90, and CD105 respectively, and lack expression of CD34, CD45, and HLA-DR (human leukocyte antigen-antigen D related) respectively.

14. A method for transporting mesenchymal stem cells, The step of transporting mesenchymal stem cells in a mesenchymal stem cell storage or transport formulation. Includes, The mesenchymal stem cells are mesenchymal stem cells from the amniotic membrane of the umbilical cord, the preparation contains 500,000 to 10,000,000 mesenchymal stem cells, and the mesenchymal stem cell storage or transport preparation is a) A step of suspending mesenchymal stem cells in a predetermined volume of crystalloid solution to obtain a first cell suspension, wherein the crystalloid solution is PlasmaLyte®, each 100 mL containing 526 mg of sodium chloride; 502 mg of sodium gluconate (C₆H₁₁NaO₂); 368 mg of sodium acetate trihydrate (C₂H₃NaO₂・3H₂O); 37 mg of potassium chloride (KCl); and 30 mg of magnesium chloride (MgCl₂・6H₂O), but without antibacterial agents, and its pH is adjusted to 7.4 (6.5–8.0) with sodium hydroxide, and the PlasmaLyte® further contains 0.5%–3% (w / v) of serum albumin. b) A step of determining the concentration of mesenchymal stem cells in the first cell suspension and determining the volume of the first cell suspension required to prepare a formulation containing 500,000 to 10,000,000 mesenchymal stem cells. c) A step of mixing the determined volume of the first cell suspension with a certain volume of liquid carrier, wherein after mixing the mesenchymal stem cell storage or transport preparation contains 20% or less of PlasmaLyte®, and the liquid carrier 0.5% to 3% (w / v) serum albumin and the following: i) Torolox, ii) Na+, iii) K+, iv) Ca²⁺, v) Mg²⁺, vi) Cl - , vii) H 2 PO 4 - , viii) HEPES, ix) Lactobionate, x) Sucrose, xi) Mannitol, xii) Glucose, xiii) Dextran-40, xiv) Adenosine, and xv) Glutathione stages Prepared by a method including, method.

15. The method according to claim 14, wherein the transport takes place over a period of seven days or less.

16. The method according to any one of claims 14 to 15, wherein the transport is carried out at a temperature of -5°C to 15°C.

17. Use of a mesenchymal stem cell storage or transport preparation prepared by any method of claim 1 to 13 for the manufacture of a pharmaceutical composition for treating a subject having a disease, wherein the disease is a skin disease or a wound, The use wherein the pharmaceutical composition comprises mesenchymal stem cells stored or transported in a mesenchymal stem cell storage or transport formulation as defined in claim 1, and is administered topically to the subject, and the mesenchymal stem cells are isolated from the mesenchymal stem cell storage or transport formulation.

18. The use according to claim 17, wherein the isolation of mesenchymal stem cells from a mesenchymal stem cell storage or transport preparation comprises centrifugation.

19. The use according to claim 17 or 18, wherein the isolation of mesenchymal stem cells from a mesenchymal stem cell storage or transport preparation comprises removing a cell population from a vial using a syringe.

20. The use according to any one of claims 17 to 19, wherein mesenchymal stem cells are administered in doses of 3 million, 5 million, or 10 million cells.

21. The use according to any one of claims 17 to 20, wherein the mesenchymal stem cell population is applied within 72 hours, 48 ​​hours, 24 hours, 12 hours, 6 hours, or less from the time the mesenchymal stem cell population is collected.

22. The use according to any one of claims 17 to 21, wherein the wound is caused by a burn, bite, trauma, surgery, or disease.

23. The use according to claim 22, wherein the wound is caused by diabetes, and preferably the wound is a diabetic wound.

24. The use according to claim 23, wherein the wound is a diabetic foot ulcer.

25. The use according to any one of claims 17 to 24, wherein a dose of 10 million cells, 5 million cells, 4 million cells, 3 million cells, 2 million cells, 1 million cells, 500,000 cells, 250,000 cells, or less than 250,000 cells is administered once or twice a week.

26. The use according to claim 25, wherein a dose of 10 million cells, 5 million cells, 4 million cells, 3 million cells, 2 million cells, 1 million cells, 500,000 cells, 250,000 cells, or fewer than 250,000 cells is administered once or twice per week over a period of 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 10 weeks, or more.

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