WO2025026877A1

WO2025026877A1 - Cell culture methods and composition

Info

Publication number: WO2025026877A1
Application number: PCT/EP2024/071164
Authority: WO
Inventors: Alan STITT; Reinhold MEDINA; Christina O'NEILL
Original assignee: Vascversa Ltd
Current assignee: Vascversa Ltd
Priority date: 2023-07-28
Filing date: 2024-07-25
Publication date: 2025-02-06
Anticipated expiration: 2026-01-28

Abstract

The invention relates to a culture method for culturing a population of umbilical cord blood derived endothelial colony forming cells (ECFC), the method comprising: (a) maintaining an umbilical cord blood sample obtained from a subject for between 24 to 72 hours at a temperature of between 4°C to 15°C; (b) isolating the mononuclear cell from the blood sample; (c) seeding the mononuclear cell on a culture substrate; (d) culturing the seeded adherent mononuclear cell in a culture medium for about 5 to about 21 days to form a colony comprising cells; (e) culturing the cells that express CD31, CD34, CD105, CD144, CD146, CD157, VEGFR2, but do not express CD45, CD14 and CD90.

Description

CELL CULTURE METHODS AND COMPOSITION

FIELD OF THE INVENTION

The present invention relates to methods of culturing a high proliferative potential- endothelial colony forming cell and their uses thereof in the treatment of disease.

BACKGROUND

Cell based therapies are at the forefront of medicine and are emerging as a new way to treat disease. Stem cells have been considered appealing due to their plastic nature allowing them to become any cell necessary for treatment. Despite this unique ability, the use of stem or induced pluripotent stem cells for cell therapy remains challenging. Difficulties with clinical grade production, laborious differentiation processes, reduced efficacy, safety risks and uncontrolled nature of differentiation in vivo remain major hurdles. Cell therapies using progenitor cells, cells which have committed themselves to a particular cell lineage but still display a certain plasticity and enhanced regenerative potential are therefore an exciting prospect for cell therapies.

Endothelial Progenitor Cell (EPC) is a term used to describe a highly heterogeneous population of cells extracted from either peripheral or umbilical cord blood. First isolated and described by Asahara et al, (Science, 1997; 275(5302):964-7) this population of cells was found to contribute to and enhance vascular regeneration. Later work identified a population of EPC which is believed to be the true vasoreparative cell type, known as an Endothelial Colony forming cell (ECFC).

Various pathologies exist today that are characterized by vascular insufficiency, often these conditions have no available therapeutic options, with treatments instead aimed at the management of symptoms. These conditions, such as chronic non-healing wounds, peripheral limb ischaemia, ischaemic retinopathies and dry AMD affect millions of patients worldwide each year, leading to a significant impact on patient quality of life. Accordingly, there is a need in the art for improved therapies to address conditions caused by vascular insufficiency. SUMMARY OF INVENTION

The inventors of the present invention have developed a method for culturing a high proliferative potential-endothelial colony forming cell (herein referred to as “Angicytes”), such that cells with improved properties can be obtained. The isolation method results in a highly pure and potent vasoreparative population of Angicytes, which can be efficiently and consistently amplified at scale for therapeutic applications. Such cells can subsequently be used in a variety of therapeutic applications, particularly those diseases which are associated with a lack of blood/oxygen flow.

Once generated, the Angicytes disclosed herein display characteristics that make them highly favorable candidates for the treatment of a number of diseases characterised by vascular dysfunction/insufficiency. One of the most advantageous characteristics they possess is a proliferative capacity many times superior to that of mature endothelial subtypes which proliferate slowly, and begin showing signs of senescence after only a few passages. In contrast, Angicytes proliferate rapidly, and are capable of reaching over 60 population doubling levels (PDLs), optionally up to 100 population doubling levels, before showing signs of senescence. This trait lends itself well to the scalability of the cell. Angicyte cells are vasoreparative in nature, having the ability to make new blood vessels and promoting vascular repair leading to 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 an umbilical cord blood sample for between 24 to 72 hours at a temperature of between 4°C to 15°C prior to isolating a mononuclear cell from the blood sample; (b) isolating the mononuclear cell from the blood sample; (c) seeding the mononuclear cell on a culture substrate; (d) culturing the seeded adherent mononuclear cell in a culture medium for about 5 to about 21 days to form a colony comprising cells; (e) culturing cells that express CD31 , CD34, CD105, CD144, CD146, CD157 and VEGFR2, but do not express CD45, CD14 and CD90. The cells have median cell size of 18 micrometers. In a second 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) isolating a mononuclear cell from an umbilical cord blood sample obtained from a subject; (b) seeding the mononuclear cell on a culture substrate; (c) culturing the seeded adherent mononuclear cell in a culture medium for about 5 to about 21 days to form a colony 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) treating the resulting cells with an anti-oxidant, such that the cells have a reparative phenotype. Median cell size is 18 micrometres.

In a third 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) isolating a mononuclear cell from an umbilical cord blood sample obtained from a subject; (b) seeding the mononuclear cell on a culture substrate; (c) culturing the seeded adherent mononuclear cell in a culture medium for about 5 to about 21 days to form a colony 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. The cells have a Median cell size of 18 micrometres.

In a fourth aspect of the invention a population of umbilical cord blood-derived endothelial colony forming cells (ECFC) obtainable by the methods herein disclosed, and therapeutic uses thereof, are provided.

In a fifth aspect, a wound dressing comprising a population of umbilical cord blood derived endothelial colony forming cells (ECFC), _obtained by the methods herein disclosed, and a substrate, is provided.

In a sixth aspect, a method of treating, inhibiting, preventing recurrence, or controlling an ischemic disease, neoplastic disease, bone disease or skin injury, wherein the method comprises administering to a subject in need thereof a cell according to the present invention is provided. In a seventh aspect, a use of the cell according to the present invention in the manufacture of a medicament for the treatment, prevention of recurrence or control of an ischemic disease, neoplastic disease, bone disease or skin injury in a subject in need thereof is provided.

In an eighth aspect, a device for the delivery of the cells herein disclosed is provided, said device comprising a syringe, wherein said syringe contains the cells herein disclosed and a delivery medium.

BRIEF DESCRIPTION OF DRAWINGS

The invention is described with reference to the accompanying figures wherein:

Figure 1 : shows the generation of Angicyte cells from umbilical cord blood. Blood was collected and mixed with anticoagulant, then held for a period of at least 35 - 72 hours before processing.

Figure 2: shows the advantage of red blood cell lysis over standard density gradient centrifugation. This diagram shows a comparability study against a Ficol density gradient centrifugation method. The red blood cell lysis approach increased cell yield (A) and increased numbers of generated Angicyte colonies (B).

Figure 3: A. shows the characteristic cobblestone morphology of a confluent monolayer of Angicyte cells. B. Representative image of Angicyte colony formation in a 6-well plate. After 7 days in culture, single cells proliferate to produce Angicyte colonies. C(1). Graph depicting the growth kinetics of 3 representative clones. C(2). The maximum population doubling levels (PDLs) of Angicyte clones is shown. Angicytes can undergo up to 100 PDLs. D(1). Representative image of the tube formation assay performed in Matrigel. These are live cells stained with Calcein AM. D(2). A skeletonized view of the same tube formation assay depicted in D(1). E. Barrier formation (cell index) is assessed in real-time using the xCELLigence system. The graph depicts barrier formation of 3 clones over 24 hours. Angicytes can reach a cell index value of 11 . Figure 4: Flow cytometry analysis to assess Angicyte phenotype. In this example, Angicyte cells have been harvested at P3, suspended in FACs buffer and stained with antibodies against CD31 , CD105, VEGFR2, CD34, CD45 and CD90. After staining, samples were washed and analysed using an Attune flow cytometer. The figure shows expression of a representative Angicyte colony.

