Generation of vascular grafts seeded with arterial endothelial cells

Abstract

The present invention provides polymeric vascular grafts seeded with human arterial endothelial cells that are suitable for replacing or bypassing native blood vessels and that exhibit increased long-term patency and reduced leukocyte adhesion compared to grafts containing venous endothelial cells. Methods for producing the human arterial endothelial cell-seeded vascular grafts and their therapeutic uses are also described.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Application No. 62 / 725,469, filed August 31, 2018, which is incorporated herein by reference as if set forth in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under HL134655 awarded by the National Institutes of Health. The Government has certain rights in the invention. [Background technology]

[0002] A routine procedure in cardiovascular surgery is to replace or bypass blood vessels to provide more adequate blood flow to downstream tissues. In coronary artery bypass surgery, arterial grafts have a much better long-term patency rate than venous grafts. However, when surgery requires multiple grafts, veins are often used because patients lack suitable additional arterial grafts. In bypass surgery to treat peripheral arterial disorders, the arteries to be bypassed are generally large, so suitable arterial grafts are not available. Again, venous grafts are often used despite a relatively high long-term occlusion rate. Many significant disadvantages are associated with transplantation of a patient's own blood vessels for bypass surgery. The time required to remove the vessels and prepare them for transplant increases the patient's exposure to anesthesia, increases the likelihood of postoperative infection, and increases the cost of the procedure. For many patients, a suitable artery or vein simply is not available for transplantation due to vascular disease or previous surgery. Due to the limitations of using the patient's own blood vessels as grafts, numerous attempts have been made to create vascular grafts from synthetic materials. Synthetic grafts generally work well in procedures involving the largest diameter vessels (e.g., the aorta), but the long-term patency rate for synthetic grafts decreases as the vessel size decreases. In cases of peripheral arterial disease of the legs, synthetic grafts are used as a last resort when suitable vein grafts are not available, as synthetic grafts in this location have a higher rate of occlusion than vein grafts. Furthermore, in cases of bypass procedures involving the smaller cardiac arteries (vessels with a diameter of approximately 3-5 mm), synthetic grafts fail at such a high rate that they are generally not used.

[0003] One group found that by lining synthetic ePTFE material with the patient's own venous endothelial cells, the long-term patency rate of ePTFE grafts in peripheral arterial disease could be improved to nearly equal that of vein grafts (Deutsch et al., 1999). However, this procedure requires harvesting the patient's vein in a separate operation, culturing and expanding the venous endothelial cells, lining the ePTFE tube with the venous endothelial cells, maturing the cells on the ePTFE during extended culture, and finally transplanting the ePTFE / venous endothelial cell graft back into the patient. The entire procedure from vein harvesting to transplantation takes about one month (Deutsch et al., 2009). Thus, the procedure is expensive and slow. Approximately one-third of patients with peripheral arterial disease require emergency intervention and cannot wait the 30 days required for the production of an ePTFE / autologous venous endothelial cell graft. And because of immune rejection, this approach can only treat individual patients and cannot be scaled up to treat a large number of patients. Finally, although the improvement in patency rates of venous endothelial cell-lined ePTFE grafts compared with standard ePTFE grafts was impressive, the rates only just approached the long-term patency rates of venous grafts but not arterial grafts. Arterial endothelial cells differ from venous endothelial cells in important biological properties such as lower leukocyte adhesion and higher nitric oxide (NO) production, which are crucial for maintaining long-term patency of vascular grafts. Polymeric grafts lined with arterial endothelial cells have not been used clinically before because it is medically not feasible to harvest primary arterial endothelial cells from patients and primary arterial endothelial cells undergo dedifferentiation during in vitro culture. Thus, there remains a need in the art for improved polymeric vascular grafts containing arterial endothelial cells that are scalable, available as an on-demand product, and suitable for a large number of patients. Summary of the Invention

[0004] In a first aspect, the present invention provides a vascular graft comprising or consisting essentially of (a) a polymeric substrate at least partially coated with an endothelial cell attachment agent and (b) human arterial endothelial cells attached to the coated polymeric substrate. The polymeric substrate can be selected from expanded polytetrafluoroethylene (ePTFE), polyvinyl chloride (PVC), PGA (polyglycolic acid), PLA (polylactic acid), PCL (polycaprolactone), PGLA (polylactic-co-glycolic acid), polyurethane, polydioxanone, polyethylene, polyethylene terephthalate (Dacron®), tetrafluoroethylene (TFE), polytetrafluoroethylene (PTFE), silk, decellularized scaffolds, extracellular matrix protein-based scaffolds, hyaluronic acid, chitosan, and polyhydroxyalkanoates. The endothelial cell attachment agent can include one or more of dopamine, fibrin glue, RGD peptide, vitronectin, and laminin. The vascular graft can exhibit reduced leukocyte adhesion compared to a polymeric substrate seeded with venous endothelial cells. The vascular graft can exhibit one or more of (a) reduced thrombus formation, (b) increased long-term patency, and (c) decreased platelet adhesion compared to a polymeric substrate not coated with human arterial endothelial cells. The human arterial endothelial cells can be generated from human pluripotent stem cells. The human pluripotent stem cells can be induced pluripotent stem cells. The induced pluripotent stem cells can be autologous to the patient. The induced pluripotent stem cells can be at least 50% HLA-matched to the patient. The human arterial endothelial cells can be non-immunogenic to the recipient of the vascular graft. The human arterial endothelial cells can include one or more genetic modifications to not express a beta2-microglobulin gene. The human arterial endothelial cells can include one or more genetic modifications to not express one or more proteins encoded by class I or class II major histocompatibility complex (MHC) genes. The human arterial endothelial cells can include one or more genetic modifications to not express a CD58 polypeptide. Human arterial endothelial cells can contain one or more modifications such that they overexpress one or both of HLA-E (Edimer) and CD47.

[0005] In another aspect, a method of forming a cell-seeded vascular graft is provided by the present invention, the method comprising or consisting essentially of the steps of: (a) coating a polymeric substrate with an endothelial cell adhesion agent; (b) seeding the coated polymeric substrate with human arterial endothelial cells; and (c) culturing the coated and seeded polymeric substrate for about 2 to about 20 days, thereby obtaining a cell-seeded vascular graft. The method can further comprise degassing the polymeric substrate prior to coating with one or more endothelial cell adhesion agents. Degassing can include washing the polymeric substrate in acetone and ethanol, washing the polymeric substrate in an organic solvent, or applying a vacuum. The polymeric substrate can be selected from expanded polytetrafluoroethylene (ePTFE), polyvinyl chloride (PVC), PGA (polyglycolic acid), PLA (polylactic acid), PCL (polycaprolactone), PGLA (polylactic-co-glycolic acid), polyurethane, polydioxanone, polyethylene, polyethylene terephthalate (Dacron®), tetrafluoroethylene (TFE), polytetrafluoroethylene (PTFE), silk, decellularized scaffolds, extracellular matrix protein-based scaffolds, hyaluronic acid, chitosan, and polyhydroxyalkanoates. The method can further include contacting the cell-seeded vascular graft with a cryopreservation solution and freezing the contacted cell-seeded vascular graft. Freezing can include storage at a temperature of 1° C. to about −196° C.

[0006] In a further aspect, a method of fabricating an AEC cell-seeded vascular graft is provided by the present invention, the method comprising or consisting essentially of the steps of: coating at least a portion of a polymeric substrate with one or more endothelial adhesion agents; and contacting the coated polymeric substrate with human arterial endothelial cells to form an AEC-seeded vascular graft that is substantially non-adhesive to leukocytes or cell fragments thereof. The polymeric substrate can be selected from expanded polytetrafluoroethylene (ePTFE), polyvinyl chloride (PVC), PGA (polyglycolic acid), PLA (polylactic acid), PCL (polycaprolactone), PGLA (polylactic-co-glycolic acid), polyurethane, polydioxanone, polyethylene, polyethylene terephthalate (Dacron®), tetrafluoroethylene (TFE), polytetrafluoroethylene (PTFE), silk, decellularized scaffolds, extracellular matrix protein-based scaffolds, hyaluronic acid, chitosan, and polyhydroxyalkanoates. The method may further include degassing the polymeric substrate prior to coating with one or more endothelial cell adhesion agents. Degassing may include washing the polymeric substrate in acetone and ethanol, washing the polymeric substrate in an organic solvent, or applying a vacuum.

