Cell culture methods and compositions

Abstract

The present invention relates to a culture method for culturing a population of endothelial colony-forming cells (ECFC) derived from umbilical cord blood, the method comprising: (a) holding an umbilical cord blood sample obtained from a subject at a temperature of 4 DEG C to 15 DEG C for 24 hours to 72 hours; (b) isolating mononuclear cells from the blood sample; (c) inoculating the mononuclear cells on a culture substrate; (d) culturing the inoculated adherent monocytes in a culture medium for about 5 days to about 21 days to form colonies comprising cells; and (e) culturing the cells which express the CD31, the CD34, the CD105, the CD144, the CD146, the CD157 and the VEGFR2 (vascular endothelial growth factor receptor 2) but do not express the CD45, the CD14 and the CD90.

Description

Technical Field

[0001] This invention relates to a method for culturing endothelial colony-forming cells with high proliferative potential and their use in the treatment of diseases. Background Technology

[0002] Cell-based therapies are at the forefront of medicine and are emerging as new approaches to treating diseases. Stem cells are considered attractive because their plasticity allows them to become any cell desired for treatment. 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 the uncontrolled nature of differentiation in vivo remain major obstacles. Therefore, cell therapies using progenitor cells represent an exciting prospect for cell therapy; progenitor cells have already oriented themselves to specific cell lineages but still exhibit a degree of plasticity and enhanced regenerative potential.

[0003] Endothelial progenitor cells (EPCs) are a term used to describe a highly heterogeneous population of cells extracted from peripheral blood or umbilical cord blood. First isolated and described by Asahara et al. (Science, 1997; 275(5302):964-7), this cell population was found to contribute to and enhance angiogenesis. Subsequent work identified an EPC population that was considered a true vascular repair cell type, termed endothelial colony-forming cells (ECFCs).

[0004] Currently, there are various pathological conditions characterized by vascular insufficiency, for which there are often no available treatment options, and treatment aims to control symptoms. These conditions (such as chronic non-healing wounds, peripheral limb ischemia, ischemic retinopathy, and dry AMD) affect millions of patients worldwide each year, significantly impacting their quality of life. Therefore, there is a need in this field for improved therapies to address conditions caused by vascular insufficiency. Summary of the Invention

[0005] The inventors of this invention have developed a method for culturing endothelial colony-forming cells with high proliferative potential (referred to herein as "Angicyte"), thereby obtaining cells with ameliorative properties. This isolation method yields a high-purity and potent population of vascular-repairing Angieyte, which can be efficiently and consistently expanded on a large scale for therapeutic applications. These cells can then be used for a variety of therapeutic applications, particularly those related to diseases associated with insufficient blood / oxygen flow.

[0006] Once generated, the Angicyte cells disclosed herein exhibit characteristics that make them highly promising candidates for the treatment of many diseases characterized by vascular dysfunction / insufficiency. One of their most advantageous features is their proliferative capacity, which is many times greater than that of mature endothelial subtypes, which proliferate slowly and begin to show signs of senescence after only a few passages. In contrast, Angicyte cells proliferate rapidly and are able to reach population doubling levels (PDL) of over 60 times, optionally up to 100 times, before showing signs of senescence. This characteristic is well-suited to the scalability of the cells. Angicyte cells are inherently vascular repair cells, possessing the ability to create new blood vessels and promote vascular repair, thereby leading to reperfusion, reoxygenation, and wound healing.

[0007] In a first aspect of the invention, a method for culturing a population of endothelial colony-forming cells (ECFCs) derived from umbilical cord blood is provided, the method comprising: (a) maintaining the umbilical cord blood sample at a temperature of 4°C to 15°C for 24 to 72 hours prior to isolating monocytes from the blood sample; (b) isolating monocytes from the blood sample; (c) seeding the monocytes onto a culture medium substrate; (d) culturing the seeded adherent monocytes in the culture medium for approximately 5 to approximately 21 days to form cell-containing colonies; and (e) culturing cells expressing CD31, CD34, CD105, CD144, CD146, CD157, and VEGFR2 but not CD45, CD14, and CD90. The cells have a mid-cell size of 18 micrometers.

[0008] In a second aspect of the invention, a method for culturing a population of endothelial colony-forming cells (ECFCs) derived from umbilical cord blood is provided, the method comprising: (a) isolating monocytes from an umbilical cord blood sample obtained from a subject; (b) seeding the monocytes onto a culture substrate; (c) culturing the seeded adherent monocytes in the culture medium for about 5 days to about 21 days to form cell-containing colonies; (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 to impart a repair phenotype to the cells. The cell mid-size is 18 micrometers.

[0009] In a third aspect of the invention, a method for culturing a population of endothelial colony-forming cells (ECFCs) derived from umbilical cord blood is provided, the method comprising: (a) isolating monocytes from an umbilical cord blood sample obtained from a subject; (b) seeding the monocytes onto a culture substrate; (c) culturing the seeded adherent monocytes in the culture medium for about 5 days to about 21 days to form cell-containing colonies; (d) culturing cells expressing CD31, CD34, CD105, CD144, CD146, CD157, and VEGFR2 but not CD45, CD14, and CD90; and (e) exposing the resulting cells to a hypoxic environment, thereby giving the cells a reparative phenotype. The cellular mid-size of the cells is 18 micrometers.

[0010] In a fourth aspect of the invention, a population of endothelial colony-forming cells (ECFCs) derived from umbilical cord blood, obtainable by the methods disclosed herein, and their therapeutic uses are provided.

[0011] In the fifth aspect, a wound dressing is provided comprising an endothelial colony-forming cell (ECFC) population and a substrate derived from umbilical cord blood obtained by the methods disclosed herein.

[0012] In a sixth aspect, methods are provided for treating, inhibiting, preventing recurrence, or controlling ischemic diseases, neoplastic diseases, bone diseases, or skin lesions, wherein the methods include applying cells according to the invention to a subject in need of such treatment.

[0013] In a seventh aspect, the use of the cells according to the invention in the preparation of medicaments for treating, preventing recurrence of, or controlling ischemic diseases, neoplastic diseases, bone diseases, or skin injuries in subjects with such needs is provided.

[0014] In an eighth aspect, an apparatus for delivering the cells disclosed herein is provided, the apparatus comprising a syringe containing the cells disclosed herein and a delivery medium. Attached Figure Description

[0015] The present invention is described with reference to the accompanying drawings, wherein:

[0016] Figure 1 This shows the generation of Angiogenic cells from umbilical cord blood. Blood is collected and mixed with an anticoagulant, then kept for at least 35 to 72 hours before processing.

[0017] Figure 2 The figure shows the advantages of erythrocyte lysis compared to standard density gradient centrifugation. It also shows a comparability study with Ficol density gradient centrifugation. The erythrocyte lysis method increased cell yield (A) and the number of Angieyte colonies generated (B).

[0018] Figure 3: A. Showing the typical cobblestone morphology of confluent monolayers of Angicyte cells. B. Representative image of Angicyte colony formation in a 6-well plate. Single cells proliferated to produce Angicyte colonies after 7 days of culture. C(1). A graph depicting the growth kinetics of 3 representative clones. C(2). Showing the maximum population multiplication level (PDL) of Angicyte clones. Angicyte can undergo up to 100 PDLs. D(1). Representative image of a lumen formation assay performed in Matrigel. These are live cells stained with calcein AM. D(2). Skeletalized view of the same lumen formation assay described in D(1). E. Real-time assessment of barrier formation (cell index) using the xCELLigence system. This figure depicts barrier formation of 3 clones over 24 hours. Angicyte can reach a cell index value of 11.

[0019] Figure 4 Flow cytometry analysis was used to assess the Angieyte phenotype. In this example, Angieyte cells were harvested at passage 3, suspended in FACs buffer, and stained with antibodies against CD31, CD105, VEGFR2, CD34, CD45, and CD90. After staining, the samples were washed and analyzed using an Attune flow cytometer. The figure shows the expression of representative Angieyte colonies.

[0020] Figure 5: Angiogenic cells rapidly form dense vascular-like networks within fibrin-based 3D gel structures. When seeded in fibrin gels, Angiogenic cells form dense 3D tubular networks that are sensitive to the addition of pro-angiogenic and anti-angiogenic factors. Multilayer images of these gels were captured and then analyzed via AI-based online analysis software (called IKOSA) (A). IKOSA analyzed several factors of the tubular networks, such as lumen area (highlighted area), number of branch points (circles), and the number and size of annular structures within the network (multicolored areas). (B) The total lumen area % provides a reading of the net size of the tubular networks generated by the cells and thus their angiogenic capacity. The addition of pro-angiogenic factors resulted in an increase in lumen area, while the addition of anti-angiogenic factors resulted in a significant decrease in total lumen area. The number of branch points (C) follows a trend significantly similar to that of the total lumen area % of the tubular networks. This indicates that the cells respond to stimuli as expected for real vascular networks in vivo.

[0021] Figure 6Before performing the lumen formation assay in Matrigel, Angiete cells were treated for 60 minutes with either the antioxidant α-tocopherol or a control (culture medium) or a solvent control (ethanol). A. Representative images of clones taken 2 days after the onset of lumen formation, processed and analyzed using ImageJ. B. Lumen coverage area measured as a percentage. Pretreatment with α-tocopherol improves tubular formation.

[0022] Figure 7 Flow cytometry analysis revealed the Angieyte phenotype. Representative Angieyte cells were stained with antibodies against CD144, CD146, CD157, and CD14. After staining, the samples were washed and analyzed using an Attune flow cytometer. This figure shows the expression of representative Angieyte clones.

[0023] Figure 8 Immunocytochemical analysis revealed the Angieyte phenotype. Angieyte cells were fixed and stained with antibodies against CD144 (VE-cadherin) and vWF, and counterstained with the nuclear staining agent DAPI. This figure shows the expression of representative Angieyte clones.