Figure 5: Angicyte cells form dense Pseudovascular networks rapidly within a fibrin-based 3D gel structure. When seeded in a Fibrin gel, Angicytes form dense, 3D tubular networks, these networks are susceptible to the addition of pro- angiogenic and anti-angiogenic factors. Multi-layered images of these gels are captured and then analysed via an online, Al-based analysis software known as IKOSA (A). I KOSA analyses multiple factors of the tubular networks such as tube area (highlighted areas), the number of branching points (circles) and the number and size of ring structures within the network (multi-coloured areas). (B) The total area % of tubes provides a readout for the net size of the tubular networks produced by the cells, and thus their angiogenic capacity. The addition of pro- angiogenic factors results in increases in tube areas, whilst the addition of anti- angiogenic factors resulted in significant decreases in total tube area. The number of branching points (C) follow a trend remarkably similar to that of the total area % of the tubular networks. This indicates that the cells are responsive to stimuli as would be expected of true vascular networks within the body.

Figure 6: Angicyte cells treated with the antioxidant a-tocopherol or control (media) or a vehicle control (ethanol) for 60 minutes prior to passing for a tube formation assay in Matrigel. A. Images of a representative clone taken 2 days after initiation of tube formation and processed and analysed in Imaged. B. Area of tube coverage was measured in percent. Priming with a-tocopherol improves tubulogenesis.

Figure 7: Flow cytometry analysis showing Angicyte phenotype. Representative Angicyte cells have been stained with antibodies against CD144, CD146, CD157, and CD14. After staining, samples were washed and analysed using an Attune flow cytometer. The figure shows expression of a representative Angicyte clone. Figure 8: Immunocytochemistry analysis showing Angicyte phenotype. Angicyte cells have been fixed and stained with antibodies against CD144 (VE-cadherin) and vWF, and counterstained with the nuclei stain DAPI. The figure shows expression of a representative Angicyte clone.

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 have been stained with antibodies against CD31 , CD105, VEGFR2, CD90, and CD45. After staining, samples were washed and analysed using an Attune flow cytometer. The Figure shows expression in a representative Angicyte clone.

Figure 10. Flow cytometry analysis to assess yield of CD45^negatlve /CD31 ^p0Sltlve Angicyte from umbilical cord blood processed as fresh, or 35 hours post-collection, or 72 hours post-collection. Cells were stained with CD45 and CD31 antibodies, and analysed by flow cytometry. Cells were gated as single cells, then gated for the live population, and then gated for the CD45 negative population. Cells were gated for the CD31 positive population and the number of CD45^negatlve/CD31 ^p0Sltlve cells were counted.

Figure 11 : The vascular network forming capacity of Angicyte from 4 donors of umbilical cord blood processed fresh, or 72 hours post-collection was assessed using the fibrin-based angiogenesis assay. Angicyte were harvested from flasks and seeded in a fibrin-based gel in a 15-well p-angiogenesis slide. After 48 hours of incubation, each well was imaged using an EVOS microscope. Images were analysed using the IKOSA software, and the percentage area of tube coverage was measured.

Figure 12: The vascular network-forming capacity of 4 Angicyte clones was assessed using the fibrin-based angiogenesis assay. Angicyte clones were incubated either under standard normoxic conditions or 10% hypoxia for 24 hours, before harvesting and seeding into fibrin-based gel in 15-well p-angiogenesis slides. After 48 hours, each well was imaged using an EVOS microscope. Images were analysed using the IKOSA software, and the percentage area of tube coverage was measured.

Figure 13: Expression analysis of Angicyte cells produced using 1) GMP conditions (Angicyte protocol as described herein), using GMP-compliant and xeno-free reagents, or 2) non-GMP conditions, using xeno-containing reagents (e.g., fetal bovine serum, rat tail collagen). Data from 9 replicates. Analysis of data showed that the samples clustered according to the GMP vs non-GMP variable, which was confirmed by principle-component analysis. 13B. The table lists significantly upregulated genes in GMP vs non-GMP cells that are associated with improved endothelial functionality, specifically the ability to form mature vascular networks by angiogenesis.

Figure 14: The vascular network forming capacity of Angicytes cultured under GMP conditions was compared to that cultured in non-GMP conditions. Angicytes were harvested from flasks and seeded in a fibrin-based gel in a 15-well p- angiogenesis slide. After 48 hours of incubation, each well was imaged using an EVOS microscope. Images were analysed using the IKOSA software, and the percentage area of tube coverage was measured.

Figure 15: The bioenergetics of Angicytes from either GMP or non-GMP grade conditions were assessed using the Seahorse XF Bioanalyzer. Cells were harvested from flasks and seeded in specialized plates per the manufacturer’s guidelines for the bioanalyzer. Mitochondrial respiration (OCR, oxygen consumption rate) and glycolytic capacity (ECAR, extracellular acidification rate) were measured, and the data plotted in an energy map.

Figure 16: Angicytes increase wound healing in a murine model of diabetic wounds. Diabetic male mice (BKS-Lepr db/Rj genetic background) at 14-15 weeks of age were used in this study. Wounds of equal size were initiated using a trephine punch. To better mimic the human condition, a silicone ring surrounding the wound was sutured in place to reduce healing by contraction. Wounds were treated with PBS (sham control) (n=6), or a CMC gel (n=9), or CMC gel containing 500,000 Angicyte (n=11), or CMC gel containing 1 million Angicytes (n=4). Wounds were photographed over time and healing assessed by measuring wound area as a percentage of the original wound area. One-way ANOVA testing showed significant differences among means at days 5, 8, 10, 12 and 14, at which point the study concluded.

DETAILED DESCRIPTION

In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

As used herein, the terms “population of umbilical cord blood derived endothelial colony forming cells (ECFC)” and “Angicyte(s)”, are used interchangeably and refer to a population of highly reparative endothelial-forming cells. Said cells express cell surface antigens that are characteristic of endothelial cells, such as CD31 , CD15, CD105, CD146, CD144, VEGFR2, CD157 and CD34; and do not express cell surface antigens that are characteristic of hematopoietic cells, such as CD45, CD14 and CD90.

As used herein, the term “mononuclear cell” refers to a mononuclear cell found within a mononuclear fraction of a whole blood sample obtained from an umbilical cord blood sample and includes all blood cells with a single nucleus, for example, lymphocytes, monocytes and stem cells. Mononuclear cells may be isolated from the blood sample via density gradient centrifugation.

As used herein, the term “subject” is intended to include human and non-human animals. Preferred subjects include human patients suffering from ischaemic disease or diseases where there is vascular dysfunction, for example, patients suffering from chronic non-healing wounds. Preferred subjects also include human patients suffering from dry age-related macular degeneration. The methods are particularly suitable for treating human patients having a disorder that can be treated by augmenting angiogenesis. In a particular embodiment, the methods are particularly suitable for treatment of an ischemic disease. In another embodiment, the methods herein are particularly suitable for treatment of dry age- related macular degeneration and/or for the treatment of ischaemic retinopathies. In another embodiment, the methods herein disclosed are particularly suitable for the treatment of a skin injury, bone disease or a neoplastic disease.

As used herein, the terms “hypoxic” and “hypoxic environment” are used interchangeably and refer to cell culture conditions wherein the cells are exposed to a low oxygen environment. Preferably, the concentration of oxygen that the cells of the invention are exposed to is between 5-10% oxygen. The skilled person will be aware of the different ways in which a cell culture hypoxic environment can be initiated and maintained, which include both physical and chemical means. For example, a hypoxic environment may be induced via the use of a hypoxia incubator chamber or chemically, for example, the use of sodium dithionite (Na₂S2C>4), cobalt chloride (C0CI2) and (NaN₃). In the context of the present invention, such an environment is considered as especially beneficial due to the preferred use of the cells in ischaemic disease, or in diseases wherein there is a lack of blood supply, and therefore a lack of oxygen. In these cases, the cells have already been primed to be in the conditions that they will most likely experience in vivo, thus resulting in a higher chance of success.

As used herein, the term “reparative phenotype” refers to a mononuclear cell which has enhanced proliferative capabilities and the ability to produce new blood vessels, as measured in a range of in vitro, ex vivo and in vivo assays. As such, cells with this phenotype are envisaged to be particularly useful in the context of treatment of disease, especially those wherein a lack of blood supply is an important factor. The in vitro, ex vivo and in vivo assays used to determine this particular phenotype include, but are not limited to in vitro assays, such as tube formation assays, clonogenic assays, bead sprouting assays, Xcelligence barrier assays, immunocytochemistry assays and migration assays, ex vivo assays such as choroid sprouting assays and aortic ring assays, and in vivo assays such as Matrigel plug assays, models of ischaemic retinopathy and skin wound healing models. The skilled person working in this field would readily understand how to both perform and interpret the results generated by the above listed assays. As used herein, the term “anti-oxidant” refers to a substance that is capable of protecting cells from the damage caused by free radicals. Said anti-oxidant may be a natural substance or a synthetic substance. Preferably, the anti-oxidants of the present invention include, but are not limited to, N-Acetyl Cysteine (NAC), Quercetin, Myricetin or any combination thereof. However, it is understood that any substance having the properties of an anti-oxidant, as defined above, are suitable for use in the present invention.