[0007] These and other features, aspects, and advantages of the present invention will be better understood from the following description, in which references made to preferred embodiments are not intended to limit the invention, and therefore reference should be made to the appended claims to interpret the scope of the invention. The following drawings form part of the present specification and are included to further illustrate certain aspects of the compositions and methods provided herein. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief description of the drawings]

[0008] [Figure 1A-1B]Images showing CD31+ AECs seeded on adhesive-coated expanded polytetrafluoroethylene (ePTFE). (A) AECs were seeded on ePTFE at cell densities of 1×106 cells / ml to 1.5×106 cells / ml. Staining was performed to detect live cells (green; stained with the cell-permeable dye calcein AM). Cell confluency statistics are shown as mean ± SD. n=3. (B) CD31+ (red) AECs seeded on ePTFE coated with collagen I, RGD (Arg-Gly-Asp) peptide, fibronectin (FN), laminin, Matrigel, and VTN (vitronectin). Nuclei were visualized using DAPI (4',6-diamidino-2-phenylindole) fluorescent staining. Cell confluency statistics are shown as mean ± SD. n=3. [Figures 2A-2D] Figure 1 shows that dopamine coating improves human arterial endothelial cell (AEC) adhesion to expanded polytetrafluoroethylene (ePTFE) substrates. (A) ePTFE with or without dopamine coating. (B) AECs were seeded on ePTFE at cell densities of 1×106 cells / ml to 1.5×106 cells / ml. Staining was performed to detect and count live cells (green; stained with the cell permeable dye calcein AM) and dead cells (red; stained with the nucleic acid stain ethidium homodimer-1). (C) Statistics of live and dead AECs on control and dopamine-coated substrates. Data are shown as mean ± SD. *: P<0.05, n=3. (D) Comparison of AEC adhesion to ePTFE coated with dopamine and fibrin glue. [Figure 3A-3B]The results of the leukocyte adhesion assay are shown. (A) AEC and human umbilical vein endothelial cells (HUVEC) seeded on ePTFE coated with dopamine. The seeded ePTFE substrates were cultured for 3 days and then treated with 10 ng / ml TNFα or control for 4-5 hours. Leukocytes were stained by exposure to 2 μM calcein AM for approximately 15 minutes. The calcein AM-labeled leukocytes were then added to the ePTFE seeded with AEC and HUVEC at a cell density of approximately 1 × 106 cells / ml. Both the calcein AM-labeled leukocyte cell suspension and ePTFE were placed in a 0.5 ml tube and the tube was rotated at 60 rpm for 1 hour. After 1 hour, the cell-seeded ePTFE was gently washed three times with fresh medium and then fixed and stained with DAPI for imaging. (B) Statistics of the leukocyte adhesion assay. Leukocytes were detected by immunostaining and counted for each image. Data are shown as mean ± SD. *: P<0.05, n=3. [Figure 4A-4C] Figure 1 shows that degassing of ePTFE suppresses cell aggregate formation and improves CD31 expression and cell density. (A) Arterial endothelial cells were seeded on degassed or non-degassed ePTFE. Two types of ePTFE, 1 and 2, were used. Samples were collected for immunostaining 2 and 8 days after cell seeding. Arrows indicate cell aggregates that could not be detected after degassing. (B) Cell density statistics. Cell density on seeded substrates was determined by comparing the number of nuclei before and 8 days after degassing. Data are presented as mean ± SD. *: P<0.05, n=4. (C) Statistics of relative mean fluorescence intensity (MFI) of CD31. Data are presented as mean ± SD. *: P<0.05, n=4. [Figure 5A-5C]Analysis of freezing media is shown. (A) Glycerol is not suitable for freezing AEC-ePTFE. (B) Comparison of Recovery™ Cell Culture Freezing Medium (Thermofisher), 10% DMSO, and serum for freezing AEC-ePTFE. (C) Optimization of DMSO concentration. Serum-free and Xeno-free medium was used to freeze AEC-ePTFE, which was made by E5 medium supplemented with 100 ng / mL FGF2, 50 ng / mL VEGFA, 10 μM SB431542, 5 μM RESV, and 20 μg / ml insulin. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] All publications, including but not limited to patents and patent applications, cited herein are hereby incorporated by reference as if fully set forth in this application. The present invention is based, at least in part, on the inventors' development of a polymeric vascular graft seeded with arterial endothelial cells, where the vascular graft and the method for obtaining it are scalable, available as an on-demand product, and suitable for a variety of patients.

[0010] I. Composition In a first aspect, the present invention provides a polymeric vascular graft comprising arterial endothelial cells. The graft can comprise or consist essentially of a polymeric substrate at least partially coated with an endothelial cell adhesion agent, and arterial endothelial cells (AECs) attached to the coated polymeric substrate. As described herein, the AEC-seeded polymeric graft of the present disclosure exhibits increased patency compared to conventional polymeric vascular grafts. In some cases, the human arterial endothelial cells used with the polymeric vascular graft of the present disclosure are modified so that they can serve as universal donor cells for transplantation into subjects in need thereof, regardless of the HLA- and blood type of the graft recipient. For example, in some embodiments, the native genome of the cells is engineered so that the engineered cells do not express certain cell surface markers, such as proteins encoded by major histocompatibility complex genes, either class II or both class I and class II. In this way, the engineered cells are more likely to avoid attack by the graft recipient's T cells, and as a result, are non-immunogenic to the recipient.

[0011] The terms "graft" and "vascular graft" are used interchangeably herein and refer to any conduit or portion thereof intended as a prosthesis for conveying fluid (e.g., blood) and thus having a surface in contact with the fluid (i.e., "luminal"). Although primarily tubular forms are intended, grafts may also be incomplete tubes or sheets of material useful for repairing peripheral portions of living blood vessels (these materials are commonly referred to as cardiovascular patches). Similarly, the term vascular graft includes intraluminal grafts used within living blood vessels. For example, the vascular grafts provided by the present invention may be used as a sheath or other covering on the exterior surface, the luminal surface, or both the luminal and exterior surfaces of an implantable vascular stent.

[0012] As used herein, the term "AEC-seeded" and grammatical variations thereof refer to a substrate that is supplied with arterial endothelial cells. Preferably, the term refers to a polymeric vascular graft having arterial endothelial cells (e.g., human AECs) and an endothelial cell adhesive, whereby the seeded polymeric vascular graft is suitable for implantation into a subject. As used herein, the term "patency" refers to the degree of openness of a vessel, such as a blood vessel or vascular graft. A vascular graft with 100% patency is free of any blockage or obstruction. As the degree of blockage or obstruction increases, the patency of the blood vessel or vascular graft decreases. Thus, the patency of a blood vessel or vascular graft is a surrogate for the success or failure of the graft. In some cases, patency is evaluated at a particular time point, including, without limitation, patency at days, weeks, months, or years after implantation of the vascular graft. Preferably, the polymeric vascular graft of the present disclosure exhibits an increased long-term patency rate compared to conventional polymeric vascular grafts. As used herein, "long-term patency" means that the blood vessel or graft remains patent in a physiological environment after implantation for more than one year, preferably more than three years, more preferably more than five years, and most preferably more than ten years. In some cases, the AEC-seeded polymeric vascular graft of the present disclosure exhibits patency comparable to, and preferably superior to, autografts.