[0024] Figure 9: Flow cytometry analysis to assess Angicyte phenotype: (A) isolated from fresh umbilical cord blood (UCB) (processed within 6 hours) and (B) isolated from UCB processed 72 hours after collection. Angicyte cells were stained with antibodies against CD31, CD105, VEGFR2, CD90, and CD45. After staining, samples were washed and analyzed using an Attune flow cytometer. This figure shows expression in representative Angicyte clones.

[0025] Figure 10 Flow cytometry analysis was used to evaluate CD45 levels in umbilical cord blood from freshly processed, processed 35 hours after collection, or processed 72 hours after collection. 阴性 / CD31 阳性 Angicyte cell yield. Cells were stained with CD45 and CD31 antibodies and analyzed by flow cytometry. Cells were gated to single cells, then to a live cell population, and then to a CD45-negative cell population. Cells were also gated to a CD31-positive population and subjected to CD45 antibody staining. 阴性 / CD31 阳性 The number of cells is counted.

[0026] Figure 11The vascular network formation capacity of Angiecyte cells from four freshly processed or 72-hour post-collection umbilical cord blood donors was evaluated using a fibrin-based angiogenesis assay. Angiecyte cells were harvested from culture flasks and seeded in fibrin-based gel on 15-well μ-angiogenesis slides. After 48 hours of incubation, each well was imaged using an EVOS microscope. Images were analyzed using IKOSA software, and the percentage of luminal coverage was measured.

[0027] Figure 12 The vascular network-forming capacity of four Angiete clones was evaluated using a fibrin-based angiogenesis assay. Angiete clones were incubated for 24 hours under standard normoxic or 10% hypoxic conditions, then harvested and seeded into fibrin-based gels on 15-well μ-angiogenesis slides. Forty-eight hours later, each well was imaged using an EVOS microscope. Images were analyzed using IKOSA software, and the percentage of luminal coverage was measured.

[0028] Figure 13: Expression analysis of Angieyte cells generated under the following conditions: 1) GMP conditions (as described in this paper's Angieyte protocol), using GMP-compliant and heterologous reagents; or 2) non-GMP conditions, using reagents containing heterologous components (e.g., fetal bovine serum, rat tail collagen). Data were obtained from 9 replicates. Data analysis showed that samples clustered according to GMP versus non-GMP variables, which was confirmed by principal component analysis. 13B. This table lists genes that were significantly upregulated in GMP cells relative to non-GMP cells, and these genes are associated with improved endothelial function, particularly the ability to form mature vascular networks through angiogenesis.

[0029] Figure 14: Comparison of the vascular network formation ability of Angiogenesis cultured under GMP conditions with that cultured under non-GMP conditions. Angiogenesis was harvested from culture flasks and seeded into fibrin-based gel on 15-well μ-angiogenesis slides. After 48 hours of incubation, each well was imaged using an EVOS microscope. Images were analyzed using IKOSA software, and the percentage of luminal coverage was measured.

[0030] Figure 15 Bioenergetics of Angielyte cells from GMP or non-GMP conditions were assessed using a Seahorse XF Bioanalyzer. Cells were harvested from culture flasks and seeded into dedicated culture plates according to the manufacturer's bioanalyzer guidelines. Mitochondrial respiration (OCR, oxygen consumption) and glycolytic capacity (ECAR, extracellular acidification rate) were measured, and the data were plotted on an energy graph.

[0031] Figure 16Angiocyte promotes wound healing in a mouse model of diabetic trauma. This study used 14- to 15-week-old diabetic male mice (BKS-Lepr db / Rj genetic background). Wounds of the same size were created using a trephine punch. To better mimic human conditions, a silicone ring around the wound was sutured in place to reduce healing caused by contraction. Wounds were treated with PBS (sham-operated control) (n=6), CMC gel (n=9), CMC gel containing 500,000 Angiocyte units (n=11), or CMC gel containing 1 million Angiocyte units (n=4). Changes in wound size over time were photographed, and healing was assessed by measuring the percentage of the original wound area. One-way ANOVA showed significant differences between the means at days 5, 8, 10, 12, and 14, at which point the study was terminated. Detailed Implementation

[0032] To facilitate understanding of this invention, certain terms are defined first. Additional definitions are set forth throughout the detailed description.

[0033] As used herein, the terms “cord blood-derived endothelial colony-forming cell (ECFC) population” and “Angicyte” are used interchangeably and refer to a highly reparative endothelial colony-forming cell population. These cells express endothelial cell-specific cell surface antigens (e.g., CD31, CD15, CD105, CD146, CD144, VEGFR2, CD157, and CD34) and do not express hematopoietic cell-specific cell surface antigens (e.g., CD45, CD14, and CD90).

[0034] As used herein, the term "monocyte" refers to a mononuclear cell found in the mononuclear fraction of a whole blood sample obtained from umbilical cord blood samples, and includes all blood cells with a mononuclear nucleus (e.g., lymphocytes, monocytes, and stem cells). Monocytes can be isolated from blood samples by density gradient centrifugation.

[0035] As used herein, the term "object" is intended to include both humans and non-human animals. Preferred objects include patients with ischemic diseases or diseases involving vascular dysfunction, such as patients with chronic non-healing wounds. Preferred objects also include patients with dry age-related macular degeneration. This method is particularly suitable for treating patients with conditions that can be treated by enhancing angiogenesis. In one specific embodiment, the method is particularly suitable for treating ischemic diseases. In another embodiment, the method herein is particularly suitable for treating dry age-related macular degeneration and / or treating ischemic retinopathy. In yet another embodiment, the method disclosed herein is particularly suitable for treating skin lesions, bone diseases, or tumors.

[0036] As used herein, the terms “hypoxia” and “hypoxic environment” are used interchangeably and refer to cell culture conditions in which cells are exposed to a hypoxic environment. Preferably, the cells of the present invention are exposed to an oxygen concentration of 5% to 10%. Those skilled in the art will recognize that different ways can be used to initiate and maintain a hypoxic environment for cell culture, including physical and chemical means. For example, a hypoxic environment can be induced by using a hypoxic culture chamber or by chemical methods (e.g., using sodium dithionite (Na₂S₂O₄), cobalt chloride (CoCl₂), and (NaN₃)). In the context of the present invention, such an environment is considered particularly advantageous because the cells are preferably used for ischemic diseases, or diseases in which blood supply is insufficient and therefore oxygen is lacking. In these cases, the cells have been pretreated to adapt to the conditions they are most likely to experience in vivo, resulting in a higher chance of success.

[0037] As used herein, the term "reparative phenotype" refers to monocytes with enhanced proliferative capacity and the ability to generate new blood vessels, as measured in a range of in vitro, ex vivo, and in vivo assays. Therefore, cells with this phenotype are envisioned to be particularly useful in treating diseases, especially where insufficient blood supply is a significant factor. In vitro, ex vivo, and in vivo assays used to determine this particular phenotype include, but are not limited to, in vitro assays (e.g., lumen formation assay, colony formation assay, microbead budding assay, Xcelligence barrier assay, immunocytochemistry assay, and migration assay), ex vivo assays (e.g., choroidal budding assay and aortic ring assay), and in vivo assays (e.g., matrix plug assay, ischemic retinopathy model, and skin wound healing model). Those skilled in the art will readily understand how to perform and interpret the results produced by the assays listed above.

[0038] As used herein, the term "antioxidant" refers to a substance that can protect cells from damage caused by free radicals. The antioxidant can be a natural or synthetic substance. Preferably, the antioxidants of the present invention include, but are not limited to, N-acetylcysteine ​​(NAC), quercetin, myricetin, or any combination thereof. However, it should be understood that any substance having antioxidant properties as defined above is suitable for the present invention.

[0039] As used herein, the term "treatment" or "therapy" means the application of an active pharmaceutical agent to cure, heal, alleviate, reduce, alter, remedy, improve, enhance, or influence a condition (e.g., a disease), symptoms of a symptom, or to prevent or delay the onset of symptoms, complications, or biochemical indicators of a disease, or to statistically significantly halt or inhibit the further development of a disease, condition, or disorder. For example, in the context of this invention, the term "treatment" or "therapy" may refer to enhancing blood supply to a target area of ​​the body by generating new blood vessels or regenerating existing blood vessels.

[0040] The use of alternatives (e.g., the term "or") should be understood to mean any one, two, or any combination of the alternatives. As used herein, the indefinite article "an" or "a" should be understood to mean "one or more" of any of the stated or enumerated components.

[0041] As used herein, “about” means within an acceptable margin of error for a particular value as determined by a person skilled in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” may mean within one or more standard deviations according to practice in the art. Alternatively, “about” may mean a range of up to 20%. When a particular value is provided in this application and claims, unless otherwise stated, it should be assumed that “about” means within an acceptable margin of error for that particular value.

[0042] Therefore, in a first aspect of the invention, a method for culturing a population of endothelial colony-forming cells (ECFCs) derived from umbilical cord blood is provided, the method comprising: (a) maintaining an umbilical cord blood sample at a temperature of 4°C to 15°C for 24 to 72 hours; (b) isolating monocytes from the blood sample; (c) seeding the monocytes onto a culture medium substrate; (d) culturing the seeded adherent monocytes in the culture medium for about 5 to about 21 days to form cell-containing colonies; and (e) culturing cells expressing CD31, CD34, CD105, CD144, CD146, CD157, and VEGFR2 but not CD45, CD14, and CD90. The cell mid-size is 18 micrometers.