As used herein, the term “treatment” or “therapy” refers to administering an active agent with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect a condition (e.g., a disease), the symptoms of the condition, or to prevent or delay the onset of the symptoms, complications, biochemical indicia of a disease, or otherwise arrest or inhibit further development of the disease, condition, or disorder in a statistically significant manner. For example, in the context of the present invention, the term “treatment” or “therapy” may refer to enhancing blood supply to a target area of the body via the generation of new blood vessels or the regeneration of already existing blood vessels.

The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the indefinite articles "a" or "an" should be understood to refer to "one or more" of any recited or enumerated component.

As used herein, "about" means within an acceptable error range for the particular value as determined by one of ordinary skill 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" can mean within 1 or more than 1 standard deviation per the practice in the art. Alternatively, "about" can mean a range of up to 20%. When particular values are provided in the application and claims, unless otherwise stated, the meaning of "about" should be assumed to be within an acceptable error range for that particular value. Accordingly, in a first aspect of the present 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 an umbilical cord blood sample for between 24 to 72 hours at a temperature of between 4°C to 15°C; (b) isolating the mononuclear cell from the blood sample; (c) seeding the mononuclear cell on a culture substrate; (d) culturing the seeded adherent mononuclear cell in a culture medium for about 5 to about 21 days to form a colony comprising cells; (e) culturing cells that express CD31 , CD34, CD105, CD144, CD146, CD157, and VEGFR2, but do not express CD45, CD14 and CD90. Median cell size is 18 micrometres.

The Angicyte cells of the present invention are generated from ethically sourced, and consented for, umbilical cord blood that would otherwise have been disposed of as a clinical waste product. The cells of the present invention can be isolated from a range of blood sample volumes, demonstrating the flexibility of the method herein disclosed. The cells of the present invention can be isolated from a blood sample having a volume of at least 1 mL. In a preferred embodiment, the cells of the present invention are isolated from a blood sample having a volume of between 20-80mLs. Larger volumes of blood sample are preferred where possible due to enhanced colony formation.

The culture methods herein disclosed therefore disclose a method in which particular cell characteristics, for example, particular cell surface markers (CD31 , CD34, CD105, CD144, CD146, CD157, and VEGFR2) and analysis of cell morphology are used to confirm that the desired cell type i.e. an Angicyte, is produced. Likewise, the methods herein disclosed also confirm the cell type by confirming that the cells do not express other cell markers indicative of other cell types. For example, the cell surface markers CD45 and CD14 are known to be indicative of hematopoietic cells and therefore are used as an additional layer of confirmation that Angicytes are not contaminated with hematopoietic cells. CD90 may be used to determine that no stromal cells are present. The presence or absence of these cell surface markers can be confirmed via any suitable assay for this purpose, for example, flow cytometry immunophenotyping. The method of the invention requires that the mononuclear cells are isolated from the blood cells present in the umbilical cord blood. The step of isolation can be carried out in any conventional way. For example, density gradient centrifugation (eg using Ficoll or Histopaque) may be used. However, in a preferred method, isolation is carried out by treating the cord blood with a red blood cell lysis buffer such that any red blood cells are lysed. The mononuclear cells that remain may then be cultured as further described. Accordingly, “isolation” is to be interpreted not merely as physical separation, but also separation due to a treatment that allows the mononuclear cells to be discriminated within the cord blood sample, permitting selective culturing to be carried out.

In one embodiment, the cord blood is treated with a red blood cell lysis buffer for between 5 to 20 minutes, preferably approximately 10 minutes, and then a wash step is performed prior to the remaining mononuclear cells being counted and seeded at high density on an appropriate substrate. In a further embodiment, the lysis step can be performed in a closed cell processing apparatus that permits washing and concentrating steps to be performed. A suitable apparatus is the Lovo Cell Processing System (6R4900) which incorporates a spinning membrane filtration system which can remove cell debris after the lysis step.

The culture method herein disclosed provides a means by which purer cultures of cells can be produced by using blood samples which have been maintained in cool temperatures for between 24 to 72 hours compared to using blood samples that are used for culture immediately, or soon after collection. Accordingly, the blood sample may have been maintained in cool temperatures for between 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 may have been maintained in cool temperatures for between 38-72 hours.

By a “cool temperature”, we intend any temperature between 4°C and 15°C. Accordingly, the temperature may 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 a temperature of 10°C. The blood sample may have a preservative and/or anti-coagulant added to the sample to ensure it maintains its viability. For example, the preservative/anti- coagulant may be citrate-phosphate dextrose (CPD), acid-citrate dextrose (ACD) or CPD/ACD with adenine. Such a method overcomes problems with previously known culture methods, which utilise fresh blood and as a result produce cell cultures contaminated with unwanted cells, for example, mesenchymal cells.

In a second 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) isolating a mononuclear cell from an umbilical cord blood sample obtained from a subject; (b) seeding the mononuclear cell on a culture substrate; (c) culturing the seeded adherent mononuclear cell in a culture medium for about 5 to about 21 days to form a colony 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) treating the resulting cells with an anti-oxidant, such that the cells have a reparative phenotype. Median cell size is 18 micrometres.

The culture method herein disclosed may include a treatment step with an antioxidant. The anti-oxidant may be selected from any anti-oxidant that can achieve the desired effect of the mononuclear cell having a reparative phenotype. For example, the anti-oxidant may be selected from the group comprising flavonoids, flavones, catechines, polyphenols, phytoestrogens and/or carotenoids. Preferably, the anti-oxidant is selected from the group comprising N-Acetyl Cysteine (NAC), tocopherols, Quercetin, Myricetin or any combination thereof. The skilled person will readily appreciate that the concentration of the anti-oxidant is dependent on the particular anti-oxidant intended for use, for example, tocopherols may be used at a concentration of 10 to 100 pM, whilst NAC may be used at a concentration of 750 to 2000 pM. The cells may be treated with an anti-oxidant in a single one-off treatment, or with repeated treatments over a period of time. The “period of time” may span the entirety of the culture method or be at regular intervals thereof. Where the cells are treated repeatedly with the anti-oxidant, the anti-oxidant may be the same at each repeated treatment or may be a different anti-oxidant at each repeated treatment. In a preferred embodiment, the cells would be treated with an antioxidant prior to them being used in a clinical setting.

Where the cells are treated with an anti-oxidant, the cells may be treated with one or more additional compounds. Such additional compounds may further support the action of the anti-oxidant or result in the cell having a reparative phenotype via a similar mechanism to treatment with an anti-oxidant.

In a third 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) isolating a mononuclear cell from an umbilical cord blood sample obtained from a subject; (b) seeding the mononuclear cell on a culture substrate; (c) culturing the seeded adherent mononuclear cell in a culture medium for about 5 to about 25 days to form a colony comprising cells; (d) culturing cells that express CD31 , CD34, CD105, CD144, CD146, CD157, 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. Median cell size is 18 micrometres.

The culture method herein disclosed includes a hypoxic priming step or hypoxic conditioning step, thereby resulting in more robust cells on application and improved angiogenicity. By “robust” we intend that the cells are more adaptable to the in vivo environment they will be in when used in a therapeutic context and therefore have a higher chance of viability/success. Without being bound by theory, it is believed that conditioning the cells in this way at the culturing stage induces a reparative phenotype and an environment in which the cells of the invention are able to stimulate blood vessel formation. The hypoxic environment of the present invention may be defined as having 3-12% oxygen levels from culture day 1 , wherein “culture day 1” is defined as the day that the cells are first isolated and seeded. In a preferred embodiment, the hypoxic environment of the present invention may be defined as having 5-10% oxygen levels from culture day 1 . For example, the hypoxic environment of the present invention may be defined as having 5-6% oxygen levels, 5-7% oxygen levels, 5-8% oxygen levels, 5-9% oxygen levels, 6-7% oxygen levels, 6-8% oxygen levels, 6-9% oxygen levels, 6- 10% oxygen levels, 7-8% oxygen levels, 7-9% oxygen levels, 7-10% oxygen levels, 8-9% oxygen levels, 8-10% oxygen levels or 9-10% oxygen levels. To maintain a continuous hypoxic environment, prior to any culture media changes or passages, the culture medium is equilibrated to the same hypoxic conditions as the cells are being cultured in. Preferably, the culture medium is equilibrated to the same hypoxic conditions as the cells for approximately 1 hour.

