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CN-121986156-A - Cell culture methods and compositions

CN121986156ACN 121986156 ACN121986156 ACN 121986156ACN-121986156-A

Abstract

The present invention relates to a culture method for culturing a population of Endothelial Colony Forming Cells (ECFC) derived from cord blood, comprising (a) maintaining a cord blood sample obtained from a subject at a temperature of 4 ℃ to 15 ℃ for 24 hours to 72 hours, (b) isolating monocytes from the blood sample, (C) inoculating the monocytes on a culture substrate, (d) culturing the inoculated adherent monocytes in the culture medium for about 5 days to about 21 days to form colonies comprising cells, (e) culturing cells expressing CD31, CD34, CD105, CD144, CD146, CD157, VEGFR2 but not CD45, CD14 and CD 90.

Inventors

  • ALAN STITT
  • Reinhold Medina
  • Cristina. O'Neal

Assignees

  • 凡斯沃萨有限公司

Dates

Publication Date
20260505
Application Date
20240725
Priority Date
20230728

Claims (20)

  1. 1. A culture method for culturing a population of umbilical cord blood-derived Endothelial Colony Forming Cells (ECFCs), the method comprising: (a) Maintaining a cord blood sample obtained from a subject at a temperature of 4 ℃ to 15 ℃ for 24 hours to 72 hours; (b) Isolating monocytes from said cord blood sample; (c) Inoculating the monocytes on a culture substrate; (d) Culturing the inoculated adherent mononuclear cells in a medium for about 5 days to about 21 days to form colonies comprising cells; (e) Cells expressing CD31, CD34, CD105, CD144, CD146, CD157, VEGFR2 but not CD45, CD14 and CD90 were cultured.
  2. 2. A culture method for culturing a population of umbilical cord blood-derived Endothelial Colony Forming Cells (ECFCs), the method comprising: (a) Isolating monocytes from a cord blood sample obtained from a subject; (b) Inoculating the monocytes on a culture substrate; (c) Culturing the inoculated adherent mononuclear cells in a medium for about 5 days to about 21 days to form colonies comprising cells; (d) Culturing cells that express CD31, CD34, CD105, CD144, CD146, CD157 and VEGFR2 but do not express CD45, CD14 and CD90, and (E) The resulting cells are treated with an antioxidant such that the cells have a reparative phenotype.
  3. 3. A culture method for culturing a population of umbilical cord blood-derived Endothelial Colony Forming Cells (ECFCs), the method comprising: (a) Isolating monocytes from a cord blood sample obtained from a subject; (b) Inoculating the monocytes on a culture substrate; (c) Culturing the inoculated adherent mononuclear cells in a medium for about 5 days to about 21 days to form colonies comprising cells; (d) Culturing cells that express CD31, CD34, CD105, CD144, CD146, CD157 and VEGFR2 but do not express CD45, CD14 and CD90, and (E) Exposing the resulting cells to a hypoxic environment, such that the cells have a reparative phenotype.
  4. 4. The culture method according to claim 1, wherein step (b) is performed by mixing the umbilical cord blood sample with a red blood cell lysis buffer so that blood cells present in the sample are lysed, leaving intact monocytes.
  5. 5. The culture method of claim 4, wherein the sample is lysed and processed in a closed cell processing apparatus.
  6. 6. The culture method of any one of claims 1,4 or 5, wherein after step (b) of the culture method, the method further comprises the step of exposing the monocytes to a hypoxic environment and/or treating the monocytes with an antioxidant such that the monocytes have a reparative phenotype.
  7. 7. The culture method according to claim 3 or 6, wherein the hypoxic environment is 5% to 10% hypoxia from day 1 of culture.
  8. 8. The culture method according to claim 2 or 6, wherein the antioxidant is selected from flavonoids, flavones, catechins, polyphenols, phytoestrogens and/or carotenoids.
  9. 9. The culture method of claim 8, wherein the antioxidant is selected from the group consisting of N-acetyl cysteine (NAC), quercetin, myricetin, tocopherol, or any combination thereof.
  10. 10. The culture method of claim 2, wherein the cells are subjected to a single treatment with an antioxidant, or wherein the cells are subjected to repeated treatments with the antioxidant over a period of time.
  11. 11. The culture method of any one of claims 1 to 10, further comprising subculturing cells expressing CD31, CD34, CD105, CD146, CD144, CD157 and VEGFR2 but not expressing CD45, CD90 and CD14 for at least 38 population doublings.
  12. 12. The culture method according to any one of claims 1 to 11, wherein the culture medium is an endothelial growth medium.
  13. 13. The culture method according to any one of claims 1 to 12, wherein the culture medium comprises human serum, preferably wherein the culture medium comprises 5% to 20% human serum, more preferably wherein the culture medium comprises 10% human serum.
  14. 14. The culture method of any one of claims 1 to 13, wherein the culture medium is free of antibiotics.
  15. 15. The culture method according to any one of claims 1 to 14, wherein the cells are cultured under GMP-grade culture conditions.
  16. 16. The culture method according to any one of claims 1 to 15, wherein the medium comprises human epidermal growth factor in a concentration of 1ng/mL to 10ng/mL, human basic fibroblast growth factor in a concentration of 5ng/mL to 20ng/mL, human insulin-like growth factor in a concentration of 5ng/mL to 25ng/mL, human vascular endothelial growth factor in a concentration of 0.5ng/mL to 50ng/mL, optionally an antioxidant in a concentration of 1 μg/mL to 50 μg/mL and hydrocortisone in a concentration of 0.1 μg/mL to 2 μg/mL.
  17. 17. The culture method according to any one of claims 1 to 16, wherein the culture substrate has a coating comprising extracellular matrix molecules, preferably wherein the extracellular matrix molecules are selected from the group consisting of type 0 collagen, type I collagen, GMP grade collagen, biolamina or any combination thereof.
  18. 18. The culture method according to claim 17, wherein the GMP-grade collagen is GMP-grade human-derived collagen or GMP-grade heterologous-component-free collagen.
  19. 19. The culture method of any one of claims 1 to 18, further comprising analyzing cells that express CD31, CD105, CD146, CD144, and VEGFR2 but do not express CD45 and CD 90.
  20. 20. The culture method according to any one of claims 1 to 19, wherein the number of cells can be increased by up to 10 21 times in 100 days in culture.