[0043] The Angiogenesis cells of this invention are generated from ethically sourced and consent-obtained umbilical cord blood, which would otherwise be disposed of as clinical waste. The cells of this invention can be isolated from a range of blood sample volumes, demonstrating the flexibility of the method disclosed herein. The cells of this invention can be isolated from blood samples with a volume of at least 1 mL. In a preferred embodiment, the cells of this invention are isolated from blood samples with a volume of 20 mL to 80 mL. Larger volumes of blood samples are preferred where possible due to enhanced colony formation.

[0044] Therefore, the culture method disclosed herein provides a method in which specific cellular characteristics, such as specific cell surface markers (CD31, CD34, CD105, CD144, CD146, CD157, and VEGFR2), and cell morphology analysis are used to confirm the generation of the desired cell type, namely Angiogenesis. Similarly, the method disclosed herein also confirms the cell type by confirming that the cells do not express other cell markers indicating other cell types. For example, the cell surface markers CD45 and CD14 are known to indicate hematopoietic cells and are therefore used as an additional confirmatory layer to confirm that Angiogenesis is not contaminated with hematopoietic cells. CD90 can be used to determine the absence of stromal cells. The presence or absence of these cell surface markers can be confirmed by any suitable assay used for this purpose, such as flow cytometry immunophenotyping.

[0045] The method of the present invention requires the isolation of monocytes from blood cells present in umbilical cord blood. The isolation step can be performed in any conventional manner. For example, density gradient centrifugation (e.g., using Ficoll or Histopaque) can be used. However, in a preferred method, isolation is performed by treating the umbilical cord blood with a red blood cell lysis buffer to lyse any red blood cells. The remaining monocytes can then be cultured as further described. Therefore, "isolation" should be interpreted not only as physical isolation but also as isolation performed due to treatment that enables the monocytes to be distinguishable in the umbilical cord blood sample, thereby allowing for selective culture.

[0046] In one embodiment, umbilical cord blood is treated with erythrocyte lysis buffer for 5 to 20 minutes, preferably about 10 minutes, followed by a washing step, after which the remaining mononuclear cells are counted and seeded at high density onto a suitable substrate. In another embodiment, the lysis step can be performed in a closed cell processing device that allows for washing and concentration steps. A suitable device is the Lovo cell processing system (6R4900), which incorporates a rotating membrane filtration system that removes cell debris after the lysis step.

[0047] The culture method disclosed herein provides a means by which purer cell cultures can be produced by using blood samples that have been kept at low temperature for 24 to 72 hours, compared to using blood samples that are used immediately for culture or shortly after collection. Therefore, blood samples can be kept at low temperature for 24 to 30 hours, 24 to 36 hours, 24 to 42 hours, 24 to 54 hours, 24 to 72 hours, 30 to 36 hours, 30 to 42 hours, 30 to 48 hours, 30 to 54 hours, 30 to 60 hours, 30 to 66 hours, 30 to 72 hours, 36 to 42 hours, 36 to 48 hours, 36 to 54 hours, 36 to 60 hours, 36 to 66 hours, 36 to 72 hours, 42 to 48 hours, 42 to 54 hours, 42 to 60 hours, 42 to 66 hours, or 42 to 72 hours. In a preferred embodiment, the blood sample can be kept at low temperature for 38 to 72 hours.

[0048] In this document, "low temperature" refers to any temperature between 4°C and 15°C. Therefore, the temperature can be between 4°C and 11°C, 4°C and 12°C, 4°C and 13°C, 4°C and 14°C, 5°C and 12°C, 5°C and 13°C, 5°C and 14°C, 5°C and 15°C, 6°C and 13°C, 6°C and 14°C, 6°C and 15°C, 7°C and 14°C, 7°C and 15°C, or 10°C and 15°C. Preferably, the blood is maintained at 10°C. The blood sample may contain preservatives and / or anticoagulants added to the sample to ensure that the blood sample retains its viability. For example, the preservative / anticoagulant may be glucose citrate phosphate (CPD), glucose acid citrate (ACD), or CPD / ACD with added adenine. This method overcomes the problems of previously known culture methods, which utilize fresh blood and thus produce cell cultures contaminated with unwanted cells (e.g., mesenchymal cells).

[0049] In a second aspect of the invention, a method for culturing a population of endothelial colony-forming cells (ECFCs) derived from umbilical cord blood is provided, the method comprising: (a) isolating monocytes from an umbilical cord blood sample obtained from a subject; (b) seeding the monocytes onto a culture substrate; (c) culturing the seeded adherent monocytes in the culture medium for about 5 days to about 21 days to form cell-containing colonies; (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 to impart a repair phenotype to the cells. The cell mid-size is 18 micrometers.

[0050] The culture methods disclosed herein may include a step of treatment with an antioxidant. The antioxidant may be selected from any antioxidant capable of achieving the desired effect of giving monocytes a reparative phenotype. For example, the antioxidant may be selected from flavonoids, catechins, polyphenols, phytoestrogens, and / or carotenoids. Preferably, the antioxidant is selected from N-acetylcysteine ​​(NAC), tocopherol, quercetin, myricetin, or any combination thereof. Those skilled in the art will readily understand that the concentration of the antioxidant depends on the specific antioxidant intended for use; for example, tocopherol may be used at a concentration of 10 μM to 100 μM, while NAC may be used at a concentration of 750 μM to 2000 μM.

[0051] Cells can be treated with antioxidants in a single, one-time process, or repeatedly over a period of time. This "period of time" can span the entire culture process or be performed at regular intervals within it. In the case of repeated treatment with antioxidants, the antioxidant can be the same for each repeated treatment, or it can be a different antioxidant for each repeated treatment. In a preferred embodiment, the cells are treated with antioxidants prior to use in a clinical setting.

[0052] When cells are treated with antioxidants, they can be treated with one or more additional compounds. Such additional compounds can further support the action of the antioxidants or lead to a repair phenotype in the cells via mechanisms similar to those of antioxidant treatment.

[0053] In a third aspect of the invention, a method for culturing a population of endothelial colony-forming cells (ECFCs) derived from umbilical cord blood is provided, the method comprising: (a) isolating monocytes from an umbilical cord blood sample obtained from a subject; (b) seeding the monocytes onto a culture substrate; (c) culturing the seeded adherent monocytes in the culture medium for about 5 days to about 25 days to form cell-containing colonies; (d) culturing cells expressing CD31, CD34, CD105, CD144, CD146, CD157, and VEGFR2 but not CD45, CD14, and CD90; and (e) exposing the resulting cells to a hypoxic environment, thereby giving the cells a reparative phenotype. The cell mid-size is 18 micrometers.

[0054] The culture methods disclosed herein include a hypoxia pretreatment step or a hypoxia regulation step, resulting in more robust cells and improved angiogenesis upon application. In this context, "robust" means that, when used in a therapeutic context, the cells are better adapted to the in vivo environment in which they will be situated, thus having a higher survival / success rate. Without being bound by theory, it is considered that regulating the cells in this way during the culture phase induces a reparative phenotype and creates an environment that enables the cells of this invention to stimulate angiogenesis. The hypoxia environment of this invention can be defined as having an oxygen level of 3% to 12% from day 1 of culture, where "day 1 of culture" is defined as the day the cells are first isolated and seeded. In a preferred embodiment, the hypoxia environment of this invention can be defined as having an oxygen level of 5% to 10% from day 1 of culture. For example, the hypoxic environment of the present invention can be defined as having an oxygen level of 5% to 6%, 5% to 7%, 5% to 8%, 5% to 9%, 6% to 7%, 6% to 8%, 6% to 9%, 6% to 10%, 7% to 8%, 7% to 9%, 7% to 10%, 8% to 9%, 8% to 10%, or 9% to 10%. To maintain a continuous hypoxic environment, the culture medium is equilibrated to the same hypoxic conditions as the cells being cultured before any culture medium replacement or passage. Preferably, the culture medium is equilibrated to the same hypoxic conditions as the cells for approximately 1 hour.

[0055] The adherent mononuclear cells seeded by the methods disclosed herein can be cultured in a suitable culture medium for up to 21 days. For example, the adherent mononuclear cells seeded by the methods disclosed herein can be cultured in a suitable culture medium for about 5 to about 10 days, about 5 to about 15 days, about 5 to about 20 days, about 10 to about 15 days, about 10 to about 20 days, about 10 to about 21 days, about 15 to about 20 days, about 15 to about 21 days, or 20 to 21 days. Those skilled in the art will readily recognize that the resulting colonies can be cultured and further expanded to produce large quantities of the desired cell type. The scalability achieved in this way allows the use of the cells described herein in a variety of therapeutic applications.

[0056] The culture method disclosed herein may further include passage-culturing cells expressing CD31, CD34, CD105, CD146, CD144, CD157, and VEGFR2 but not expressing CD45, CD14, and CD90 at least 30 times to increase the population size. This property is a characteristic feature of the cells disclosed herein.

[0057] The culture medium for culturing cells can be endothelial growth medium (EGR). Those skilled in the art will readily understand that EGR is a medium optimized for the specific culture of endothelial cells. In the context of this invention, EGR can be Good Manufacturing Practice (GMP) grade, thus containing all relevant clinical factors (e.g., human serum) and being antibiotic-free. Such media are commercially available, such as PromoCell medium. Therefore, in a preferred embodiment, cells are cultured under GMP-grade conditions. In another preferred embodiment, the culture medium (e.g., EGR) contains human serum, preferably 5% to 20%, and even more preferably 10%. In yet another preferred embodiment, the culture medium (e.g., EGR) is antibiotic-free.