The resulting seeded adherent mononuclear cells of the methods herein disclosed are cultured in a suitable culture medium for up to 21 days. For example, the resulting seeded adherent mononuclear cells of the methods herein disclosed may be cultured in a suitable culture medium for about 5 to about 10 days, for about 5 to about 15 days, for about 5 to about 20 days, for about 10 to about 15 days, for about 10 to about 20 days, for about 10 to about 21 days, for about 15 to about 20 days, for about 15 to about 21 days or from 20 to 21 days. The skilled person will readily recognize that resulting colonies are cultured and expanded further to produce large quantities of the desired cell type. Scalability in this manner allows for the use of the cells herein described in various therapeutic applications.

The culture methods herein disclosed may further comprise subculturing the cells that express CD31 , CD34, CD105, CD146, CD144, CD157 and VEGFR2 but not CD45, CD14 and CD90 for at least 30 population doublings. Such a property is a characteristic feature of the cells herein disclosed.

The culture medium in which the cells are cultured may be an endothelial growth medium. The skilled person will readily understand that an endothelial growth medium is a medium which has been optimised for the specific cultivation of endothelial cells. In the context of the present invention, the endothelial growth medium may be of good-manufacturing practice (GMP) grade, and thus contains all the relevant clinical factors, such as human serum, and contains no antibiotics. Such mediums are available commercially, for example, PromoCell medium. As such, in one preferred embodiment, the cells are cultured in GMP grade culture conditions. In another preferred embodiment, the culture medium, for example, the endothelial growth medium, comprises human serum, preferably, wherein the culture medium comprises 5-20% human serum, even more preferably wherein the culture medium comprises 10% human serum. In yet another preferred embodiment, the culture medium, for example, the endothelial growth medium, does not contain antibiotics.

The culture medium of the methods herein disclosed may comprise the following components: human epidermal growth factor at a concentration of 1-10 ng/mL, human basic fibroblast growth factor at a concentration of 5-25 ng/mL, human insulin-like growth factor at a concentration of 5-100 ng/mL, human vascular endothelial growth factor at a concentration of 0.5-50 ng/mL, optionally an antioxidant at a concentration of 1-100 pg/mL and hydrocortisone at a concentration of 0.1-2 pg/mL. For example, the human epidermal growth factor may be at a concentration of 1-2 ng/mL, 1-3 ng/mL, 1-4 ng/mL, 1-5 ng/mL, 1-6 ng/mL, 1-7 ng/mL, 1-8 ng/mL, 1-9 ng/mL, 2-3 ng/mL, 2-4 ng/mL, 2-5 ng/mL, 2-6 ng/mL, 2-7 ng/mL, 2-8 ng/mL, 2-9 ng/mL, 2-10 ng/mL, 3-4 ng/mL, 3-5 ng/mL, 3-6 ng/mL, 3-7 ng/mL, 3-8 ng/mL, 3-9 ng/mL, 3-10 ng/mL, 4-5 ng/mL, 4-6 ng/mL, 4-7 ng/mL, 4-8 ng/mL, 4-9 ng/mL, 4-10 ng/mL, 5-6 ng/mL, 5-7 ng/mL, 5-8 ng/mL, 5-9 ng/mL, 5-10 ng/mL, 6-7 ng/mL, 6-8 ng/mL, 6-9 ng/mL, 6-10 ng/mL, 7-8 ng/mL, 7- 9 ng/mL, 7-10 ng/mL, 8-9 ng/mL, 8-10 ng/mL, or 9-10 ng/mL. The human basic fibroblast growth factor may be at a concentration of 5-10 ng/mL, 5-15 ng/mL, 10- 15 ng/mL, 10-20 ng/mL or 15-20 ng/mL. The human insulin-like growth factor may be used at a concentration of 5-10 ng/mL, 5-15 ng/mL, 5-20 ng/mL, 10-15 ng/mL, 10-20 ng/mL, 10-25 ng/mL, 15-20 ng/mL, 15-25 ng/mL or 20-25 ng/mL. The human vascular endothelial growth factor may be used at a concentration of 0.5- 5 ng/mL, 0.5-10 ng/mL, 0.5-15 ng/mL, 0.5-20 ng/mL, 0.5-25 ng/mL, 0.5-30 ng/mL, 0.5-35 ng/mL, 0.5-40 ng/mL, 0.5-45 ng/mL, 5-10 ng/mL, 5-15 ng/mL, 5-20 ng/mL, 5-25 ng/mL, 5-30 ng/mL, 5-35 ng/mL, 5-40 ng/mL, 5-45 ng/mL, 5-50 ng/mL, 10- 15 ng/mL, 10-20 ng/mL, 10-25 ng/mL, 10-30 ng/mL, 10-35 ng/mL, 10-40 ng/mL, 10-45 ng/mL, 10-50 ng/mL, 15-20 ng/mL, 15-25 ng/mL, 15-30 ng/mL, 15-35 ng/mL, 15-40 ng/mL, 15-45 ng/mL, 15-50 ng/mL, 20-25 ng/mL, 20-30 ng/mL, 20- 35 ng/mL, 20-40 ng/mL, 20-45 ng/mL, 20-50 ng/mL, 25-30 ng/mL, 25-35 ng/mL, 25-40 ng/mL, 25-45 ng/mL, 25-50 ng/mL, 30-35 ng/mL, 30-40 ng/mL, 30-45 ng/mL, 30-50 ng/mL, 35-40 ng/mL, 35-45 ng/mL, 35-50 ng/mL, 40-45 ng/mL, 40- 50 ng/mL, or 45-50 ng/mL. The antioxidant may be at a concentration of 1-5 pg/mL, 1-10 pg/mL, 1-15 pg/mL, 1-20 pg/mL, 1-25 pg/mL, 1-30 pg/mL, 1-35 pg/mL, 1-40 pg/mL, 1-45 pg/mL, 5-10 pg/mL, 5-15 pg/mL, 5-20 pg/mL, 5-25 pg/mL, 5-30 pg/mL, 5-35 pg/mL, 5-40 pg/mL, 5-45 pg/mL, 5-50 pg/mL, 10-15 pg/mL, 10-20 pg/mL, 10-25 pg/mL, 10-30 pg/mL, 10-35 pg/mL, 10-40 pg/mL, IQ- 45 pg/mL, 10-50 pg/mL, 15-20 pg/mL, 15-25 pg/mL, 15-30 pg/mL, 15-35 pg/mL, 15-40 pg/mL, 15-45 pg/mL, 15-50 pg/mL, 20-25 pg/mL, 20-30 pg/mL, 20-35 pg/mL, 20-40 pg/mL, 20-45 pg/mL, 20-50 pg/mL, 25-30 pg/mL, 25-35 pg/mL, 25- 40 pg/mL, 25-45 pg/mL, 25-50 pg/mL, 30-35 pg/mL, 30-40 pg/mL, 30-45 pg/mL, 30-50 pg/mL, 35-40 pg/mL, 35-45 pg/mL, 35-50 pg/mL, 40-45 pg/mL, 40-50 pg/mL, or 45-50 pg/mL. The hydrocortisone may be used at a concentration of 0.1 -0.2 pg/mL, 0.1 -0.4 pg/mL, 0.1 -0.6 pg/mL, 0.1 -0.8 pg/mL, 0.1-1 pg/mL, 0.1 -1.2 pg/mL, 0.1-1 .4 pg/mL, 0.1-1 .6 pg/mL, 0.1-1.8 pg/mL, 0.2-0.4 pg/mL, 0.2-0.6 pg/mL, 0.2-0.8 pg/mL, 0.2-1 pg/mL, 0.2-1 .2 pg/mL, 0.2-1 .4 pg/mL, 0.2-1 .6 pg/mL, 0.2-1.8 pg/mL, 0.2-2 pg/mL, 0.4-0.6 pg/mL, 0.4-0.8 pg/mL, 0.4-1 pg/mL, 0.4-1 .2 pg/mL, 0.4-1 .4 pg/mL, 0.4-0.6 pg/mL, 0.4-0.8 pg/mL, 0.4-2 pg/mL, 0.6-0.8 pg/mL, 0.6-1 pg/mL, 0.6-1 .2 pg/mL, 0.6-1 .4 pg/mL, 0.6-1 .6 pg/mL, 0.6-1.8 pg/mL, 0.6-2 pg/mL, 0.8-1 pg/mL, 0.8-1 .2 pg/mL, 0.8-1 .4 pg/mL, 0.8-1.6 pg/mL, 0.8-1.8 pg/mL, 0.8-2 pg/mL, 1 -1 .2 pg/mL, 1 -1 .4 pg/mL, 1-1.6 pg/mL, 1-1.8 pg/mL, 1 -2 pg/mL, 1.2- 1.4 pg/mL, 1.2-1 .6 pg/mL, 1.2-1.8 pg/mL, 1.2-2 pg/mL, 1.4-1 .6 pg/mL, 1.4-1.8 pg/mL, 1.4-2 pg/mL, 1.6-1.8 pg/mL, 1.6-2 pg/mL or 1.8-2 pg/mL.