[0013] A. Materials of the composition The polymeric vascular graft provided by the present invention comprises a polymeric substrate at least partially coated with an endothelial cell adhesion agent, and human arterial endothelial cells attached to the coated polymeric substrate. Suitable polymeric materials for the vascular graft provided by the present invention include, without limitation, polyvinyl chloride (PVC), PGA (polyglycolic acid), PLA (polylactic acid), PCL (polycaprolactone), polylactic-co-glycolic acid (PLGA), polyurethane, polydioxanone, polyethylene terephthalate (Dacron®), polyethylene, and fluoropolymers such as tetrafluoroethylene (TFE), polytetrafluoroethylene (PTFE), and expanded polytetrafluoroethylene (ePTFE). PTFE is a homopolymer of tetrafluoroethylene (TFE). When PTFE is expanded and expanded to become ePTFE, the polymeric material exhibits low thrombogenicity and is particularly suitable for vascular applications because it can be extruded into tubes, sheets, or other suitable graft shapes. In some cases, the polymeric substrate is a GORE-TEX® vascular graft.

[0014] In some cases, biological materials are suitable for the polymeric substrate of the present disclosure. For example, the polymeric substrate can include biological materials including, without limitation, silk, decellularized constructs (e.g., decellularized arteries, veins, or small intestines), extracellular matrix protein-based scaffolds (e.g., collagen, MATRIGEL™, fibrin, elastin), hyaluronic acid, chitosan, polyhydroxyalkanoates. In some cases, the polymeric substrates used in the vascular grafts provided by the present invention are biocompatible, meaning that the substrate material does not cause adverse reactions when implanted or placed in contact with the body. In some cases, the polymeric substrate is a porous substrate. Without being bound by any mechanism of action or theory, it is believed that the pores in the polymeric vascular graft allow for the recruitment and integration of host cells into the graft. For example, ePTFE exhibits high porosity and contains a matrix of nodes and fibrils. Fibrils are the thin connections between nodes and are submicron in size. Thin fibrils are used to create more tortuosity and surface area in the membrane, affecting filtration efficiency. In some cases, the geometry of the fibrils and nodes in the membrane is modified (e.g., to increase or decrease pore size, pore distribution) to customize the functionality of the material. In some cases, an intermodal spacing of about 7 μm to about 20 μm is preferred. In some cases, the polymeric substrate is microporous, meaning that the pores in the porous substrate are micrometer in size. Preferably, the pore size of suitable polymeric substrates is in the range of 2 μm to 80 μm, preferably ranging therebetween, preferably in the range of 3 μm to 40 μm, most preferably in the range of 5 μm to 35 μm, especially approximately 30 μm.

[0015] In some cases, the disclosed vascular grafts are substantially tubular in shape and have a circular or substantially circular cross-section. The disclosed vascular grafts are substantially tubular in shape and of circular or substantially circular cross-section. In some cases, the tubular structures have wall thicknesses of about 200 μm to about 500 μm (e.g., about 200, 250, 300, 350, 400, 450, 500 μm). In other cases, the polymeric substrate is a planar sheet or "patch" of polymeric material. In such cases, the thickness can vary widely, from about 0.2 mm to about 1.0 mm or more. The various dimensions of the polymeric vascular grafts of the present disclosure may vary depending on the desired application. In principle, the dimensions are similar to those of the host tissue that the vascular graft is being used to replace. Generally, tubular grafts have a lumen that extends throughout the length of the graft. The lumen of the vascular grafts provided by the present invention may be of any suitable diameter suitable for the intended surgical application of the graft. For example, the average luminal dimensions of coronary arteries, including those with a higher incidence of occlusion (anterior interventricular artery, right coronary artery, circumflex artery), are well described in the literature. In some cases, the polymeric substrate has an inner diameter of about 0.5 mm to about 10 mm (e.g., about 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mm). The vascular graft may be of any suitable length suitable for the intended surgical application of the graft. Typically, the graft should be slightly longer than the length of the artery or vein to be replaced. In some cases, the polymeric substrates used in the vascular grafts provided by the present invention are hydrophobic membranes, meaning that they resist wetting by fluids (e.g., biological fluids) and are not chemically altered or degraded by biological fluids. In some cases, hydrophobic membranes are impermeable to fluids but allow the flow of gases through the membrane.

[0016] In some cases, the polymeric substrate is at least partially coated with an endothelial cell adhesive. Endothelial cell adhesives useful in the vascular grafts of the present disclosure include, without limitation, dopamine, fibrin, RGD (Arg-Gly-Asp)-peptides, and extracellular matrix proteins such as vitronectin and laminin, or a mixture of two or more adhesives. Traditionally, a network of blood clotting protein fibrin (Fb), sometimes referred to as a "fibrin adhesive" or "fibrin glue", has been used to coat vascular grafts. For example, Zilla et al. (1989) described improved seeding of venous endothelial cells onto ePTFE using fibrin glue. However, the fibrin glue method involves multiple steps and is therefore difficult to scale up for clinical applications. With reference to Figures 2A-2B, the inventors have shown that other cell adhesives can achieve comparable levels of AEC adhesion to polymeric substrates. Advantageously, coating a polymeric substrate with these agents requires a single step. Others have reported improved venous endothelial attachment to vascular grafts coated with laminin compared to those coated with Fb (p<0.001) or a mixture of fibrin and fibronectin (p<0.05). See Chlupac et al., Physiol. Res. 63:167-177, 2014.

[0017] Preferably, endothelial cell adhesive is non-immunogenic.For example, endothelial cell adhesive is preferably free of any components derived from non-human animals, and therefore free of xenogeneic substances ("xeno-free").As used herein, the terms "free of xenogeneic substances" and "xeno-free" are used interchangeably and refer to materials (e.g., cell substrates, culture media) or cell culture conditions that do not contain any cells or cell products of species other than the species of the recipient of cultured cells or materials.

[0018] In some cases, the endothelial cell adhesive comprises dopamine, where the dopamine is dissolved in a buffer at a concentration of about 0.1 mg / ml to about 20 mg / ml (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20 mg / ml dopamine). When the endothelial cell adhesive comprises an RGD peptide, the peptide can be provided in a buffer at a concentration of about 0.5 mM to about 10 mM (e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mM RGD peptide). In some cases, the endothelial cell adhesive comprises vitronectin. Optionally, the vitronectin is provided in a buffer at a concentration of about 1 μg / ml to about 50 μg / ml (e.g., about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 μg / ml of vitronectin). As used herein, the term "vitronectin" refers to a vitronectin polypeptide or fragment or peptide thereof, including recombinant vitronectin polypeptides and peptides (e.g., recombinant human vitronectin) and vitronectin polypeptide variants, such as those described in U.S. Patent No. 9,133,266, which is incorporated herein by reference as if set forth in its entirety. Optionally, the endothelial cell adhesive comprises laminin. Optionally, the laminin is provided in a buffer at a concentration of about 1 μg / ml to about 50 μg / ml (e.g., about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 μg / ml of laminin). As used herein, the term "laminin" refers to a laminin polypeptide or fragments or peptides thereof, and includes recombinant laminin polypeptides and peptides (eg, recombinant human laminin).

[0019] Optionally, the polymeric substrate can be partially or completely coated with or immersed in a solution containing an endothelial cell adhesive for about 4 to about 24 hours. Coating by immersion in an endothelial cell adhesive solution can be performed at any suitable temperature, including, without limitation, 4° C., 25° C. (room temperature), or 37° C. Preferably, the coated polymeric substrate is rinsed with distilled water or a buffer solution prior to use. In some cases, it is advantageous to degas the polymeric substrate material before at least partially coating it with the endothelial cell adhesive. As described in the Examples below, the inventors have shown reduced cell aggregate formation, improved cell density, and improved coating with the endothelial adhesive when the polymeric substrate is degassed before use. Degassing can be done by methods known in the art. As described in the Examples, the polymeric substrate can be degassed with a series of acetone and ethanol washes. In some cases, degassing is done as described, but with an organic solvent instead of acetone. In other cases, the substrate can be degassed by applying a high vacuum before use.