[0058] The culture medium of the method disclosed herein may contain the following components: human epidermal growth factor at a concentration of 1 ng / mL to 10 ng / mL, human basic fibroblast growth factor at a concentration of 5 ng / mL to 25 ng / mL, human insulin-like growth factor at a concentration of 5 ng / mL to 100 ng / mL, human vascular endothelial growth factor at a concentration of 0.5 ng / mL to 50 ng / mL, and optionally, an antioxidant at a concentration of 1 μg / mL to 100 μg / mL and hydrocortisone at a concentration of 0.1 μg / mL to 2 μg / mL. For example, the concentration of human epidermal growth factor can be 1 ng / mL to 2 ng / mL, 1 ng / mL to 3 ng / mL, 1 ng / mL to 4 ng / mL, 1 ng / mL to 5 ng / mL, 1 ng / mL to 6 ng / mL, 1 ng / mL to 7 ng / mL, 1 ng / mL to 8 ng / mL, 1 ng / mL to 9 ng / mL, 2 ng / mL to 3 ng / mL, 2 ng / mL to 4 ng / mL, 2 ng / mL to 5 ng / mL, 2 ng / mL to 6 ng / mL, 2 ng / mL to 7 ng / mL, 2 ng / mL to 8 ng / mL, 2 ng / mL to 9 ng / mL, 2 ng / mL to 10 ng / mL, 3 ng / mL to 4 ng / mL, 3 ng / mL to 5 ng / mL, 3 ng / mL to 6 ng / mL, 3 ng / mL to 7 ng / mL, 3 ng / mL to 8 ng / mL, 3 ng / mL to 9 ng / mL. g / mL, 3ng / mL to 10ng / mL, 4ng / mL to 5ng / mL, 4ng / mL to 6ng / mL, 4ng / mL to 7ng / mL, 4ng / mL to 8ng / mL, 4ng / mL to 9ng / mL, 4ng / mL to 10ng / mL, 5ng / mL to 6ng / mL, 5ng / mL to 7ng / mL, 5ng / mL to 8ng / mL, 5ng / mL to 9ng / mL , 5ng / mL to 10ng / mL, 6ng / mL to 7ng / mL, 6ng / mL to 8ng / mL, 6ng / mL to 9ng / mL, 6ng / mL to 10ng / mL, 7ng / mL to 8ng / mL, 7ng / mL to 9ng / mL, 7ng / mL to 10ng / mL, 8ng / mL to 9ng / mL, 8ng / mL to 10ng / mL or 9ng / mL to 10ng / mL. The concentration of human basic fibroblast growth factor can be 5 ng / mL to 10 ng / mL, 5 ng / mL to 15 ng / mL, 10 ng / mL to 15 ng / mL, 10 ng / mL to 20 ng / mL, or 15 ng / mL to 20 ng / mL.Human insulin-like growth factor can be used at concentrations of 5 ng / mL to 10 ng / mL, 5 ng / mL to 15 ng / mL, 5 ng / mL to 20 ng / mL, 10 ng / mL to 15 ng / mL, 10 ng / mL to 20 ng / mL, 10 ng / mL to 25 ng / mL, 15 ng / mL to 20 ng / mL, 15 ng / mL to 25 ng / mL, or 20 ng / mL to 25 ng / mL.Human vascular endothelial growth factor can be administered at dosages ranging from 0.5 ng / mL to 5 ng / mL, 0.5 ng / mL to 10 ng / mL, 0.5 ng / mL to 15 ng / mL, 0.5 ng / mL to 20 ng / mL, 0.5 ng / mL to 25 ng / mL, 0.5 ng / mL to 30 ng / mL, 0.5 ng / mL to 35 ng / mL, 0.5 ng / mL to 40 ng / mL, 0.5 ng / mL to 45 ng / mL, 5 ng / mL to 10 ng / mL, 5 ng / mL to 15 ng / mL, 5 ng / mL to 20 ng / mL, and 5 ng / mL to 25 ng / mL. / mL, 5ng / mL to 30ng / mL, 5ng / mL to 35ng / mL, 5ng / mL to 40ng / mL, 5ng / mL to 45ng / mL, 5ng / mL to 50ng / mL, 10ng / mL to 15ng / mL, 10ng / mL to 20ng / mL, 10ng / mL to 25ng / mL, 10ng / mL to 30ng / mL, 10ng / mL to 35ng / mL, 10ng / mL to 40ng / mL, 10ng / mL to 45ng / mL, 10ng / mL to 50ng / mL, 15ng / mL to 20ng / mL mL, 15ng / mL to 25ng / mL, 15ng / mL to 30ng / mL, 15ng / mL to 35ng / mL, 15ng / mL to 40ng / mL, 15ng / mL to 45ng / mL, 15ng / mL to 50ng / mL, 20ng / mL to 25ng / mL, 20ng / mL to 30ng / mL, 20ng / mL to 35ng / mL, 20ng / mL to 40ng / mL, 20ng / mL to 45ng / mL, 20ng / mL to 50ng / mL, 25ng / mL to 30ng / mL, 25ng / mL to 30ng / mL Use at concentrations of 5 ng / mL, 25 ng / mL to 40 ng / mL, 25 ng / mL to 45 ng / mL, 25 ng / mL to 50 ng / mL, 30 ng / mL to 35 ng / mL, 30 ng / mL to 40 ng / mL, 30 ng / mL to 45 ng / mL, 30 ng / mL to 50 ng / mL, 35 ng / mL to 40 ng / mL, 35 ng / mL to 45 ng / mL, 35 ng / mL to 50 ng / mL, 40 ng / mL to 45 ng / mL, 40 ng / mL to 50 ng / mL, or 45 ng / mL to 50 ng / mL.The concentration of the antioxidant can be 1 µg / mL to 5 µg / mL, 1 µg / mL to 10 µg / mL, 1 µg / mL to 15 µg / mL, 1 µg / mL to 20 µg / mL, 1 µg / mL to 25 µg / mL, 1 µg / mL to 30 µg / mL, 1 µg / mL to 35 µg / mL, 1 µg / mL to 40 µg / mL, 1 µg / mL to 45 µg / mL, 5 µg / mL to 10 µg / mL, 5 µg / mL to 15 µg / mL, 5 µg / mL to 20 µg / mL, 5 µg / mL to 25 µg / mL, 5 µg / mL to 30 µg / mL. µg / mL, 5µg / mL to 35µg / mL, 5µg / mL to 40µg / mL, 5µg / mL to 45µg / mL, 5µg / mL to 50µg / mL, 10µg / mL to 15µg / mL, 10µg / mL to 20µg / mL, 10µg / mL to 25µg / mL, 10µg / mL to 30µg / mL, 10µg / mL to 35µg / mL, 10µg / mL to 40µg / mL, 10µg / mL to 45µg / mL, 10µg / mL to 50µg / mL, 15µg / mL to 20µg / mL, 15µg / mL to 25µg / mL, 15µg / mL to 30µg / mL, 15µg / mL to 35µg / mL, 15µg / mL to 40µg / mL, 15µg / mL to 45µg / mL, 15µg / mL to 50µg / mL, 20µg / mL to 25µg / mL, 20µg / mL to 30µg / mL, 20µg / mL to 40µg / mL, 20µg / mL to 45µg / mL, 20µg / mL to 50µg / mL, 25µg / mL to 30µg / mL, 25µg / mL to 35µg / mL µg / mL, 25µg / mL to 40µg / mL, 25µg / mL to 45µg / mL, 25µg / mL to 50µg / mL, 30µg / mL to 35µg / mL, 30µg / mL to 40µg / mL, 30µg / mL to 45µg / mL, 30µg / mL to 50µg / mL, 35µg / mL to 40µg / mL, 35µg / mL to 45µg / mL, 35µg / mL to 50µg / mL, 40µg / mL to 45µg / mL, 40µg / mL to 50µg / mL or 45µg / mL to 50µg / mL.Hydrocortisone can be used at a concentration of 0.1 µg / mL to 0.2 µg / mL, 0.1 µg / mL to 0.4 µg / mL, 0.1 µg / mL to 0.6 µg / mL, 0.1 µg / mL to 0.8 µg / mL, 0.1 µg / mL to 1 µg / mL, 0.1 µg / mL to 1.2 µg / mL, 0.1 µg / mL to 1.4 µg / mL, 0.1 µg / mL to 1.6 µg / mL, 0.1 µg / mL to 1.8 µg / mL, 0.2 µg / mL to 0.4 µg / mL, 0.2 µg / mL to 0.6 µg / mL, 0.2 µg / mL to 0.8 µg / mL, 0.2 µg / mL to 1 µg / mL, 0.2 µg / mL to 1.2 µg / mL, 0.2 µg / mL to 1.4 µg / mL, 0.2 µg / mL to 1.6 µg / mL, 0.2 µg / mL to 1.8 µg / mL, 0.2 µg / mL to 2 µg / mL, 0.4 µg / mL to 0.6 µg / mL, 0.4 µg / mL to 0.8 µg / mL, 0.4 µg / mL to 1 µg / mL, 0.4 µg / mL to 1.2 µg / mL, 0.4 µg / mL to 1.4 µg / mL, 0.4 µg / mL to 0.6 µg / mL, 0.4 µg / mL to 0.8 µg / mL, 0.4 µg / mL to 2 µg / mL, 0.6 µg / mL to 0.8 µg / mL, 0.6 µg / mL to 1 µg / mL, 0.6 µg / mL to 1.2 µg / mL, 0.6 µg / mL to 1.4 µg / mL, 0.6 µg / mL to 1.6 µg / mL, 0.6 µg / mL to 1.8 µg / mL, 0.6 µg / mL to 2 µg / mL, 0.8 µg / mL to 1 µg / mL, 0.8 µg / mL to 1.2 µg / mL, 0.8 µg / mL to 1.4 µg / mL, 0.8 µg / mL to 1.6 µg / mL, 0.8 µg / mL to 1.8 µg / mL, 0.8 µg / mL to 2 µg / mL, 1 µg / mL to 1.2 µg / mL, 1 µg / mL to 1.4 µg / mL, 1 µg / mL to 1.6 µg / mL, 1 µg / mL to 1.8 µg / mL, 1 µg / mL to 2 µg / mL, 1.2 µg / mL to 1.4 µg / mL, 1.2 µg / mL to 1.6 µg / mL, 1.2 µg / mL to 1.8 µg / mL, 1.2 µg / mL to 2 µg / mL, 1.4 µg / mL to 1.6 µg / mL, 1.4 µg / mL to 1.8 µg / mL, 1.4 µg / mL to 2 µg / mL, 1.6 µg / mL to 1.8 µg / mL, 1.6 µg / mL to 2 µg / mL or 1.8 µg / mL to 2 µg / mL.