In a most preferred embodiment, the culture medium may comprise 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 0.5ng/mL, optionally an antioxidant at a concentration of 1 pg/mL and hydrocortisone at a concentration of 0.2 pg/mL.

The cells of the present invention may be seeded on any suitable culture substrate for the purpose of growing and isolating the cells herein disclosed. The cells herein descried possess integrins; molecules responsible for mediating cell attachment. Accordingly, any culture substrate which has an affinity for any of the known endothelial integrin units, for example collagen and laminin, may be used as a suitable culture substrate. It is noted that the methods herein disclosed are suitable for culturing from both single cells and colonies up. The culture substrate may comprise a coating comprising an extracellular matrix molecule (ECM). Such an extracellular matrix molecule acts as a scaffold of various proteins and molecules that provides structural and biochemical support for cells. For example, the extracellular matrix molecule may be selected from the group comprising a GMP grade collagen, a type 0 collagen, a type I collagen, a type II collagen, a type III collagen, a type IV collagen, a type X collagen, laminin, a recombinant laminin, or any combination thereof. Preferably, the extracellular matrix molecule is selected from the group comprising a type 0 collagen, a type I collagen, a GMP grade collagen, a recombinant laminin (for example, Biolamina) or any combination thereof. In an even more preferred embodiment, the extracellular matrix molecule is a type I collagen or a GMP grade collagen, wherein the GMP grade collagen is a GMP grade human-derived collagen, ora GMP grade xenofree collagen. As used herein, the term “xenofree” refers to a product, for example, a culture medium, being free of non-human animal components.

The culture methods herein disclosed may result in cultures wherein the number of cells can increase up to over 10²¹. Accordingly, the cells of the present invention can be cultured over a prolonged period whilst maintaining their proliferative capacity, thus demonstrating the proliferative potential of these cells and their potential use in therapeutic applications wherein proliferation is key, for example, in angiogenesis.

The culture methods herein disclosed may comprise maintaining a blood sample for between 24 to 72 hours at a temperature of between 4°C to 15°C exposing the mononuclear cells to a hypoxic environment and/or treating the mononuclear cells with an anti-oxidant. In one embodiment, following step b) of the culture method of the first aspect, the method may further comprise the steps of exposing the mononuclear cell to a hypoxic environment and/or treating the mononuclear cell with an anti-oxidant, such that the mononuclear cell has a reparative phenotype. In an alternative embodiment, the step of maintaining the blood sample for between 24 to 72 hours at a temperature of between 4°C to 15°C is omitted and the culture method may comprise the steps of exposing the mononuclear cell to a hypoxic environment and treating the cells with an anti-oxidant only, such that the mononuclear cell has a reparative phenotype.

In one embodiment, the culture method of any one of the culture methods herein disclosed does not undergo a purification process. As such, the culture methods herein disclosed do not require multi-step depletion or enrichment steps to obtain the desired cells at the end of the culture period. Not only does this make the culture process more efficient, both from a time and resource perspective, but it also significantly reduces the potential risks associated with sterility. Accordingly, the culture methods herein described represent an improved methodology.

In a fourth aspect, the present invention discloses a population of umbilical cord blood derived endothelial colony forming cells (ECFC) obtainable by any one of the methods herein disclosed, and therapeutic uses thereof.

As is demonstrated in the Examples below, the inventors of the present invention have demonstrated that the resulting ECFCs from the methods herein disclosed (herein referred to as “Angicytes”), possess improved properties compared to those ECFCs that have not been produced by the methods herein described. Specifically, the inventors have demonstrated that not only do the resulting cells have a more glycolytic/energetic metabolic profile, but that the cells also show a significantly enhanced angiogenic/vasculogenic profile. As such, the resulting cells have an enhanced reparative phenotype, and are therefore envisaged to be particularly useful in therapies in which angiogenic properties are desirable. As such, the present invention also discloses the use of the Angicytes herein described for use in the treatment of disease in a subject in need thereof, or for therapeutic use. The Angicytes obtained by any of the methods herein disclosed have numerous advantageous characteristics, such as high proliferation rates, low levels of senescence and high levels of adaptability to their surrounding environment. Accordingly, such a cell type is highly advantageous in therapeutic areas in which these properties are desired, for example, to treat diseases/conditions associated with vascular dysfunction.

Accordingly, the present invention discloses the use of the Angicytes herein described for use in the treatment of ischemic disease or diseases associated with vascular dysfunction. By “ischemic disease” we intend any disease which is caused (either partially or wholly) by restricted or reduced blood flow to a specific part of the body. For example, the present invention discloses the use of the Angicytes herein described for use in the treatment of ischemic heart disease, ischemic brain disease, critical limb ischemia, mesenteric ischemia, stroke or ischemic retinopathy.

In one preferred embodiment, the ischemic disease to be treated is ischemic retinopathy, wherein the ischemic retinopathy 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 in which the macula of the retina is damaged, resulting in loss of central vision. There are two types of AMD; wet AMD and dry AMD, with the latter being the most common. Dry AMD is characterised by vascular dysfunction, retinal pigment epithelium dysfunction and photoreceptor dysfunction. Specifically, it has been found that in early onset dry AMD, degeneration of the choriocapillaris has been observed (Biesemeier et al., 2014, Neurobiology of Aging; 35(11 ):2562-2573), defining dry AMD as a vascular disease. Accordingly, it is envisaged that the cells herein disclosed may be used to effectively repair these damaged vessels. Treatment at an early stage in the disease could prevent disease progression and ultimately save a significant number of patients suffering from dry AM D from losing their vision. As such, in a preferred embodiment, the AMD is dry AMD. The treatment may also be of a skin injury, for bone repair or disease or neoplastic disease (cancer).

The present invention may be used for the treatment and/or prevention of a neoplastic disease, and/or secondary diseases associated with neoplastic disease. In one embodiment, the neoplastic disease may be a solid cancer and/or a haematological malignancy. Neoplasia, tumours 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 neoplasia, tumour, or cancer, or a neoplasia, tumour, cancer or metastasis that is progressing, worsening, stabilized or in remission. It is particularly envisaged that the present invention may be used for the treatment and/or prevention of a neoplastic disease, wherein the neoplastic disease has a high vascular need. In the context of treatment and/or prevention of a neoplastic disease, the cells herein disclosed, i.e. the Angicytes of the present invention, may be used to deliver a cargo molecule, for example, a therapeutic molecule, to a target site i.e. a tumour. As used herein, the term “cargo” will be well known to those in the art, and refers to a specific molecule of interest which is 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. Cargo molecules may be heterologous proteins which do not occur naturally to the carrier cell. The cargo peptide and/or protein may be a therapeutic peptide and/or a therapeutic protein. While it is envisaged that the cargo molecule of the present invention may be a protein or peptide, other cargo types include DNA and RNA molecules. Therefore, in another embodiment, the cargo molecule is an RNA molecule or a DNA molecule.