[0020] In some cases, arterial endothelial cells are cultured on a degassed and coated (e.g., partially or completely coated) polymeric substrate at a concentration of about 0.5×10 6 cells / ml ~ approx. 3×10 6 Cells are seeded at a cell density of about 1×10 cells / ml. 6 ~Approx. 1.5×10 6 AECs at a density of 100 cells / ml are seeded onto the prepared polymeric substrates. The AECs can be provided in any suitable cell culture medium for seeding onto the polymeric substrate. For example, the AECs can be provided in a chemically defined cell culture medium that is xeno-free, serum-free, and albumin-free. As used herein, the terms "chemically defined culture conditions," "well-defined, growth factor-free culture conditions," and "well-defined conditions" indicate that the type and amount of each medium component is known, and the type and amount of supporting surface is known. As used herein, "serum-free" means that the medium does not contain serum or is essentially serum-free. For example, an essentially serum-free medium can contain less than about 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% serum. As used herein, the term "albumin-free" indicates that the culture medium used does not contain any form of added albumin (e.g., serum substitutes), such as, without limitation, bovine serum albumin (BSA) or any form of recombinant albumin. Preferably, human AECs are seeded onto the polymeric substrate in a chemically defined cell culture medium that does not contain any xenogeneic substances, ie, does not contain any components derived from non-human animals.

[0021] Optionally, seeding is performed by injecting a suspension of AECs into the lumen of the tubular vascular graft and placing the graft in a rotating device. Optionally, seeding is followed by aging the seeded substrate in a culture flask with fresh medium without rotation at any temperature suitable for cell growth, such as room temperature or preferably 37°C. For example, seeding is optionally followed by aging in a culture flask with fresh culture medium without rotation in a humidified incubator at 37°C for 2-3 days. Optionally, the AEC-seeded graft is cultured in the presence of 5% CO2.

[0022] In some cases, it may be appropriate to include a Rho-Kinase (ROCK) inhibitor in the cell culture medium for seeding AEC on polymeric substrate.Kinase inhibitors such as ROCK inhibitors are known to increase the plating efficiency and the viability of single cells and small aggregates of cells.See, for example, U.S. Patent Application Publication No. 2008 / 0171385, which is incorporated herein by reference in its entirety; and Watanabe K, et al., "A ROCK inhibitor permits survival of dissociated human embryonic stem cells," Nat. Biotechnol. 25:681-686 (2007). Suitable ROCK inhibitors for use in the present invention include, but are not limited to, (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]homopiperazine dihydrochloride (informal name: H-1152), 1-(5-isoquinolinesulfonyl)piperazine hydrochloride (informal name: HA-100), 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (informal name: H-7), 1-(5-isoquinolinesulfonyl)-3-methylpiperazine (informal name: isoquinolinesulfonyl)-4-methylpiperazine (informal name: isoquinolinesulfonyl)-5-methylpiperazine (informal name: isoquinolinesulfonyl)-6-methylpiperazine (informal name: isoquinolinesulfonyl)-7-methylpiperazine (informal name: isoquinolinesulfonyl)-8-methylpiperazine (informal name: isoquinolinesulfonyl)-9-methylpiperazine (informal name: isoquinolinesulfonyl)-10-methylpiperazine (informal name: isoquinolinesulfonyl)-11-methylpiperazine (informal name: isoquinolinesulfonyl)-2-methylpiperazine (informal name: isoquinolinesulfonyl)-1 ... H-7), N-2-(methylamino)ethyl-5-isoquinoline-sulfonamide dihydrochloride (informal name: H-8), N-(2-aminoethyl)-5-isoquinolinesulfonamide dihydrochloride (informal name: H-9), N-[2-p-bromo-cinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (informal name: H-89), N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride (informal name: HA-1004), 1-(5-isoquinolinesulfonyl)homopiperazine dihydrochloride (informal name: HA-1077), (S)-(+)-2-methyl-4-glycyl-1-(4-methylisoquinolinyl-5-sulfonyl)homopiperazine dihydrochloride (informal name: glycyl H-1152) and (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride (informal name: Y-27632). Kinase inhibitors can be provided in high enough concentrations that the cells remain viable and attached to the surface.When included in AEC culture medium, the concentration of the ROCK inhibitor can be from about 3 μM to about 10 μM (e.g., about 3, 4, 5, 6, 7, 8, 9, 10 μM Y-27632).

[0023] B. Cells for Composition Because harvesting primary arterial endothelial cells from patients requiring cardiovascular intervention is medically infeasible and because primary arterial endothelial cells dedifferentiate in culture, polymeric grafts lined with arterial endothelial cells have not been used clinically to date. Thus, the AEC-seeded polymeric grafts provided by the present invention are superior to previously described cell-seeded vascular grafts. AECs can be distinguished from other cell types, including venous endothelial cells and endothelial progenitor cells, based on the cells' unique in vitro expression profile and functional attributes as described herein. In particular, arterial endothelial cells adapt to the high flow and high pressure environment of arteries and exhibit distinct physiological properties that distinguish them from venous endothelial cells. Compared to venous endothelial cells, arterial endothelial cells produce higher levels of nitrous oxide, respond more robustly to shear stress, exhibit a higher metabolic rate, and adhere less strongly to leukocytes. See, e.g., U.S. Patent Application Publication No. 2016 / 0244719, which is incorporated herein by reference in its entirety. The reduced ability of leukocytes to adhere to arterial endothelial cells compared to venous endothelial cells is particularly important because inflammation and the resulting proliferation of myointimal cells are critical factors in graft occlusion (i.e., loss of patency) and failure. These and other distinctly different properties of arterial endothelial cells make them more suitable than venous endothelial cells for seeding polymeric arterial grafts and improving long-term patency rates, but due to lack of availability, to date none have been able to use them clinically for this purpose. The process of harvesting an artery is much more invasive than harvesting a vein. In addition, the arteries available are few and small. Once one of the few and small available arteries is harvested for endothelial culture, it is no longer available for any future cardiac bypass surgery.

[0024] In some cases, the human arterial endothelial cells used with the polymeric vascular grafts of the present disclosure are modified so that they can serve as universal donor cells. In some cases, the cells are genetically modified. The term "modified" as used herein refers to an altered state or structure of a molecule or cell of the present disclosure. A molecule can be modified in many ways, for example, chemically, structurally, and functionally. A cell can be modified by the introduction of a nucleic acid. In some embodiments, a modified cell can be "genetically modified" or "genetically edited," in which one or more nucleic acids in the cell are altered. The terms "genetic engineering," "gene editing," and "genetically modified" are used interchangeably herein and refer to a cell (e.g., a prokaryotic or eukaryotic cell) in which one or more nucleic acids in the cell have been altered, or a cell that has been modified to include a non-naturally occurring nucleic acid molecule that has been created or modified by the hand of man (e.g., using recombinant DNA technology) or a molecule derived from such a molecule (e.g., by transcription, translation, etc.).

[0025] Arterial endothelial cells containing exogenous, recombinant, synthetic, and / or otherwise modified polynucleotides are considered genetically modified cells, and therefore do not naturally occur relative to their naturally occurring counterparts. In some cases, genetically modified cells contain one or more recombinant nucleic acids. In other cases, genetically modified cells contain one or more synthetic or genetically engineered nucleic acids (e.g., nucleic acids containing at least one artificially created insertion, deletion, inversion, or substitution relative to the sequence found in its naturally occurring counterpart). Procedures for producing genetically engineered cells are generally known in the art and are described in Sambrook et al, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989), which is incorporated herein by reference.