[0059] In the most preferred embodiment, the culture medium may contain the following components: human epidermal growth factor at a concentration of 5 ng / mL, human basic fibroblast growth factor at a concentration of 10 ng / mL, human insulin-like growth factor at a concentration of 20 ng / mL, human vascular endothelial growth factor at a concentration of 0.5 ng / mL, an antioxidant at a concentration of 1 μg / mL, and hydrocortisone at a concentration of 0.2 μg / mL.

[0060] The cells of this invention can be seeded on any suitable culture substrate for the purpose of growing and isolating the cells disclosed herein. The cells described herein possess integrins; molecules responsible for mediating cell attachment. Therefore, any culture substrate with an affinity for any known endothelial integrin unit (e.g., collagen and laminin) can be used as a suitable culture substrate. It should be noted that the methods disclosed herein are applicable to both single-cell and colony cultures. The culture substrate may have a coating containing extracellular matrix molecules (ECM). Such ECM molecules act as a scaffold for various proteins and molecules that provide structural and biochemical support to the cells. For example, ECM molecules may be selected from GMP-grade collagen, type O collagen, type I collagen, type II collagen, type III collagen, type IV collagen, type X collagen, laminin, recombinant laminin, or any combination thereof. Preferably, the ECM molecules are selected from type O collagen, type I collagen, GMP-grade collagen, recombinant laminin (e.g., Biolamina), or any combination thereof. In one or even a more preferred embodiment, the extracellular matrix molecule is type I collagen or GMP-grade collagen, wherein the GMP-grade collagen is GMP-grade human collagen or GMP-grade heterologous-free collagen. As used herein, the term "heterologous-free" means a product (e.g., culture medium) that does not contain non-human animal components.

[0061] The culture method disclosed in this paper can produce cells in which the number of cells can be increased to more than 10. 21 The cells of this invention can be cultured for extended periods while maintaining their proliferative capacity, thus demonstrating their proliferative potential and potential use in therapeutic applications where cell proliferation is a key factor, such as in angiogenesis.

[0062] The culture method disclosed herein may include maintaining a blood sample at a temperature of 4°C to 15°C for 24 to 72 hours, exposing monocytes to a hypoxic environment, and / or treating monocytes with an antioxidant. In one embodiment, after step b) of the culture method of the first aspect, the method may further include the steps of exposing monocytes to a hypoxic environment and / or treating monocytes with an antioxidant, thereby endowing the monocytes with a reparative phenotype. In an alternative embodiment, the step of maintaining the blood sample at a temperature of 4°C to 15°C for 24 to 72 hours is omitted, and the culture method may consist only of the steps of exposing monocytes to a hypoxic environment and treating the cells with an antioxidant, thereby endowing the monocytes with a reparative phenotype.

[0063] In one embodiment, none of the culture methods disclosed herein involve a purification process. Therefore, the culture methods disclosed herein do not require multiple consumption or enrichment steps to obtain the desired cells at the end of the culture period. This not only improves the efficiency of the culture process from a time and resource perspective but also significantly reduces the potential risks associated with aseptic handling. Therefore, the culture methods described herein represent an improved methodology.

[0064] In a fourth aspect, the present invention discloses umbilical cord blood-derived endothelial colony-forming cell (ECFC) populations obtainable by any of the methods disclosed herein and their therapeutic uses.

[0065] As demonstrated in the following examples, the inventors of the present invention have shown that ECFCs obtained by the methods disclosed herein (referred to herein as "Angicyte") have improved properties compared to ECFCs not produced by the methods described herein. Specifically, the inventors have demonstrated that the resulting cells not only possess a stronger glycolytic / energy metabolism profile, but also exhibit a significantly enhanced angiogenesis / angiogenesis profile. Therefore, the resulting cells possess an enhanced reparative phenotype and are thus envisioned for use in therapies desiring angiogenesis properties.

[0066] Therefore, this invention also discloses the use of Angichyte described herein for treating diseases in subjects with such needs or for therapeutic purposes. Angichyte obtained by any of the methods disclosed herein possesses numerous advantageous characteristics, such as a high proliferation rate, low levels of senescence, and a high level of adaptability to its surrounding environment. Therefore, this cell type is highly advantageous in therapeutic areas where these properties are required, for example, for treating diseases / conditions associated with vascular dysfunction.

[0067] Therefore, this invention discloses the use of Angichyte, as described herein, for the treatment of ischemic diseases or diseases related to vascular dysfunction. In this context, "ischemic disease" refers to any disease caused by (partially or wholly) restricting or reducing blood flow to a specific part of the body. For example, this invention discloses the use of Angichyte, as described herein, for the treatment of ischemic heart disease, ischemic cerebral disease, severe limb ischemia, mesenteric ischemia, stroke, or ischemic retinopathy.

[0068] In a preferred embodiment, the ischemic disease to be treated is ischemic retinopathy, which is retinopathy of prematurity (ROP), diabetic retinopathy (DR), or age-related macular degeneration (AMD). In a further preferred embodiment, the ischemic retinopathy is AMD. AMD is a condition of damage to the macula of the retina, resulting in central vision loss. There are two types of AMD; wet AMD and dry AMD, the latter being the most common. Dry AMD is characterized by vascular dysfunction, retinal pigment epithelial dysfunction, and photoreceptor dysfunction. Specifically, choroidal capillary degeneration has been observed in early-onset dry AMD (Biesemeier et al., 2014, Neurobiology of Aging; 35(11):2562-2573), defining dry AMD as a vascular disease. Therefore, it is envisioned that the cells disclosed herein can be used to effectively repair these damaged blood vessels. Treatment at an early stage of the disease can prevent disease progression and ultimately save a large number of patients with dry AMD from vision loss. Therefore, in a preferred embodiment, the AMD is dry AMD.

[0069] Treatment can also involve skin lesions for bone repair or to treat bone diseases or neoplastic diseases (cancer).

[0070] This invention can be used to treat and / or prevent neoplastic diseases and / or secondary diseases associated with neoplastic diseases. In one embodiment, a neoplastic disease can be a solid carcinoma and / or a hematologic malignancy. Tumor formation, tumors, and cancers include benign, malignant, metastatic, and non-metastatic types, and include any stage (I, II, III, IV, or V) or grade (G1, G2, G3, etc.) of a tumor formation, tumor, or cancer, or a tumor formation, tumor, cancer, or metastasis that is progressing, worsening, stable, or in remission. The invention is particularly envisioned for use in treating and / or preventing neoplastic diseases where the neoplastic disease has a high vascular demand. In the case of treating and / or preventing neoplastic diseases, the cells disclosed herein (i.e., the Angichyte of this invention) can be used to deliver cargo molecules (e.g., therapeutic molecules) to a target site (i.e., a tumor). As used herein, the term "cargo" is well known to those skilled in the art and refers to a specific target molecule intended to be translocated, delivered, transported, or exported from one place to another. In one embodiment, the cargo molecule is a protein and / or peptide. The cargo molecule can be a heterologous protein that is not naturally present for the carrier cell. The cargo peptides and / or proteins may be therapeutic peptides and / or therapeutic proteins. While the cargo molecules of the present invention are envisioned to be proteins or peptides, other cargo types include DNA and RNA molecules. Thus, in another embodiment, the cargo molecule is an RNA molecule or a DNA molecule.

[0071] Cancers that can be treated according to the present invention include, but are not limited to, cells or tumors of the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, cancer can specifically be, but is not limited to, the following histological types: malignant tumor; carcinoma; undifferentiated carcinoma; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatal carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; hepatocellular carcinoma with cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenomatous polyposis adenocarcinoma; familial adenomatous polyposis adenocarcinoma; solid carcinoma; malignant carcinoid tumor; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; eosinophilic cell carcinoma; eosinophilic adenocarcinoma; basophilic cell carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; non-capsulated sclerosing carcinoma; adrenocortical carcinoma; endometrioid carcinoma; skin appendage carcinoma; Apocrine gland adenocarcinoma; sebaceous gland adenocarcinoma; ceruminous gland carcinoma; mucoepidermoid carcinoma; cystic adenocarcinoma; papillary cystic adenocarcinoma; papillary serous cystic adenocarcinoma; mucinous cystic adenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease of the breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant theca cell tumor; malignant granulosa cell tumor; malignant androgenoma; Sertoli cell carcinoma; malignant stromal cell tumor; malignant Lipocytoma; Malignant paraganglioma; Malignant extramammary paraganglioma; Pheochromocytoma; Balloon sarcoma; Malignant melanoma; Amelanotic melanoma; Superficial diffuse melanoma; Giant melanocytic nevus; Epithelioid cell melanoma; Malignant blue nevus; Sarcoma; Fibrosarcoma; Malignant fibrous histiocytoma; Myxosarcoma; Liposarcoma; Leiomyosarcoma; Rhabdomyosarcoma; Embryonic rhabdomyosarcoma; Alveolar rhabdomyosarcoma; Stromal sarcoma; Mixed tumor; Müllerian mixed tumor Nephroblastoma; Hepatoblastoma; Carcinosarcoma; Malignant mesenchymal tumor; Malignant Brenner's tumor; Malignant phyllodes tumor; Synovial sarcoma; Malignant mesothelioma; Dysgerminoma; Embryonic carcinoma; Malignant teratoma; Malignant ovarian goiter; Choriocarcinoma; Malignant mesonephroma; Angiosarcoma; Malignant hemangioendothelioma; Kaposi's sarcoma; Malignant hemangiopericytoma; Lymphangiosarcoma; Osteosarcoma; Paracortical osteosarcoma; Chondrosarcoma; Malignant chondroblastoma; Mesenchymal chondrosarcoma; Giant cell tumor of bone; Ewing's sarcoma; Malignant odontogenic tumors; ameloblastic odontosarcoma; malignant ameloblastoma; ameloblastic fibrosarcoma; malignant pineal tumor; chordoma; malignant glioma; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrous astrocytoma; astrocytoma; glioblastoma; oligodendroglioma; oligodendroglioma; primitive neuroectodermal tumors; cerebellar sarcoma; ganglioneuroma; neuroblastoma; retinoblastoma; olfactory neurogenic tumors; malignant meningioma;Neurofibrosarcoma; malignant schwannoma; malignant granular cell tumor; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; small lymphocytic malignant lymphoma; diffuse large cell malignant lymphoma; follicular malignant lymphoma; mycosis fungoides; other specific non-Hodgkin's lymphoma; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small bowel disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and piloblastic leukemia.