Cancers that may be treated according to the invention include, but are not limited to, cells or neoplasms of the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestines, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to the following: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumour, malignant; bronchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; ovarian stromal tumour, malignant; thecoma, malignant; granulosa cell tumour, malignant; androblastoma, malignant; Sertoli cell carcinoma; Leydig cell tumour, malignant; lipid cell tumour, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumour; Mullerian mixed tumour; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; Brenner tumour, malignant; phyllodes tumour, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; Kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumour of bone; Ewing's sarcoma; odontogenic tumour, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumour; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumour, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

Where the treatment is of a skin injury, the treatment may be of any injury in which there is damage to the skin or underlying tissue. For example, the skin injury may be a wound (cuts, lacerations, gashes, tears, ulcers, scrapes, abrasions or scratches), a bruise, an avulsion or a bum. In a preferred embodiment, the skin injury may be a wound or a burn. Preferably, where the skin injury is a wound, the wound is a wound ulcer and where the skin injury is a bum, the bum is a bum caused by radiotherapy. In yet a further preferred embodiment, the wound ulcer is a foot wound ulcer. In another preferred embodiment, the wound ulcer to be treated is a chronic wound ulcer. In a further preferred embodiment, the wound/ ulcer to be treated is a diabetic ulcer, preferably wherein the diabetic ulcer is a diabetic leg ulcer or diabetic foot ulcer. It will be readily understood that the route of administration of the cells herein disclosed will depend on the disease to be treated. The cells may be administered topically, intravenously, intramuscularly, intra-articularly, subcutaneously, orally, intraarterially or transdermally. The cells may therefore may be administered systemically, locally to the target site, or a combination of the two. For example, for the treatment of wound ulcers, topical administration may be most beneficial. Likewise, for the treatment of dry AMD, direct supra-choroidal administration may be most beneficial.

The cells may 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 the skilled person.

It is understood that in addition to the administration of the Angicytes for the treatment of the above diseases/conditions, one or more additional therapeutic may be given in combination to maximise the end clinical results (i.e. either via an additive or synergistic effect). As such, Angicytes of the present invention may be administered to the subject in combination with one or more additional therapeutics. Accordingly, the additional therapeutic may include, but is not limited to, cell therapies, aspirin, nitrates, beta-blockers, calcium channel blockers, cholesterol-lowering medications, angiotensin converting enzyme (ACE) inhibitors, alginate dressings, hydrocolloid dressings, other wound dressings, antimicrobials, antibiotics, anti-VEGF medicines, photodynamic therapy or any combination thereof. Such additional therapeutics may be given in combination i.e. at the same time, or sequentially. The term “sequentially” covers both the scenarios in which the Angicytes may be given before the additional therapeutic(s) and where the Angicytes may be given after the additional therapeutics(s). It is understood that the particular additional therapy is dependent on the disease to be treated. It is envisaged that a combination of the Angicytes herein disclosed with any one of the one or more additional therapeutics listed above will be particularly beneficial in any disease or therapy that requires a vascular supply. Target sites that are considered to particularly benefit from an enhanced vascular supply include, but are not limited to, the heart, brain, eyes, skin, liver, pancreas and kidney.

In a preferred embodiment, the additional therapeutic may be a cell therapy. The term “cell therapy or therapies” refers to any therapy in which a cellular material is injected or otherwise transplanted into a subject in need thereof.

It is envisaged that the Angicytes produced as a result of the methods herein disclosed are particularly advantageous in the treatment of any disease in which the lack of a blood supply is a contributing factor. Furthermore, it is envisaged that the Angicytes produced as a result of the methods herein disclosed are advantageous as a supportive treatment for any therapy which has sub-optimal efficacy due to the lack of an appropriate blood supply. As such, in one embodiment, the Angicytes of the present invention may be a supportive blood supply element. The term “supportive blood supply element” in the context of the present invention refers to the ability of the Angicytes to enhance vascular networks via angiogenesis. It is envisaged that the target cells may be within IQ- 200 pm of a bloody supply to survive.

In a fifth aspect, a wound dressing comprising a population of umbilical cord blood derived endothelial colony forming cells (ECFC), obtainable by the methods herein disclosed, and a substrate is provided.

It is envisaged that a wound dressing incorporating the Angicytes herein disclosed will facilitate faster wound healing, and as a result reduce incidences of infection.

The substrate of the wound dressing may be any suitable structural material in which the Angicytes of the present invention can be incorporated, thereby providing a physical medium on or in which the Angicytes may be administered. However, in a preferred embodiment, the substrate may be a polymer-based material, a supportive matrix, a hydrogel, a scaffold, or any combination thereof. The polymer-based material may be a synthetic or natural polymer. 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, hyaluronic acid. The substrate may comprise more than one polymer-based material, wherein the polymer-based materials may be solely synthetic, solely natural, or a combination of the two. In yet a further preferred embodiment, the substrate of the wound dressing is a hydrogel. The hydrogel may be a synthetic hydrogel, for example, a PEG or PVA hydrogel, or a natural-based hydrogen, for example, a collagen- based hydrogel, a gelatin-based hydrogel, a chitosan-based hydrogel, a fibrin- based hydrogel or any polysaccharide-based hydrogel. The hydrogel may be a hybrid hydrogel, comprising more than one type of hydrogel. The hydrogel may comprises a synthetic hydrogel and a natural-based hydrogel. The hydrogel may be an injectable hydrogel, such that the hydrogel is formed upon administration to the subject in need thereof. The wound dressing of the present invention is intended for topical administration to a subject in need thereof.

For topical wounds, it is envisaged that the Angicytes of the present invention are delivered to the target site via the use of a wound dressing. In one embodiment, the Angicytes are combined with a gel prior to application to a wound to provide for the correct consistency, which may be adapted to the size and shape of the wound to be treated. Any appropriate gel may be used. In one embodiment, the gel may be a carboxymethyl cellulose gel. The skilled person will readily understand that the number of Angicytes 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 Angicyte and gel product may contain 100,000-10 million Angicytes, for example, 100,000 to 1 million Angicytes, 100,000 to 2 million Angicytes, 100,000 to 3 million Angicytes, 100,000 to 4 million Angicytes, 100,000 to 5 million Angicytes, 100,000 to 6 million Angicytes, 100,000 to 7 million Angicytes, 100,000 to 8 million Angicytes, 100 or 100,000 to 9 million Angicytes, In a preferred embodiment, the Angicyte and gel product may contain 500,000-1 million Angicytes. In a preferred embodiment, the combined Angicyte and gel product is applied via a syringe system. In a further preferred embodiment, the syringe system is a dual syringe system, wherein the Angicytes of the present invention and the gel are mixed within the syringe. Such a system allows for the gel and Angicytes to be kept under separate storage conditions until mixing of the two components is desirable and for the mixing of the two components to be conducted under sterile conditions.

It is further envisaged that the Angicytes herein disclosed may be delivered using a point-of-care delivery system. Such a point-of-care system may use the syringe system immediately described above. In said system, the Angicytes herein disclosed may be combined with a hypothermic solution, allowing for enhanced accessibility to the treatment, for example, in non-hospital based clinics. Such a delivery system would allow for the Angicytes herein disclosed to be stored at 4°C, instead of -80°C, the latter requiring specialist equipment.

The Angicytes herein disclosed may also be particularly advantageous in the context of surgery, for example, delivery of Angicytes to a target site in need of enhanced blood supply. The skilled person will readily understand that the mode of administration and concentration of the Angicytes to be delivered in the context of surgical application will depend on the purpose of the surgery. As such, in some embodiments, the Angicytes herein disclosed may be delivered directly to the target site. In other embodiments, the Angicytes herein disclosed may be delivered systemically, for example, via an IV drip. The Angicytes herein disclosed may be administered in combination with one or more additional therapies. In a preferred embodiment, the Angicytes herein disclosed may be administered prior to any additional therapy so as to maximise the beneficial effects of the administered Angicytes.

In a sixth aspect, a method of treating, inhibiting, preventing recurrence, or controlling an ischemic disease, neoplastic disease, bone disease or skin injury, wherein the method comprises administering to a subject in need thereof a cell according to the present invention is provided.