[0026] In some cases, the genome of the cells is modified (e.g., genetically engineered) so that the modified cells are "universally acceptable" for therapeutic applications. As used herein, the term "universally acceptable" refers to the total immunological acceptance of the cell product, such that no cross-matching of the patient and the cells is required and no immunosuppression is required. In some cases, the cells are modified so that no functional proteins encoded by major histocompatibility complex genes, either class II or both class I and class II, are expressed on the cell surface. In this way, the modified cells are more likely to avoid attack by the T cells of the graft recipient. In some cases, human arterial endothelial cells are genetically modified (engineered) as described in U.S. Pat. No. 6,916,654. In other cases, it may be advantageous to produce immune-non-responsive cells from iPS cells by disrupting beta-2 microglobulin as described by U.S. Patent Application Publication No. 2014 / 0134195. For example, the cells can be modified to contain a genetically engineered division of the cell's endogenous beta-2 microglobulin (B2M) gene. As described in US Patent Application Publication No. 2014 / 0134195, the genetically engineered division can include introducing one or more polynucleotide sequences capable of encoding a single-chain fusion human leukocyte antigen (HLA) class I protein comprising at least a portion of a B2M protein covalently linked directly or via a linker sequence to at least a portion of a human leukocyte antigen (HLA)-I chain. However, it will be understood that the method of obtaining "universal" human AECs is not limited to modifying HLA proteins. In some cases, the AECs are derived from induced pluripotent stem cells that are at least 50% HLA-matched to the patient to receive the vascular graft.

[0027] Cells can also be genetically modified using other strategies to minimize immune responses. For example, Riolobos et al. (Molecular Therapy 2013, 21(6):1232-1241) describe the production of stable HLA-I negative human pluripotent cells by creating targeted disruptions in both alleles of the beta-2 microglobulin (B2M) gene using recombinant adeno-associated virus (rAAV)-mediated gene editing. The resulting B2M - / - Pluripotent stem cells could be differentiated into human AECs according to the chemically defined method of US Patent Publication No. 2016 / 0244719 to produce non-immunogenic "universal" human AECs for use with the polymeric vascular grafts of the present disclosure. In another example, genetic modification to totally or partially abolish cell surface expression of CD58 has been shown to increase evasion from immune recognition by both arms of cellular immunity. See, e.g., Challa-Malladi et al. (Cancer Cell 2011; 20(6):728-740). Pluripotent stem cells expressing HLA-E (Edimer cells) also avoid allogeneic immune responses and lysis by NK cells (Gornalusse et al., Nat Biotechnol. 2017; 35(8):765-772).

[0028] In some cases, the cells for the vascular graft provided by the present invention are modified without introducing a transgene into the genome of the cells. In some cases, for example, AECs are genetically edited to regulate the expression of endogenous genes (e.g., increase or decrease expression compared to control unmodified cells). Any suitable gene editing means can be used. Various gene editing techniques are known to those skilled in the art. Gene editing techniques include, without limitation, homing endonucleases, zinc finger nucleases (ZFNs), transcription activator-like effector (TALE) nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein (e.g., Cas9) genome editing system, and CRISPR-Cpf1 genome editing system. Homing endonucleases generally cleave DNA substrates as dimers and do not have distinct binding and cleavage domains. ZFNs recognize a target site consisting of two zinc finger binding sites flanked by a 5-7 base pair (bp) spacer sequence recognized by a FokI cleavage domain. TALENs recognize a target site consisting of two TALE DNA binding sites flanked by a 12-20 bp spacer sequence recognized by a FokI cleavage domain. Cas9 nuclease targets DNA sequences complementary to a targeting sequence within a single guide RNA (gRNA) located immediately upstream of a compatible protospacer adjacent motif (PAM) that may be present on either strand of the DNA helix. Thus, one skilled in the art would be able to select an appropriate gene editing technique for the present invention.

[0029] In some cases, gene editing techniques (e.g., CRISPR-Cas9 gene editing system) can be used to modify human pluripotent stem cells to make these cells functionally "invisible" to the immune system. Such "universally acceptable" pluripotent stem cells can be differentiated into AECs used in the vascular grafts of the present disclosure. In some cases, AECs are modified to induce overexpression of CD47 as a means of avoiding detection by the innate immune system. CD47 is highly expressed in tumor cells and is known as a critical immune checkpoint that renders tumor cells resistant to host immune surveillance. In some cases, overexpression of CD47 is induced by the introduction of a virus containing the CD47 gene, which delivers additional copies of the gene to the cells. See, e.g., Deuse et al., Nature Biotechnology 37, 252-258 (2019). As used herein, the terms "overexpress" and "overexpression" refer to increasing expression of a gene product (e.g., mRNA, protein) to a level higher than that normally produced by the cell. These terms are intended to encompass overexpression of endogenous as well as exogenous proteins. In some cases, overexpression is determined relative to a reference standard.

[0030] In some cases, the AECs are genetically modified to express a selectable marker. In such cases, expression of the selectable marker is used to identify and / or isolate the modified cells from unmodified cells. In this manner, expression of the selectable marker is used to obtain a pure or substantially pure population of modified cells for use in the vascular grafts of the present disclosure. In some cases, the AECs are modified to include a selectable marker cassette. The selectable marker cassette can confer resistance to an agent such as an antibiotic. As will be appreciated by those skilled in the art, additional selectable markers or combinations of selectable markers can also be used. Other forms of selectable markers may be used, such as markers that provide a growth advantage other than antibiotic resistance or colorimetric selection. Preferably, the selectable marker cassette comprises a polynucleotide encoding the selectable marker operably linked to a promoter or regulatory region that can induce transcription of the selectable marker specifically in endothelial cells, more preferably. The use of a promoter specific to endothelial cells allows for purification of endothelial or arterial endothelial cells from contaminating cells that do not express the gene. Promoters useful in the practice of the present invention include, but are not limited to, constitutive, inducible, temporally regulated, and chemically regulated promoters. Preferably, the promoter is inducible.

[0031] Following modification, AECs can be applied to the prepared polymeric substrate to prepare an AEC-seeded vascular graft for immediate use, later use, or storage. As used herein, the term "prepared" refers to a polymeric substrate that has been treated in preparation for constructing an AEC-seeded vascular graft. "Prepared" encompasses polymeric substrates that have been previously coated (partially or completely) and / or degassed. In some cases, AECs can be stored, for example, in a liquid nitrogen tank, until needed for treatment of a particular patient. For short-term storage (e.g., about 6-12 months), AECs can be stored at -80°C or below (e.g., -80°, -90°, -100°, -110°, -120°C, -130°C, -140°C, -150°C, -160°C, -170°C, -180°C, -190°C, -196°C, or below). In some cases, the AECs are maintained at a temperature above 0° C., such as, without limitation, 4° C., room temperature (about 25° C.), and about 37° C., prior to seeding onto the prepared polymeric substrate. The ability to prepare a polymeric vascular graft containing universal AECs in advance and store it until needed is an important advantage, especially for treating patients in acute need. In such cases, the AEC-seeded polymeric vascular graft is suitable for transplantation or implantation into a subject, where the graft induces reduced or no graft rejection in the subject.

[0032] In some cases, the AECs for the vascular grafts provided by the present invention are obtained from a cell bank. Generally, a cell bank collects samples of cells from multiple sources, catalogs them according to at least one predetermined characteristic, and stores the cells under conditions that keep the cells viable. Thus, stored or "banked" cells with a particular predetermined characteristic are available upon request. Preferably, banked cells representing many HLA types from healthy individuals are stored in a cell bank to provide haplotype matching for all potential recipients in a particular population. In some cases, the banked cells useful for the vascular grafts and methods provided by the present invention are induced pluripotent stem cells (iPSCs) derived from screened and HLA-typed donors. In other cases, the banked cells are AECs derived from HLA-typed iPSCs. For example, AECs can be obtained from HLA-typed iPSCs according to the methods described in US Patent Application Publication No. 2016 / 0244719. Each HLA type comprises a pair of co-expressed haplotypes, each consisting of HLA-A, HLA-B, HLA-C, HLA-DQ, HLA-DP, and HLA-DR. In other cases, HLA-matched human embryonic stem cells (hESCs) are used. Depending on the racial composition of a given population of individuals, the frequency of certain HLA allele combinations varies. Using algorithms, it is possible to determine the frequency of each HLA allele combination in a pool of tissue donors and the number of homozygous and heterozygous HLA types required in a cell bank to provide HLA matching for 100% of potential recipients. For a general review, see, for example, de Rham & Villard, J. Immunology Res. 2014. Preferably, the banked cells are generated from healthy donors with blood type O to reduce the potential risk of anti-ABO agglutinin-mediated alloimmune reactions (Zimmermann et al., Stem Cells and Development 2012; 21(13):2364-2373).