[0072] In cases where the treatment involves a skin injury, the treatment can be any injury that causes damage to the skin or subcutaneous tissue. For example, a skin injury can be a trauma (cut, laceration, deep laceration, tear, ulcer, scrape, abrasion, or scratch), bruise, avulsion, or burn. In a preferred embodiment, the skin injury can be a trauma or a burn. Preferably, in the case of a traumatic injury, the trauma is a traumatic ulcer, and in the case of a burn, the burn is a burn caused by radiation therapy. In yet another preferred embodiment, the traumatic ulcer is a foot traumatic ulcer. In another preferred embodiment, the traumatic ulcer to be treated is a chronic traumatic ulcer. In yet another preferred embodiment, the trauma / ulcer to be treated is a diabetic ulcer, preferably wherein the diabetic ulcer is a diabetic leg ulcer or a diabetic foot ulcer.

[0073] It is readily understood that the routes of cell administration disclosed herein will depend on the disease to be treated. Cells can be administered locally, intravenously, intramuscularly, intra-articularly, subcutaneously, orally, intra-arterially, or transdermally. Therefore, cells can be administered systemically, locally to the target site, or a combination of both. For example, for the treatment of traumatic ulcers, local application may be most beneficial. Similarly, for the treatment of dry AMD, direct choroidal administration may be most beneficial.

[0074] The cells can be administered in any suitable therapeutic formulation. In one embodiment, the cells are prepared for delivery in a gel or gel matrix. Other suitable formulations will be apparent to those skilled in the art.

[0075] It should be understood that, in addition to administering Angichyte for the treatment of the aforementioned diseases / conditions, one or more additional therapeutic agents may be given in combination to maximize the final clinical outcome (i.e., through additive or synergistic effects). Thus, the Angichyte of the present invention can be administered to the subject in combination with one or more additional therapeutic agents. Therefore, additional therapeutic agents may include, but are not limited to, cell therapies, aspirin, nitrates, beta-blockers, calcium channel blockers, cholesterol-lowering drugs, angiotensin-converting enzyme (ACE) inhibitors, alginate dressings, hydrocolloid dressings, other wound dressings, antimicrobial agents, antibiotics, anti-VEGF drugs, photodynamic therapy, or any combination thereof. Such additional therapeutic agents may be given in combination, i.e., simultaneously or sequentially. The term "sequentially" covers situations where Angichyte may be given before or after the additional therapeutic agent. It should be understood that the specific additional therapy depends on the disease to be treated. It is envisioned that the combination of the Angichyte disclosed herein with any of the one or more additional therapeutic agents listed above will be particularly beneficial in any disease or therapy requiring vascular supply. Target sites believed to particularly benefit from enhanced vascular supply include, but are not limited to, the heart, brain, eyes, skin, liver, pancreas, and kidneys.

[0076] In a preferred embodiment, the additional therapeutic agent may be a cell therapy. The term "one or more cell therapies" refers to any therapy in which cellular material is injected or otherwise transplanted into the subject in need.

[0077] It is envisioned that Angizyte produced by the methods disclosed herein will be particularly advantageous in treating any disease in which insufficient blood supply is a contributing factor. Furthermore, it is anticipated that Angizyte cells produced by the methods disclosed herein will be advantageous as a supportive treatment for any therapy that is ineffective due to a lack of adequate blood supply. Therefore, in one embodiment, the Angizyte of the present invention can be a supportive blood supply element. In the context of the present invention, the term "supportive blood supply element" refers to the ability of Angizyte to enhance vascular networks via angiogenesis. It is envisioned that target cells can survive within a blood supply range of 10 μm to 200 μm.

[0078] In a fifth aspect, a wound dressing is provided comprising a population of endothelial colony-forming cells (ECFCs) derived from umbilical cord blood and a substrate, which can be obtained by the methods disclosed herein.

[0079] It is envisioned that wound dressings incorporating Angietyte disclosed herein will promote faster wound healing and thus reduce the incidence of infection.

[0080] The base of the wound dressing can be any suitable structural material (in which the Angiete of the present invention may be incorporated), thereby providing a physical matrix on which or in which Angiete can be applied. However, in a preferred embodiment, the base can be a polymer-based material, a supporting matrix, a hydrogel, a scaffold, or any combination thereof. Polymer-based materials can be synthetic or natural polymers. For example, synthetic polymer-based materials include, but are not limited to, PEG, PLA, PLGA, PU, ​​and PEG. Natural polymer-based materials include, but are not limited to, chitosan, collagen, gelatin, elastin, cellulose, alginate, and hyaluronic acid. The base can include more than one polymer-based material, wherein the polymer-based material can be purely synthetic, purely natural, or a combination of both. In yet another preferred embodiment, the base of the wound dressing is a hydrogel. The hydrogel can be a synthetic hydrogel (e.g., PEG or PVA hydrogel) or a natural-based hydrogel (e.g., collagen-based hydrogel, gelatin-based hydrogel, chitosan-based hydrogel, fibroin-based hydrogel, or any polysaccharide-based hydrogel). The hydrogel can be a hybrid hydrogel comprising more than one type of hydrogel. The hydrogel can include synthetic and natural-based hydrogels. The hydrogel can be an injectable hydrogel, which forms when applied to an object in need. The wound dressing of the present invention is intended for topical application to an object in need.

[0081] For localized wounds, it is envisioned that the Angicyte of the present invention be delivered to the target site using a wound dressing. In one embodiment, the Angicyte is combined with a gel prior to application to the wound to provide the correct consistency, which can be adapted to the size and shape of the wound to be treated. Any suitable gel can be used. In one embodiment, the gel may be a carboxymethyl cellulose gel. Those skilled in the art will readily understand that the amount of Angicyte to be applied to the wound will depend on various factors, including the size, depth, and complexity of the wound. However, in some embodiments, the Angiocyte and gel product may contain 100,000 to 10 million Angiocyte, such as 100,000 to 1 million Angiocyte, 100,000 to 2 million Angiocyte, 100,000 to 3 million Angiocyte, 100,000 to 4 million Angiocyte, 100,000 to 5 million Angiocyte, 100,000 to 6 million Angiocyte, 100,000 to 7 million Angiocyte, 100,000 to 8 million Angiocyte, or 100 or 100,000 to 9 million Angiocyte. In a preferred embodiment, the Angiocyte and gel product may contain 500,000 to 1 million Angiocyte. In a preferred embodiment, the combined Angiocyte and gel product is administered via a syringe system. In a further preferred embodiment, the syringe system is a dual-syringe system, wherein the Angiete and the gel of the present invention are mixed within the syringe. Such a system allows the gel and Angiete to remain under separate storage conditions until the need to mix the two components, and allows the mixing of the two components to be carried out under sterile conditions.

[0082] Furthermore, it is envisioned that the Angieyte disclosed herein could be delivered using an on-demand delivery system. Such an on-demand system could utilize the syringe system just described above. In this system, the Angieyte disclosed herein could be combined with a cryogenic solution, thereby allowing for enhanced accessibility to treatment, for example, in non-hospital primary care clinics. Such a delivery system would allow the Angieyte disclosed herein to be stored at 4°C (instead of -80°C), where storage at -80°C requires specialized equipment.

[0083] The Angiogenesis disclosed herein can also be particularly advantageous in the context of surgery, for example, delivering Angiogenesis to target sites requiring enhanced blood supply. Those skilled in the art will readily understand that the administration modality and concentration of Angiogenesis to be delivered in the context of surgical application will depend on the purpose of the surgery. Therefore, in some embodiments, the Angiogenesis disclosed herein can be delivered directly to the target site. In other embodiments, the Angiogenesis disclosed herein can be delivered systemically, for example, via intravenous infusion. The Angiogenesis disclosed herein can be administered in combination with one or more additional therapies. In a preferred embodiment, the Angiogenesis disclosed herein can be administered prior to any additional therapies to maximize the beneficial effects of the administered Angiogenesis.

[0084] In a sixth aspect, methods are provided for treating, inhibiting, preventing recurrence, or controlling ischemic diseases, neoplastic diseases, bone diseases, or skin lesions, wherein the methods include applying cells according to the invention to a subject in need of such treatment.

[0085] The method according to the sixth aspect may also include any feature relating to the cells of the present invention.

[0086] In a seventh aspect, the use of the cells according to the invention in the preparation of medicaments for treating, preventing recurrence of, or controlling ischemic diseases, neoplastic diseases, bone diseases, or skin injuries in subjects with such needs is provided.