The method according to the sixth aspect may further comprise any one of the features pertaining to the cell(s) of the present invention.

In a seventh aspect, a use of the cell according to the present invention in the manufacture of a medicament for the treatment, prevention of recurrence or control of an ischemic disease, neoplastic disease, bone disease or skin injury in a subject in need thereof is provided.

The use of the cell(s) according to the seventh aspect may further comprise any one of the features pertaining to the cell(s) of the present invention.

In an eighth aspect, a device for the delivery of the cells herein disclosed is provided, said device comprising a syringe, wherein said syringe contains the cells herein disclosed and a delivery medium. In a preferred embodiment, the delivery medium may be a gel, for example, a solidified gel, a semi-solid gel, a flowable gel or a scaffold gel.

The invention is further described with reference to the following non-limiting examples:

EXAMPLES

Example 1 : Isolation of clinical grade Angicyte cells.

Fresh umbilical cord blood was collected and mixed with anticoagulant (citratephosphate dextrose (CPD) for storage. The cord blood was stored cooled for a period of at least 35 -72 hours before processing. Cord blood was mixed with red blood cell lysis buffer for 10 minutes, then sample washed, this resulted in red blood cell lysis leaving leukocytes intact. Cells are counted and seeded at high density on human collagen 1 culture substrate coated plasticware and maintained in EGM growth media with 10% human serum (Figure 1). Cultures are maintained for up to 21 days. Cultures are monitored daily for appearance of ANGICYTE vascular colonies which emerge between D3 and D21 after seeding. Cell and colony yield is shown in Figure 2A & B.

Isolated Angicyte cells appear as a homogenous population of cells with a cobblestone like morphology (Figure 3A), they form colonies of cells when seeded as single cells (Figure 3B). They possess a remarkable capacity for expansion, reaching up to 78 PDLs in 100 days (Figure 3C). Angicyte cells form vascular networks in Matrigel (Figure 3D) and form tight barriers with a high cell index (9- 11 Cl) (Figure 3E).

Angicyte cells demonstrate a clear endothelial phenotype. Here we have characterized a population of Angicyte cells harvested from P3 and stained with antibodies against CD31 , CD105, VEGFR2, CD34, CD45 and CD90. Samples analyzed on Attune flow cytometer showed Angicyte cells express high CD31 , CD105, VEGFR2 and are negative for CD90 and CD45 (Figure 4).

We use a fibrin-based assays to allow the complexity of the vascular networks formed by Angicytes to be assessed. Angicyte cells are resuspended in Fibrin and allowed to form primitive blood vessels over a 48-hour period. Figure 5 shows a complex 3D multilayer networks at 48 hours. This network was then assessed using IKOSA platform for analysis (Figure 5).

Priming of Angicyte cells with antioxidants leads to increased vascular function and network formation (Figure 6) shows representative Angicyte cells pretreated with Alpha Tocopherol for 1 hour prior to embedding in a 3D Matrigel assay. Figure 6A reveals enhanced vascular network formation.

Example 2: Confirmation of Angicyte identity

As already highlighted above, Angicyte cells have a clear endothelial phenotype, as demonstrated by cell marker expression. A population of Angicyte cells was harvested at P5 and stained with antibodies against CD144, CD146, CD157, and CD14. Samples analyzed on Attune flow cytometer showed Angicyte cells express CD144, CD146, and CD157, but are negative for CD14 (see Figure 7). Angicyte cells cultured in tissue-culture treated dishes on human collagen were fixed and stained with antibodies against CD144 and vWF by immunocytochemistry, and imaged using a fluorescent DMi8 microscope. Angicyte cells are shown to express CD144 (VE-cadherin), which localizes to cell membranes, and vWF which is found in the cytosol (Figure 8).

Example 3: Holding blood for 72 hours results in enhanced vascular networks Fresh umbilical cord blood was collected and mixed with anticoagulant for storage/transport. Per sample, umbilical cord blood was processed as fresh either (within 6 hours of collection) or stored cooled and processed at 35 or 72 hours post-collection. For processing, umbilical cord blood was mixed with red blood cell lysis buffer to lyse red blood cells and leave leukocytes intact. Cells were counted and seeded at high density (2x10⁶ (±20%) cm²) on human collagen-1 substrate in plastic tissue culture flasks and maintained in endothelial growth media supplemented with 10% human serum. Cultures were monitored daily for the appearance of Angicyte colonies. Angicyte cells were isolated and expanded by passaging and the endothelial phenotype confirmed by flow cytometry. Cells harvested at P5 were stained with antibodies against CD31 , CD105, VEGFR2, CD90, and CD45 and analyzed using an Attune flow cytometer. There was no change in phenotype between Angicyte from ‘fresh’ umbilical cord blood and Angicyte from umbilical cord blood processed 72 hours after collection (Figure 9A and 9B). Angicyte cells retained their endothelial phenotype and expression of stem or hematopoietic marker expression was not increased.

Flow cytometry was used to assess the yield of Angicyte from umbilical cord bloods processed fresh, 35 hours post-collection (35h), or 72 hours post collection (72h). After processing and maintaining in culture for several days, cells were harvested at P0 and stained with antibodies against CD45 and CD31. Using a flow cytometer, live single cells were analyzed, and the numbers of CD45-CD31 + cells were calculated by gating for these populations. Any appropriate software may be used to analyze the results. There was a trend towards increased yield of CD45- CD31+ cells in samples from the 35h and 72h groups (n=6) (Figure 10).

A fibrin-based angiogenesis assay was used to assess the formation and complexity of vascular networks formed by Angicytes in vitro from samples from either freshly processed cord blood or 72h post-collection. Angicytes were harvested from passaged cells in flasks at P5 and resuspended in a fibrin gel and incubated for 48 hours to allow the formation of primitive blood vessels. These vascular networks were imaged using an EVOS microscope and the images analyzed using the IKOSA platform. 4 samples were assessed and the percentage area of vascular network coverage was measured (Figure 11). It was observed that vessel network formation was greater in the 72h samples compared to the fresh samples. In 2 out of 4 clones (Y and Z), processing the blood after 72 hours significantly improved tube formation. In clones V and X, there was a trend towards increased tube formation.

Example 4: Exposure to hypoxia improves functionality

The vascular network-forming capacity of 4 Angicyte clones was assessed using the fibrin-based angiogenesis assay. Angicyte cells in human collagen-coated flasks were incubated under standard normoxic conditions or in 10% O2 for 24 hours before harvesting and resuspending in fibrin gel to allow formation of primitive blood vessels. After 48 hours, the vascular networks were imaged using an EVOS microscope and the images analyzed using the IKOSA platform, to measure the area of vascular network coverage (%). It was observed that exposure to hypoxia primed Angicytes to form more complex vascular structures. In 3 out of 4 clones (1 , 2 and 4) assessed, this was a statistically significant increase (Figure 12). A trend towards increased tube formation in hypoxia was observed in clone 3. Therefore, priming of Angicyte cells by exposing to 10% O2 (hypoxia) for 24 hours leads to improved vascular network formation in vitro.

Example 5: GMP manufacture yields cells with improved endothelial functionality

Fresh umbilical cord blood was collected and mixed with anticoagulant and processed to generate Angicyte cells. Umbilical cord blood was mixed with red blood cell lysis buffer and washed, and the cells counted. Per sample, half of the cells were processed using GMP-grade conditions (human collagen 1 culture substrate with GMP-grade endothelial growth media phenol red-free, xeno-free, and supplemented with human serum). The other half of the cells were processed at the same density using non GMP conditions (rat tail collagen I culture substrate with non GMP-endothelial growth media and fetal bovine serum) Cultures were monitored daily for the appearance of Angicyte colonies. Angicyte colonies were passaged and expanded for various analyses. At P4, Angicytes from substrate-coated flasks were harvested and RNA isolated using a Maxwell instrument for automated RNA extraction. Analysis of RNA showed that the samples clustered according to their treatment status (GMP vs. non-GMP). This is shown in a heatmap dendogram in Figure 13A. GMP Angicytes have a transcriptomic signature that is distinct from non-GMP cultured cells. Figure 13B lists significantly upregulated genes in GMP vs non-GMP cells that are associated with improved endothelial functionality, specifically the ability to form mature vascular networks by angiogenesis. It is therefore clear that the GMP process of production generates a distinct cell type.