[0033] Preparations containing AEC cells useful for clinical applications must be obtained in compliance with regulations imposed by government agencies such as the United States Food and Drug Administration. Thus, in a representative embodiment, the methods provided by the present invention are performed in accordance with Good Manufacturing Practice (GMP), Good Tissue Practice (GTP), and Good Laboratory Practice (GLP). No reagents containing animal-derived components are used, and all reagents are purchased from GMP-compliant sources. In the context of clinical manufacturing of cell therapy products, such as the in vitro population of human arterial endothelial cells for vascular grafts provided by the present invention, GTP governs donor consent, traceability, and infectious disease screening, while GMP relates to facilities, processes, tests, and practices to produce a product that is consistently safe and effective for human use. See Lu et al., Stem Cells 27: 2126-2135 (2009). Where appropriate, informed consent is obtained; product safety, bioactivity, appropriate dose, and efficacy are investigated stepwise; results are statistically significant; and ethical guidelines are adhered to, and oversight of patient protocols by institutions and panels of institutions is contemplated.

[0034] In some cases, human arterial endothelial cells can be obtained according to the methods described in US Patent Application Publication No. 2016 / 0244719. AECs obtained according to such methods are characterized by high levels of expression of arterial endothelial markers such as EphrinB2, DLL4, Hey-2, jagged-1, and jagged-2. AECs are also characterized by low leukocyte adhesion, high NO production and oxygen consumption, response to shear stress, and the ability to form vascular networks in vitro and in vivo while maintaining expression of intraarterial markers in such networks. The method includes or consists essentially of culturing mesodermal cells in a serum-free, albumin-free, chemically defined culture medium that is substantially insulin-free and includes fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and at least one of a Notch agonist, a TGF-beta inhibitor, and an inhibitor of inositol monophosphatase, wherein the culturing is performed for a sufficient length of time to differentiate the cultured mesodermal cells into arterial endothelial cells. Amounts of FGF, VEGF, Notch agonist, TGF-beta inhibitor, and inhibitor of inositol monophosphatase useful for differentiating human mesodermal cells (including mesodermal cells derived from pluripotent stem cells) into AECs are described in US Patent Application Publication No. 2016 / 0244719. In some embodiments, the cell culture medium used in the AEC differentiation methods described herein comprises each of these components. In other cases, the culture medium is substantially free of one or more of these components. Culturing can be performed on any suitable surface (e.g., in two-dimensional or three-dimensional culture).

[0035] AECs are characterized by the expression profile: CD31 + / CD144 + / CD41 - / CD45 -Preferably, the AECs express one or more of the arterial endothelial cell markers: Ephrin B2 (EFNB2), Neuropilin-1 (NRP-1) / CD304, Delta-like 4 (DLL4), and CD184 (cluster of differentiation 184). The Ephrin B2 (EFNB2) gene encodes an EFNB class Ephrin that binds to the EPHB4 and EPHA3 receptors. Neuropilin-1 (NRP1), also known as the vascular endothelial growth factor 165 receptor (VEGF165R), is expressed primarily in arterial endothelial cells. DLL4 is a Notch ligand expressed in arterial endothelial cells (Shutter et al., Genes & Dev. 14:1313-18 (2000)). CD184 is also known as CXCR4 (CXC chemokine receptor type 4) or fusin. Any suitable method can be used to detect the expression of biological markers characteristic of the cell types described herein. For example, the presence or absence of one or more biological markers can be detected using, for example, RNA sequencing (e.g., RNA-seq), immunohistochemistry, polymerase chain reaction, quantitative real-time PCR (qRT-PCR), or other gene expression detection or measurement techniques. RNA-seq is a high-throughput sequencing technique that provides genome-wide assessment of the RNA content of an organism, tissue, or cell. Alternatively, or in addition, the presence or absence or level of one or more biological markers of AEC can be detected using, for example, fluorescent in situ hybridization; (FISH; see WO 98 / 45479, published October 1998), Southern blotting, Northern blotting, or polymerase chain reaction (PCR) techniques, such as qRT-PCR. Quantitative methods for assessing the expression of markers at the protein level in a cell population are also known in the art. For example, flow cytometry is used to determine the percentage of cells that express or do not express a biological marker of interest in a given cell population.

[0036] Preferably, the AEC population comprises at least 80% arterial endothelial cells. Optionally, at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 99%, or more) of the cells in the resulting cell population are arterial endothelial cells. The mesodermal cells can express one or more mesodermal markers selected from the group consisting of Brachyury(T), EMOS, FOXA2, MIXL1, MSX1, and MSX2. For the methods described herein, the mesodermal cells are typically cultured in a culture medium that is free, substantially free, or essentially free of insulin, albumin, or any components derived from a non-human animal (i.e., xeno-free). As used herein, the term "substantially free" refers to cell culture conditions that are substantially devoid of a certain component or reagent. Substantially free of insulin means that the medium contains less than 1% of the original concentration of insulin by mass, or less than 2×10 -5 % insulin, preferably less than 1×10 -5 Less than 0.5 x 10 -5 Less than 0.2 x 10 -5 Less than % or 0.1 x 10 -5 % insulin.

[0037] Suitable TGFβ receptor inhibitors for use in the methods of the present invention include, without limitation, SB-431542, SB-525334, A83-01, LY2157299, LY210976, RepSox, SB-505124, D4476, GW788388, SD208, and EW-7197. Preferably, the inhibitor of TGF-beta signaling is SB-431542, a small molecule inhibitor of endogenous activin and type I receptor (TGFβ receptor I) (Inman et al., Mol Pharmacol. 62(1):65-74 (2002). Notch is a single-pass cell-surface receptor that binds to a family of cell-surface ligands, including the Delta-like and Jagged families. As used herein, the terms "Notch agonist" and "Notch activator" refer to molecules (e.g., biomolecules, small molecules, chemicals) that bind to Notch receptors and initiate or mediate the signaling events associated with Notch activation. Resveratrol (3,4',5-trihydroxystilbene) belongs to a class of polyphenolic compounds called stilbenes and is an activator (agonist) of Notch signaling. Other Notch agonists suitable for use according to the method of promoting arterial differentiation provided by the present invention include valproic acid and suberoyl bishydroxamic acid. In addition, immobilized or multimerized soluble Notch ligands, such as immobilized DLL4 and immobilized Jagged-1 peptide, can also be used as Notch activators.

[0038] Inositol monophosphatase (IMPase) catalyzes the hydrolysis of myo-inositol monophosphate to myo-inositol, which is required in the phosphoinositide cell signaling pathway. In some cases, the inhibitor of IMPase is the biphosphonate L-690,330 ([1-(4-hydroxyphenoxy)ethylidene]bisphosphonic acid). Lithium also inhibits IMPase to attenuate phosphoinositide signaling (Berridge et al., Cell 59:411-419 (1989)). Other inhibitors of the phosphoinositide signaling pathway include, without limitation, the phosphoinositide 3-kinase (PI3K) inhibitors Ly294002, pictilisib, HS-173, GSK2636771, duvelisib, TG100-115, GSK1059615, PF-04691502, PIK-93, BGT226, AZD6482, SAR245409, BYL719, CUDC-907, IC-87114, TG100713, gedatricisib, CH5132799, PKI-402, BAY 80-6946, XL147, PIK-90, PIK-293, PIK-294, quercetin, wortmannin, ZSTK474, AS-252424, AS-604850, and apitolisib.

[0039] Suitable working concentration ranges for the chemical inhibitors of IMPase, TGFβ receptor, and others described herein are from about 0.1 μM to about 100 μM, e.g., about 2 μM, 5 μM, 7 μM, 10 μM, 12 μM, 15 μM, 18 μM, or another working concentration of one or more of the above chemical inhibitors of about 0.1 μM to about 100 μM. Preferably, the mesodermal cells are cultured in AEC differentiation medium until at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 98%, or more) of the cells in the resulting cell population are arterial endothelial cells.