[0087] According to the seventh aspect regarding the use of the cells, any feature relating to the cells of the present invention may also be included.

[0088] In an eighth aspect, an apparatus for delivering the cells disclosed herein is provided, the apparatus comprising a syringe containing the cells disclosed herein and a delivery medium. In a preferred embodiment, the delivery medium may be a gel, such as a solidified gel, a semi-solid gel, a flowable gel, or a scaffold gel.

[0089] The invention is further described with reference to the following non-limiting embodiments.

[0090] Example

[0091] Example 1: Isolation of clinical-grade Angieyte cells.

[0092] Fresh cord blood was collected and mixed with an anticoagulant (citrate phosphate glucose (CPD)) for storage. The cord blood was stored under cooling for at least 35 to 72 hours prior to processing. The cord blood was mixed with red blood cell lysis buffer for 10 minutes, followed by sample washing, which resulted in red blood cell lysis, leaving intact white blood cells. Cells were counted and seeded at high density on plastic dishes coated with human collagen 1 medium and maintained in EGM growth medium containing 10% human serum. Figure 1 The cultures were maintained for up to 21 days. The cultures were monitored daily to observe the appearance of ANGICYTE vascular colonies between day 3 (D3) and day 21 (D21) post-inoculation. Cell and colony yields were... Figure 2 A and Figure 2 It is shown in B.

[0093] Isolated Angicyte cells presented as a homogeneous cell population with a cobblestone-like morphology (Fig. 3A), forming cell colonies when seeded as single cells (Fig. 3B). Angicyte cells exhibited significant proliferative capacity, reaching up to 78 PDLs within 100 days (Fig. 3C). Angicyte cells formed a vascular network in the matrix gel. Figure 3D ), and form a tight barrier with a high cell index (9 CI to 11 CI). Figure 3E ).

[0094] Angicyte cells exhibited a distinct endothelial cell phenotype. At this point, we had characterized the Angicyte cell population harvested from passage 3 and stained it with antibodies against CD31, CD105, VEGFR2, CD34, CD45, and CD90. Samples analyzed on Attune flow cytometry showed that Angicyte cells expressed high levels of CD31, CD105, and VEGFR2, and were negative for CD90 and CD45. Figure 4 ).

[0095] We used a fibrin-based assay to assess the complexity of the vascular network formed by Angietyte. Angietyte cells were resuspended in fibrin and allowed to form primitive vessels within 48 hours. Figure 5 shows the complex 3D multilayer network after 48 hours. This network was then evaluated and analyzed using the IKOSA platform (Figure 5).

[0096] Pretreatment of Angiete cells with antioxidants led to increased vascular function and network formation. Figure 6 This shows representative Angiete cells pretreated with α-tocopherol for 1 hour before being embedded in the 3D matrix gel assay. Figure 6 A revealed the formation of an enhanced vascular network.

[0097] Example 2: Angieyte Identity Verification

[0098] As described above, Angieyte cells exhibit a well-defined endothelial phenotype, as indicated by the expression of cellular markers. A fifth-generation Angieyte cell population was harvested and stained with antibodies against CD144, CD146, CD157, and CD14. Samples analyzed on an Attune flow cytometer showed that Angieyte cells expressed CD144, CD146, and CD157, but were negative for CD14 (see [link to Attune flow cytometer]). Figure 7 Angicyte cells cultured in tissue culture dishes coated with human collagen were fixed and stained with antibodies against CD144 and vWF by immunocytochemistry, and imaged using a fluorescent DMi8 microscope. Angicyte cells showed expression of CD144 (VE-cadherin) localized to the cell membrane and vWF (VE-cadherin) found in the cytosol. Figure 8 ).

[0099] Example 3: Strengthening the vascular network by keeping blood in the blood for 72 hours

[0100] Collect fresh cord blood and mix it with an anticoagulant for storage / transport. Each cord blood sample is processed as a fresh sample (within 6 hours of collection) or stored chilled and processed 35 or 72 hours after collection. For processing, the cord blood is mixed with red blood cell lysis buffer to lyse red blood cells and keep white blood cells intact. Cells are counted and processed at a high density (2 × 10⁻⁶). 6 (±20%) cm 2 Human collagen-1 substrates were inoculated into plastic tissue culture flasks and maintained in endothelial growth medium supplemented with 10% human serum. Cultures were monitored daily for the appearance of Angieyte colonies. Angieyte cells were isolated and expanded by passage, and the endothelial phenotype was confirmed by flow cytometry. Harvested passage 5 cells were stained with antibodies against CD31, CD105, VEGFR2, CD90, and CD45 and analyzed using Attune flow cytometry. There was no phenotypic difference between Angieyte cells from "fresh" umbilical cord blood and those from umbilical cord blood processed 72 hours after collection. Figure 9A and Figure 9B Angicyte cells retained their endothelial phenotype, and the expression of stem cells or hematopoietic markers was not increased.

[0101] Flow cytometry was used to assess the yield of Angiocyte from freshly processed cord blood, processed 35 hours (35h) or 72 hours (72h) after collection. After processing and holding in culture for several days, passage 0 cells were harvested and stained with antibodies against CD45 and CD31. Live single cells were analyzed using flow cytometry, and the number of CD45-CD31+ cells was counted by gating these populations. Any suitable software could be used to analyze the results. A trend of increasing CD45-CD31+ cell yield was observed in samples from the 35h and 72h groups (n=6). Figure 10 ).

[0102] The formation and complexity of vascular networks formed in vitro by Angieyte cells from freshly processed umbilical cord blood or samples processed 72 hours after collection were evaluated using a fibrin-based angiogenesis assay. Angieyte cells were harvested from passage 5 cells in culture flasks and resuspended in fibrin gels for 48 hours to allow for the formation of primitive vessels. These vascular networks were imaged using an EVOS microscope, and the images were analyzed using the IKOSA platform. Four samples were evaluated, and the percentage of vascular network coverage was measured. Figure 11 Observations showed that larger vascular networks formed in 72-hour samples compared to fresh samples. In two of the four clones (Y and Z), blood processing after 72 hours significantly improved lumen formation. In clones V and X, there was a trend toward increased lumen formation.

[0103] Example 4: Functional Improvement for Hypoxia Exposure

[0104] The vascular network formation capacity of four AngieCyte clones was assessed using a fibrin-based angiogenesis assay. AngieCyte cells in culture flasks coated with human collagen were incubated under standard normoxic conditions or in 10% O2 for 24 hours, then harvested and resuspended in fibrin gels to allow for the formation of primitive vessels. After 48 hours, the vascular networks were imaged using an EVOS microscope, and the images were analyzed using the IKOSA platform to measure the vascular network coverage area (%). Pretreatment with hypoxia was observed to induce the formation of more complex vascular structures in AngieCyte. Of the four clones evaluated, three (clones 1, 2, and 4) showed a statistically significant increase (…). Figure 12 In clone 3, an increased trend in lumen formation was observed under hypoxic conditions. Therefore, pretreatment of Angiete cells with 10% O2 (hypoxia) for 24 hours resulted in improved in vitro vascular network formation.

[0105] Example 5: Cells produced by GMP manufacturing process have improved endothelial function.

[0106] Fresh umbilical cord blood was collected and mixed with an anticoagulant, and then processed to generate Angieyte cells. The cord blood was mixed with erythrocyte lysis buffer and washed, and the cells were counted. In each sample, half of the cells were processed under GMP-grade conditions (human collagen 1 medium substrate, supplemented with GMP-grade endothelial growth medium, phenol red-free, heterologous components-free, and supplemented with human serum). The other half of the cells were processed at the same density under non-GMP conditions (rat tail collagen 1 medium substrate, supplemented with non-GMP endothelial growth medium and fetal bovine serum). Cultures were monitored daily for the appearance of Angieyte colonies. Angieyte colonies were passaged and amplified for various analyses.

[0107] In the fourth generation, Angielyte was harvested from substrate-coated culture flasks, and RNA was isolated using a Maxwell instrument for automated RNA extraction. RNA analysis showed that the samples clustered according to their treatment status (GMP vs. non-GMP). This was in Figure 13A The dendritic heatmap shows that GMP Angicyte exhibits transcriptomic characteristics distinct from those of non-GMP cultured cells. Figure 13B The study listed genes that were significantly upregulated in GMP cells compared to non-GMP cells. These genes are associated with improved endothelial function, particularly the ability to form mature vascular networks through angiogenesis. Therefore, it is clear that the GMP manufacturing process produces a distinct cell type.

[0108] The vascular network formation ability of Angicyte cells cultured under GMP conditions was compared with that of cells cultured under non-GMP conditions. Angicyte cells were harvested from substrate-coated culture flasks and resuspended in fibrin gel to assess the ability of Angicyte cells to form vascular networks in vitro. After 48 hours, the original vessels were imaged using an EVOS microscope and analyzed using the IKOSA platform. The vascular network coverage area (%) was measured. It was observed that GMP Angicyte cells formed more vascular networks compared to non-GMP cells (Figure 14). Statistically significant differences were observed in 4 out of the 5 clones evaluated (A, B, C, and E). Figure 14A An increased trend in vascular network formation was also observed in clone D. Figure 14B Representative images from the IKOSA platform are also shown. Compared to non-GMP cells, GMP Angicyte forms a more complex network of interconnected blood vessels, indicated by a greater number of “rings”.

[0109] The bioenergetic properties of Angiete cells from GMP or non-GMP conditions were evaluated. Angiete cells were harvested from substrate-coated culture flasks and reseeded into Seahorse FX Analyzer 96-well plates to assess cellular bioenergeticity, using the Mito stress test and Glyco stress kit according to the manufacturer's instructions. Mitochondrial respiration and glycolysis were measured, and the data were plotted on an energy graph. Figure 15 Observations have shown that GMP-grade Angie cells are more vigorous and have greater glycolytic capacity than non-GMP cells. This translates into improved angiogenesis capacity and a more pronounced characteristic profile.