Description

Cell culture methods and compositions Technical Field The present invention relates to a method of culturing endothelial colony forming cells of high proliferative potential and their use in the treatment of diseases. Background Cell-based therapies are at the front of medicine and are becoming new approaches to treating diseases. Stem cells are considered attractive because their plasticity enables them to become any cells that are required for therapy. Despite this unique ability, cell therapies using stem cells or induced pluripotent stem cells remain challenging. Difficulties in clinical grade production, laborious differentiation processes, reduced efficacy, safety risks and uncontrolled nature of in vivo differentiation remain major hurdles. Thus, cell therapies using progenitor cells that have committed themselves to a particular cell lineage, but still exhibit some plasticity and enhanced regenerative potential, are exciting prospects for cell therapies. Endothelial Progenitor Cells (EPCs) are terms used to describe highly heterogeneous populations of cells extracted from peripheral blood or umbilical cord blood. This cell population was first isolated and described by Asahara et al (Science, 1997; 275 (5302): 964-7) and was found to aid in and enhance revascularization. Later work identified a population of EPCs that were considered to be truly vascular repair cell types, termed Endothelial Colony Forming Cells (ECFCs). There are various pathological states characterized by vascular insufficiency, and these conditions often have no available treatment options, the purpose of which is to control symptoms. These conditions (e.g., chronic non-healing wounds, peripheral limb ischemia, ischemic retinopathy, and dry AMD) affect millions of patients worldwide each year, with a significant impact on the quality of life of the patient. Thus, there is a need in the art for improved therapies to address conditions caused by vascular insufficiency. Disclosure of Invention The inventors of the present invention have developed a method for culturing endothelial colony forming cells (herein referred to as "Angicyte") of high proliferative potential, whereby cells with improved properties can be obtained. The isolation method produces a highly pure and potent population of vascular reparative Angicyte that can be efficiently and consistently amplified on a large scale for therapeutic applications. Such cells may then be used in a variety of therapeutic applications, particularly those diseases associated with lack of blood/oxygen flow. Once generated, angicyte disclosed herein shows features that make it a very advantageous candidate for treating many diseases characterized by vascular dysfunction/insufficiency. One of the most advantageous features they have is that the proliferation capacity is many times higher than that of the mature endothelial subtype, which proliferates slowly and begins to show signs of aging after only a few passages. In contrast Angicyte proliferate rapidly and can reach more than 60 Population Doubling Levels (PDL), optionally up to 100 population doubling levels, before signs of aging are shown. This feature is well suited to cell scalability. Angicyte cells are vascular in nature, with the ability to make new blood vessels and promote vascular repair, resulting in reperfusion, reoxygenation, and wound healing. In a first aspect of the invention, a culture method for culturing a population of umbilical cord blood-derived Endothelial Colony Forming Cells (ECFC) is provided, the method comprising (a) maintaining a sample of umbilical cord blood at a temperature of from 4 ℃ to 15 ℃ for from 24 hours to 72 hours prior to isolation of monocytes from the blood sample, (b) isolating monocytes from the blood sample, (C) inoculating the monocytes on a culture substrate, (d) culturing the inoculated adherent monocytes in the culture medium for from about 5 days to about 21 days to form colonies comprising cells, (e) culturing cells expressing CD31, CD34, CD105, CD144, CD146, CD157 and VEGFR2 but not CD45, CD14 and CD 90. The cells had a median cell size of 18 microns. In a second aspect of the invention, there is provided a culture method for culturing a population of Endothelial Colony Forming Cells (ECFC) derived from cord blood, the method comprising (a) isolating monocytes from a cord blood sample obtained from a subject, (b) seeding the monocytes on a culture substrate, (c) culturing the seeded adherent monocytes in a medium for about 5 days to about 21 days to form colonies comprising cells, (d) culturing cells expressing CD31, CD34, CD105, CD144, CD146, CD157 and VEGFR2 but not CD45, CD14 and CD90, and (e) treating the resulting cells with an antioxidant such that the cells have a reparative phenotype. The median cell size was 18 microns. In a third aspect of the invention, there is provided a culture method for culturing a population of umbilical cord blood-derived Endothelial Colony For