[0040] For some of the biological markers described herein, the levels of expression are lower or intermediate. Although it is common to refer to cells as "positive" or "negative" for a particular marker, the actual expression level is a quantitative attribute. The number of molecules on the cell surface can vary by several logs, yet still be characterized as "positive". Thus, characterization of the staining level allows for subtle distinctions between cell populations. Expression levels can be detected or monitored by flow cytometry, where a laser detects the quantitative level of a fluorescent dye (proportional to the amount of cell surface antigen bound by the antibody). Flow cytometry or fluorescence activated cell sorting (FACS) can be used to separate cell populations based on the intensity of antibody staining, as well as other parameters such as cell size and light scattering. Although the absolute level of staining may vary with a particular fluorescent dye and antibody preparation, the data can be normalized to controls. In some cases, the arterial endothelial cells are derived from human pluripotent stem cells. As described in US Patent Application Publication No. 2016 / 0244719, the human pluripotent stem cells are cultured in a serum-free, albumin-free, chemically defined cell culture medium containing bone morphogenetic protein (BMP), activin A, and an activator of Wnt / β-catenin signaling for a period of about 2 days, resulting in a cell population containing mesodermal cells. The mesodermal cells can express one or more mesodermal markers selected from the group consisting of Brachyury (T), EMOS, FOXA2, MIXL1, MSX1, and MSX2.

[0041] Human pluripotent stem cells (hPSCs), whether embryonic or induced, provide access to the earliest stages of human development and provide a platform for deriving large numbers of cells for cell therapy and tissue engineering. Thus, in a representative embodiment, the method provided by the present invention further comprises differentiating human pluripotent stem cells under conditions that promote differentiation of mesodermal stem cells into arterial endothelial cells. Optionally, the method for producing arterial endothelial cells comprises culturing human pluripotent stem cells in a serum-free, albumin-free, chemically defined culture medium that promotes mesodermal differentiation. Mesodermal cells derived from the pluripotent stem cells are then differentiated according to AEC differentiation methods (e.g., those described in U.S. Patent Application Publication No. 2016 / 0244719), thus producing pluripotent stem cell-derived AECs. In a representative embodiment, the serum-free, albumin-free, chemically defined culture medium that promotes mesodermal differentiation comprises activin A, bone morphogenetic protein 4 (BMP4), FGF2, and an activator of Wnt / β-catenin signaling. The pluripotent stem cells can be human embryonic stem cells or human induced pluripotent stem cells. As used herein, a "pluripotent stem cell" suitable for use according to the methods of the invention is a cell that has the ability to differentiate into cells of all three germ layers. Suitable pluripotent cells for use in the present invention include human embryonic stem cells (hESCs) and human induced pluripotent stem (iPS) cells. As used herein, "embryonic stem cells" or "ESCs" refers to a pluripotent cell or population of pluripotent cells derived from the inner cell mass of a blastocyst. See Thomson et al., Science 282:1145-1147 (1998). These cells express Oct-4, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. Pluripotent stem cells appear as compact colonies containing cells with high nuclear to cytoplasmic ratios and prominent nucleoli. ESCs are commercially available from sources such as WiCell Research Institute (Madison, Wis.).

[0042] In some cases, arterial endothelial cells are derived from human induced pluripotent stem cells. For example, in the case of patients who do not have an urgent need, induced pluripotent stem cells can be derived from the patient to produce arterial endothelial cells specific to the patient. As used herein, the term "induced pluripotent stem cells" ("iPS cells") refers to a pluripotent cell or a population of pluripotent cells that may vary in terms of origin of the original differentiated somatic cell, may vary in terms of a specific set of efficacy determinants, and may vary in terms of the culture conditions used to isolate, but are nevertheless genetically substantially identical to the original respective differentiated somatic cell origin and exhibit similar characteristics to higher efficacy cells such as ESCs described herein. See, for example, Yu et al., Science 318:1917-1920 (2007).

[0043] Induced pluripotent stem cells exhibit similar morphological (e.g., round shape, large nucleoli and scant cytoplasm) and growth characteristics (e.g., doubling time of about 17-18 hours) as ESCs. In addition, iPS cells express markers specific for pluripotent cells (e.g., Oct-4, SSEA-3, SSEA-4, Tra-1-60 or Tra-1-81, but not SSEA-1). However, induced pluripotent stem cells are not directly derived from an embryo. As used herein, "not directly derived from an embryo" means that the starting cell type for producing iPS cells is a non-pluripotent cell, e.g., a multipotent cell or a terminally differentiated cell, e.g., a somatic cell obtained from a postnatal individual. Human iPS cells can be used according to the methods described herein to obtain AECs with the genetic complement of a particular human subject. For example, it may be advantageous to obtain AECs that exhibit one or more specific phenotypes associated with or resulting from a particular disease or disorder of a particular mammalian subject. In such cases, iPS cells are obtained by reprogramming somatic cells of a particular human subject according to methods known in the art. See, for example, Yu et al., Science 324(5928):797-801 (2009); Chen et al., Nat. Methods 8(5):424-9 (2011); Ebert et al., Nature 457(7227):277-80 (2009); Howden et al., Proc. Natl. Acad. Sci. USA 108(16):6537-42 (2011). Subject-specific somatic cells for reprogramming into iPS cells can be obtained or isolated from a target tissue of interest by biopsy or other tissue sampling methods. In some cases, the subject-specific cells are manipulated or modified in vitro prior to use. For example, the subject-specific cells can be expanded, differentiated, chemically treated, genetically modified, contacted with polypeptides, nucleic acids, or other factors, cryopreserved, or otherwise modified prior to reprogramming, and then the reprogrammed cells can be directed to differentiate to produce subject-specific AECs.

[0044] An important difference between arterial endothelial cells produced from iPS cells derived from a particular individual and primary arterial endothelial cells isolated from the same individual is that cells derived from iPS cells are infinitely scalable and can surpass Hayflick's limit (a fixed number of cell divisions). As used herein, the term "Hayflick's limit" refers to a finite number of population doublings in vitro before cells can no longer proliferate and enter senescence (Hayflick L. Exp Cell Res 37:614-36, 1965). Although the inherent self-renewal capacity of primary cultured arterial endothelial cells is limited, an almost inexhaustible supply of arterial endothelial cells can be obtained from a single source (e.g., an individual's somatic cells) according to the methods provided by the present invention. Thus, in one embodiment of the present invention, AECs can be expanded in a tissue culture laboratory and the number of cells obtained is sufficient to treat more than one patient and is suitable for cell banking in a preferred embodiment.

[0045] The defined medium and substrate conditions for culturing pluripotent stem cells used in the methods described herein are known in the art.Preferably, the medium used in the present invention is chemically defined, albumin-free and xeno-free.Optionally, the pluripotent stem cells differentiated according to the methods disclosed herein are cultured in a chemically defined serum-free albumin-free medium. In some embodiments, the proportion of arterial endothelial cells in the population of cells is enriched using cell separation, cell sorting, or other enrichment methods, such as fluorescence-activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), magnetic beads, magnetic activated cell sorting (MACS), laser-targeted ablation of non-endothelial cells, and combinations thereof. Preferably, FACS is used to identify and separate cells based on cell surface antigen expression. Optionally, after obtaining a cell population containing human AECs according to the methods described herein, the human AEC population can be expanded in a suitable culture medium for growing human AECs, including, without limitation, human endothelial serum-free medium (Life Technologies, catalog number 11111-044), EGM-2 (Lonza, catalog number CC-3162), and endothelial cell culture medium (BD Biosciences, catalog number 355054).