[0110] Example 6: Use of Angiocyte for diabetic foot ulcers

[0111] In this study, diabetic male mice aged 14 to 15 weeks were used to assess diabetic wound healing. Figure 16 Wounds of the same size were created using a trephine punch. To better mimic human conditions, a silicone ring around the wound was sutured in place to reduce contraction-induced healing. Wounds were treated with PBS (sham-operated control), carboxymethyl cellulose (CMC) gel, or CMC gel containing 500,000 Angilide or 1 million Angilide, respectively. Wounds were photographed over time, and healing was assessed by measuring the percentage of the original wound area. At days 12 and 14, both doses of Angilide in CMC significantly promoted wound healing compared to the control group (PBS and CMC alone). On days 5, 8, and 10, CMC containing 500,000 Angilide showed stronger healing capacity than CMC containing 1 million Angilide; however, this was only statistically significant on day 5. In conclusion, Angilide promotes wound healing in diabetic mice.

Claims

1. A method for culturing a population of endothelial colony-forming cells (ECFCs) derived from umbilical cord blood, the method comprising: (a) The cord blood samples obtained from the subjects were kept at a temperature of 4°C to 15°C for 24 to 72 hours; (b) Isolate mononuclear cells from the said umbilical cord blood sample; (c) The mononuclear cells are seeded on the substrate of the culture medium; (d) Inoculate adherent mononuclear cells in culture medium for about 5 to about 21 days to form cell-containing colonies; (e) Culture cells that express CD31, CD34, CD105, CD144, CD146, CD157, and VEGFR2 but not CD45, CD14, and CD90.

2. A method for culturing a population of endothelial colony-forming cells (ECFCs) derived from umbilical cord blood, the method comprising: (a) Isolation of mononuclear cells from umbilical cord blood samples obtained from subjects; (b) Inoculate the mononuclear cells onto the substrate of the culture medium; (c) Inoculate adherent mononuclear cells in culture medium for about 5 to about 21 days to form cell-containing colonies; (d) Culture cells expressing CD31, CD34, CD105, CD144, CD146, CD157, and VEGFR2 but not CD45, CD14, and CD90; and (e) Treat the resulting cells with an antioxidant to give the cells a repair phenotype.

3. A method for culturing a population of endothelial colony-forming cells (ECFCs) derived from umbilical cord blood, the method comprising: (a) Isolation of mononuclear cells from umbilical cord blood samples obtained from subjects; (b) Inoculate the mononuclear cells onto the substrate of the culture medium; (c) Inoculate adherent mononuclear cells in culture medium for about 5 to about 21 days to form cell-containing colonies; (d) Culture cells expressing CD31, CD34, CD105, CD144, CD146, CD157, and VEGFR2 but not CD45, CD14, and CD90; and (e) Expose the resulting cells to a hypoxic environment to give the cells a repair phenotype.

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 to lyse the blood cells present in the sample, leaving intact mononuclear cells.

5. The culture method according to claim 4, wherein the sample is lysed and processed in a closed cell processing device.

6. The culture method according to any one of claims 1, 4 or 5, wherein after step (b) of the culture method, the method may further include the steps of exposing the monocytes to a hypoxic environment and / or treating the monocytes with an antioxidant, thereby giving the monocytes a reparative phenotype.

7. The cultivation method according to claim 3 or 6, wherein the hypoxic environment is 5% to 10% hypoxic from day 1 of cultivation.

8. The cultivation method according to claim 2 or 6, wherein the antioxidant is selected from flavonoids, flavonoids, catechins, polyphenols, phytoestrogens and / or carotenoids.

9. The cultivation method according to claim 8, wherein the antioxidant is selected from N-acetylcysteine ​​(NAC), quercetin, myricetin, tocopherol, or any combination thereof.

10. The culture method according to claim 2, wherein the cell receiver is treated with an antioxidant once, or wherein the cell receiver is treated with the antioxidant repeatedly over a period of time.

11. The culture method according to any one of claims 1 to 10, further comprising passage-culturing cells expressing CD31, CD34, CD105, CD146, CD144, CD157 and VEGFR2 but not expressing CD45, CD90 and CD14 at least 38 times to increase the population size.

12. The culture method according to any one of claims 1 to 11, wherein the culture medium is an endothelial growth medium.

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. The culture method according to any one of claims 1 to 13, wherein the culture medium is free of antibiotics.

15. The culture method according to any one of claims 1 to 14, wherein the cells are cultured under GMP-grade culture conditions.

16. The culture method according to any one of claims 1 to 15, wherein the culture medium comprises the following components: human epidermal growth factor at a concentration of 1 ng / mL to 10 ng / mL, human basic fibroblast growth factor at a concentration of 5 ng / mL to 20 ng / mL, human insulin-like growth factor at a concentration of 5 ng / mL to 25 ng / mL, human vascular endothelial growth factor at a concentration of 0.5 ng / mL to 50 ng / mL, optionally an antioxidant at a concentration of 1 μg / mL to 50 μg / mL, and hydrocortisone at a concentration of 0.1 μg / mL to 2 μg / mL.

17. The culture method according to any one of claims 1 to 16, wherein the culture substrate has a coating containing extracellular matrix molecules, preferably wherein the extracellular matrix molecules are selected from type O collagen, type I collagen, GMP grade collagen, Biolamina, or any combination thereof.

18. The cultivation method according to claim 17, wherein the GMP-grade collagen is GMP-grade human collagen or GMP-grade collagen without heterologous components.

19. The culture method according to any one of claims 1 to 18, further comprising analyzing cells expressing CD31, CD105, CD146, CD144 and VEGFR2 but not expressing CD45 and CD90.

20. The culture method according to any one of claims 1 to 19, wherein during culture, the number of cells can increase by up to 100 over a period of 100 days. 21 times.

21. The culture method according to any one of claims 1 to 20, wherein the umbilical cord blood sample has a volume of at least 1 mL, preferably wherein the umbilical cord blood sample has a volume between 20 mL and 80 mL.

22. The culture method according to any one of claims 1 to 21, wherein the cells are not purified.

23. A cell that can be obtained by any one of claims 1 to 22.

24. The cell of claim 23, used to treat a disease of the subject, or the cell of claim 23, used for therapeutic purposes.

25. The cell for use according to claim 24, wherein the disease is an ischemic disease.

26. The cell for use according to claim 25, wherein the ischemic disease is ischemic heart disease, ischemic cerebral disease, severe limb ischemia, mesenteric ischemia, stroke, or ischemic retinopathy.

27. The cell for use according to claim 26, wherein the ischemic disease is ischemic retinopathy.

28. The cell for use according to claim 27, wherein the ischemic retinopathy is retinopathy of prematurity (ROP), diabetic retinopathy (DR), or age-related macular degeneration (AMD), preferably wherein the ischemic retinopathy is AMD.

29. The cell for use according to claim 28, wherein the age-related macular degeneration is wet age-related macular degeneration or dry age-related macular degeneration, preferably wherein the age-related macular degeneration is dry age-related macular degeneration.

30. The cell for use according to claim 24, wherein the disease is skin damage.

31. The cell for use according to claim 24, wherein the disease is a bone disease.

32. The cell according to claim 23, used for treating neoplastic diseases.

33. The cell for use according to claim 32, wherein the skin injury is a trauma or burn.

34. The cell for use according to claim 33, wherein the trauma is a traumatic ulcer, preferably wherein the traumatic ulcer is a foot traumatic ulcer, and wherein the burn is a burn caused by radiotherapy.

35. The cell for use according to claim 34, wherein the traumatic ulcer is a diabetic traumatic ulcer, preferably wherein the diabetic traumatic ulcer is a diabetic leg traumatic ulcer or a diabetic foot traumatic ulcer.

36. The cells for use according to any one of claims 24 to 35, wherein the cells are administered topically, intravenously, intramuscularly, intra-articularly, subcutaneously, or orally, preferably wherein the cells are administered topically.

37. The cell for use according to any one of claims 24 to 36, wherein the cell is administered to the subject in combination with one or more additional therapeutic agents.

38. The cell for use according to claim 37, wherein the one or more additional therapeutic agents are selected from the list including: cell therapy, aspirin, nitrates, beta-blockers, calcium channel blockers, cholesterol-lowering drugs, angiotensin-converting enzyme (ACE) inhibitors, alginate dressings, hydrocolloid dressings, wound dressings, antimicrobial agents, antibiotics, antiVEGF drugs, photodynamic therapy, or any combination thereof.

39. The cell for use according to any one of claims 24 to 38, wherein the cell is a supporting blood supply element.

40. A wound dressing comprising cells and a substrate obtained by the method of any one of claims 1 to 23.

41. The wound dressing of claim 39, wherein the substrate is a polymer-based material, a supporting matrix, a hydrogel, or a scaffold.

42. A method for treating, inhibiting, preventing recurrence, or controlling ischemic diseases, neoplastic diseases, bone diseases, or skin lesions, wherein the method comprises applying the cells of claim 23 to a subject in need of such treatment.

43. The method of claim 42, wherein the method comprises cells used according to any one of claims 26 to 29 or 33 to 39.

44. Use of the cell according to claim 23 in the preparation of a medicament for treating, preventing recurrence of, or controlling ischemic diseases, neoplastic diseases, bone diseases, or skin injuries in subjects with such need.

45. The use according to claim 44, wherein the cell used is the cell according to any one of claims 26 to 29 or 33 to 39.

46. ​​An apparatus for delivering cells according to claim 23, wherein the apparatus comprises a syringe containing cells and a delivery medium according to any one of claims 26 to 29.

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