The vascular network forming capacity of Angicytes cultured under GMP conditions was compared to that of cells cultured in non-GMP conditions. Angicytes from substrate-coated flasks were harvested and resuspended in fibrin gel to assess their ability to form vascular networks in vitro. After 48 hours, primitive blood vessels were imaged using an EVOS microscope and analyzed using the IKOSA platform. The area of vascular network coverage was measured (%). It was observed that GMP Angicytes formed more vascular networks compared to non-GMP cells (Figure 14). This was statistically significant in 4 out of 5 clones assessed (A, B, C and E) (Figure 14A). A trend for increased vascular network formation was also observed for clone D. Figure 14B also shows representative images from the IKOSA platform. GMP Angicytes formed more complex interconnecting vascular networks than non-GMP cells, as indicated by the greater number of ‘loops’.

The bioenergetic properties of Angicytes from either GMP or non-GMP grade conditions was assessed. Angicytes from substrate-coated flasks were harvested and replated into Seahorse FX Analyzer 96 well plates to assess cell bioenergetics, using the Mito Stress Test and Glyco Stress Kits as per the manufacturer’s instructions. Mitochondrial respiration and glycolysis were measured, and the data plotted in an energy map (Figure 15). It was observed that GMP-grade Angicytes were more energetic and glycolytic than non-GMP counterparts. This translates to an improved angiogenic capacity and profile.

Example 6: Use of Angicytes for diabetic foot ulcers Diabetic male mice at 14-15 weeks of age were used in this study to assess diabetic wound healing (Figure 16). Wounds of equal size were initiated using a trephine punch. To better mimic the human condition, a silicone ring surrounding the wound was sutured in place to reduce healing by contraction. Wounds were treated with PBS (sham control), a carboxymethyl cellulose (CMC) gel, or CMC gel containing 500,000 Angicytes or CMC gel containing 1 million Angicytes. Wounds were photographed over time and healing assessed by measuring wound area as a percentage of the original wound area. At days 12 and 14, both CMC containing Angicyte doses significantly increased wound healing compared to both PBS and CMC alone control groups. There was an observation that CMC with 500,000 Angicytes had greater healing capacity than with 1 million Angicytes at days 5, 8 and 10, however, this was only significant at day 5. In conclusion, Angicytes increased wound healing in diabetic mice.

Claims

1. A culture method for culturing a population of umbilical cord blood derived endothelial colony forming cells (ECFC), the method comprising:

(a) maintaining an umbilical cord blood sample obtained from a subject for between 24 to 72 hours at a temperature of between 4°C to 15°C;

(b) isolating the mononuclear cell from the umbilical cord blood sample;

(c) seeding the mononuclear cell on a culture substrate;

(d) culturing the seeded adherent mononuclear cell in a culture medium for about 5 to about 21 days to form a colony comprising cells;

(e) culturing cells that express CD31 , CD34, CD105, CD144, CD146, CD157, VEGFR2, but do not express CD45, CD14 and CD90.

2. A culture method for culturing a population of umbilical cord blood derived endothelial colony forming cells (ECFC), the method comprising:

(a) isolating a mononuclear cell from an umbilical cord blood sample obtained from a subject;

(b) seeding the mononuclear cell on a culture substrate;

(c) culturing the seeded adherent mononuclear cell in a culture medium for about 5 to about 21 days to form a colony 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) treating the resulting cells with an anti-oxidant, such that the cells have a reparative phenotype.

3. A culture method for culturing a population of umbilical cord blood derived endothelial colony forming cells (ECFC), the method comprising: (a) isolating a mononuclear cell from an umbilical cord blood sample obtained from a subject;

(b) seeding the mononuclear cell on a culture substrate;

(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. The culture method of claim 1 , wherein step (b) is carried out by mixing the umbilical cord blood sample with red blood cell lysis buffer such that blood cells present in the sample are lysed leaving intact the mononuclear cells.

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

6. The culture method of any one of claims 1 , 4 or 5, wherein following step (b) of the culture method, the method may further comprise the steps of exposing the mononuclear cell to a hypoxic environment and/or treating the mononuclear cell with an anti-oxidant, such that the mononuclear cell has a reparative phenotype.

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

8. The culture method of claim 2 or 6, wherein the anti-oxidant is selected from the group comprising flavonoids, flavones, catechines, polyphenols, phytoestrogens and/or carotenoids.

9. The culture method of claim 8, wherein the anti-oxidant is selected from the group comprising N-Acetyl Cysteine (NAC), Quercetin, Myricetin, tocopherols or any combination thereof.

10. The culture method of claim 2,, wherein the cells undergo a single treatment with the antioxidant or wherein the cells undergo repeated treatment with the antioxidant over a period of time.

11 . The culture method of any one of claims 1 to 10 further comprising subculturing the cells that express CD31 , CD34, CD105, CD146, CD144, CD157 and VEGFR2 but not CD45, CD90 and CD14 for at least 38 population doublings.

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

13. The culture method of claim any one of claims 1 to 12, wherein the culture medium comprises human serum, preferably wherein the culture medium comprises 5-20% human serum, more preferably wherein the culture medium comprises 10% human serum.

14. The culture method of any one of claims 1 to 13, wherein the culture medium does not contain antibiotics.

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

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

17. The culture method of any one of claims 1 to 16, wherein the culture substrate comprises a coating comprising an extracellular matrix molecule, preferably wherein the extracellular matrix molecule is selected from the group comprising a type 0 collagen, a type I collagen, a GMP grade collagen, biolamina or any combination thereof.

18. The culture method of claim 17, wherein the GMP grade collagen is a GMP grade human-derived collagen or a GMP grade xenofree collagen.

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

20. The culture method of any one of claims 1 to 19, wherein the number of cells can increase up to 10²¹ fold over a period of 100 days in culture.

21. The culture method of 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 of between 20-80 m Ls.

22. The culture method of any one of claims 1 to 21 , wherein the cells do not undergo a purification process.

23. A cell obtainable by the method of any one of claims 1 to 22.

24. The cell of claim 23 for use in the treatment of disease in a subject or the cell of claim 23 for therapeutic use.

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

26. The cell for use of claim 25, wherein the ischemic disease is ischemic heart disease, ischemic brain disease, critical limb ischemia, mesenteric ischemia, stroke or ischemic retinopathy.

27. The cell for use of claim 26, wherein the ischemic disease is an ischemic retinopathy.

28. The cell for use of 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 of 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 of claim 24, wherein the disease is a skin injury.

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

32. The cell of claim 23 for use in the treatment of a neoplastic disease.

33. The cell for use of claim 32, wherein the skin injury is a wound or a bum.

34. The cell for use of claim 33, wherein the wound is a wound ulcer, preferably wherein the wound ulcer is a foot wound ulcer and wherein the bum is a bum caused by radiotherapy.

35. The cell for use of claim 34, wherein the wound ulcer is a diabetic wound ulcer, preferably wherein the diabetic wound ulcer is a diabetic leg wound ulcer or diabetic foot wound ulcer.

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

38. The cell for use according to claim 37, wherein the one or more additional therapeutic is selected from the list comprising: cell therapies, aspirin, nitrates, beta-blockers, calcium channel blockers, cholesterol-lowering medications, angiotensin converting enzyme (ACE) inhibitors, alginate dressings, hydrocolloid dressings, wound dressings, antimicrobials, antibiotics, anti-VEGF medicines, 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 supportive blood supply element.

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

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

42. A method of treating, inhibiting, preventing recurrence, or controlling an ischemic disease, neoplastic disease, bone disease or skin injury, wherein the method comprises administering to a subject in need thereof a cell according to claim 23.

43. The method of claim 42, wherein the method comprises the cell for use of any one of claims 26 to 29 or 33 to 39.

44. Use of the cell according to claim 23 in the manufacture of a medicament for the treatment, prevention of recurrence or control of an ischemic disease, neoplastic disease, bone disease or skin injury in a subject in need thereof.

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

46. A device for the delivery of the cell according to claim 23, wherein said device comprises a syringe, said syringe containing the cell according to any one of claims 26 to 29 and a delivery medium.