[0046] C. Additional Vascular Graft Components Depending on the particular use to which the polymeric vascular graft described herein is applied, it may be advantageous for the graft to further include one or more bioactive agents. As used herein, the term "bioactive agent" or "active agent" refers to a therapeutic, prophylactic, and / or diagnostic agent, including, without limitation, a biologically, physiologically, or pharmacologically active substance that acts locally or systemically in the human or animal body. Examples include, without limitation, small molecule drugs, peptides, proteins, antibodies, saccharides, polysaccharides, nucleotides, oligonucleotides, aptamers, siRNA, nucleic acids, and combinations thereof. "Bioactive agent" includes a single agent or multiple bioactive agents, including, for example, a combination of two or more bioactive agents. Suitable bioactive agents for use with the polymeric implants of the present disclosure include, without limitation, pharmaceutical compositions, polypeptides (e.g., chemokines, cytokines), and / or additional therapeutic agents or drugs, such as, without limitation, anti-thrombotic agents, anti-proliferative agents, agents that prevent, inhibit, or reduce restenosis or aneurysm formation, anti-tumor / anti-proliferative / anti-mitotic agents, vascular cell growth promoters, vascular cell growth inhibitors, and vasodilators. Cytokines and chemokines include, without limitation, interleukin (IL) 1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-15, IL-17, IP-10, eotaxin, interferon gamma (IFNγ), granulocyte colony-stimulating factor (G-CSF), granulocyte / macrophage colony-stimulating factor (GM-CSF), macrophage inflammatory protein 1 alpha (MIP-1 alpha), RANTES, tumor necrosis factor-alpha (TNF-α), platelet-derived growth factor (PDGF)-AA, PDGF-AB / BB, TGF-beta, VEGF, and combinations thereof. In some cases, the bioactive agent is incorporated into or applied to the vascular graft. In some cases, the polymeric vascular graft comprises one or more additional cell types. For example, smooth muscle cells (SMCs) can be seeded onto the polymeric vascular graft in addition to AECs. The SMCs can be primary smooth muscle cells or human pluripotent stem cell-derived SMCs. The SMCs can be wild-type, genetically modified, or genetically edited.

[0047] method The polymeric vascular grafts described herein are useful as arterial or arterial-venous shunts for any vascular or cardiovascular surgical application. Representative applications include, without limitation, congenital heart surgery, coronary artery bypass surgery, and peripheral vascular surgery. Thus, in another aspect, the present invention provides a method of producing and using the polymeric vascular grafts provided by the present invention to treat a vascular defect in a subject in need thereof. Such a method may include implanting the polymeric vascular grafts disclosed herein into a subject in need thereof. The terms "individual," "host," "subject," and "patient" are used interchangeably herein. In various embodiments, the polymeric vascular grafts are implanted to replace a portion of a diseased or damaged blood vessel, for example, to replace a weakened portion of an aorta or blood vessel damaged by trauma or damaged due to a vascular disorder.

[0048] In some embodiments, the polymeric vascular grafts are used to bypass and / or replace stenosed or partially blocked portions of blood vessels, for example, in coronary or peripheral artery bypass graft surgery. For example, the polymeric vascular grafts seeded with AECs of the present disclosure are useful in cardiac or leg bypass surgery. In another example, the polymeric vascular grafts seeded with AECs of the present disclosure are useful in reconstructive surgery, for example, to correct developmental abnormalities or repair severe injuries. The vascular grafts are also well suited to provide hemodialysis access in arteriovenous shunts.

[0049] In some cases, the method of treatment includes performing an anastomosis (i.e., surgical connection of tubular portions) to implant a polymeric vascular graft. Typically, an anastomosis between an in situ artery or vein and a polymeric vascular graft is performed by sewing the graft to the in situ vessel with a suture. Commonly used suture materials include PROLENE® polypropylene suture and ePTFE. Thus, the vascular graft of the present disclosure includes a sewable material such as PTFE or ePTFE. One of the major problems with existing autologous venous endothelial cell procedures is that it takes about one month to harvest, grow, seed and culture the cells in the graft. About 30% of patients cannot undergo such treatment because their urgent medical needs do not allow them to wait 30 days to obtain an autologous venous endothelial cell graft. Thus, the present disclosure provides materials and methods that are particularly advantageous compared to conventional methods. In particular, the present invention provides a method in which an AEC-seeded polymeric vascular graft is prepared and ready for clinical use within about 10 days. Such grafts are prepared using human AECs produced on a large scale and frozen until needed. Optionally, therefore, the method includes thawing and seeding human AECs onto a polymeric substrate, preferably a polymeric substrate that is at least partially coated with one or more endothelial cell adhesives. By request, the frozen human AECs are selected based on their compatibility with the patient in need of the graft, or are "universal" AECs that are unlikely to cause an immune response in the graft recipient (patient). Preferably, the selected cells are thawed and seeded onto the prepared polymeric substrate, and the AEC-seeded polymeric substrate is cultured for less than 10 days, preferably less than 7 days (e.g., on the order of 2, 3, 4, 5, or 6 days). The cultured polymeric substrate is then delivered to a patient or provided for therapy within about 10 days, preferably within about 7 days, from the initial request. If the AECs are derived from banked iPSC cells according to the AEC differentiation protocol described, for example, in U.S. Patent Application Publication No. 2016 / 0244719, the time from the initial request to delivery of the prepared AEC-seeded vascular graft must include time to complete the differentiation process. The present application provides a directed differentiation protocol, in which optionally, human pluripotent stem cells are differentiated into mesodermal cells in about 2-3 days, and the resulting mesodermal cells are induced to differentiate into endothelial cells in approximately 3 days. Optionally, the method includes seeding cells of a universal cell line onto the polymeric vascular graft. In such cases, the seeded vascular grafts can be prepared and ready to use as a "ready to use" product upon request, in which case the AEC-seeded vascular grafts can be provided to patients in need as soon as they are needed.For example, prepared "universal" AEC-seeded vascular grafts can be provided using next-day or more rapid delivery. If produced locally, delivery of the prepared vascular grafts may take only minutes or hours. In some cases, prepared "universal" AEC-seeded vascular grafts can be purchased and stored locally as cryopreserved frozen products, in which case the AEC-seeded grafts may be available for patient use with minimal delay.

[0050] Any suitable method can be used to detect and measure functional and morphological changes after implantation of the polymeric vascular graft of the present disclosure. For example, vascular ultrasound can be performed to evaluate fluid flow in the arteries and veins of the body to detect the presence, severity, and / or specific location of disease. Vascular ultrasound is a non-invasive ultrasound method used to examine circulation in the blood vessels of the body (also called duplex ultrasound). In some cases, vascular ultrasound is used to calculate the rate of fluid flow in blood vessels before and after treatment of the blood vessel with the polymeric vascular graft described herein. In some cases, contrast-enhanced ultrasound (CEUS) is used to detect and / or monitor vascular lesions before and after therapeutic intervention. Vascular ultrasound and CEUS are particularly useful for detecting and characterizing restenosis after therapeutic intervention. "Restenosis," as defined herein, refers to the narrowing of the lumen of a blood vessel at the site of a previous stenosis (i.e., the site of balloon dilation during angioplasty), or the narrowing of the lumen of a blood vessel or synthetic graft after an interventional procedure (e.g., narrowing of the venous side of an arterio-venous anastomosis after bypass surgery using a graft). Restenosis, as used herein, encompasses occlusion. Restenosis includes any luminal narrowing that occurs after injury to the vessel wall. Injuries that cause restenosis can therefore include trauma to an atherosclerotic lesion (as seen in angioplasty), resection of the lesion (as seen in endarterectomy), external trauma (e.g., cross-clamping injury), or surgical anastomosis.

[0051] In another aspect, the present invention provides a method for fabricating a polymeric vascular graft. The method comprises or consists essentially of the steps of: coating at least a portion of a polymeric substrate with one or more endothelial adhesion agents; contacting the coated polymeric substrate with human arterial endothelial cells to form a polymeric vascular graft seeded with AECs that are substantially non-adhesive to leukocytes or cell fragments thereof. As used herein, the term "coating" refers to attaching or depositing an endothelial adhesion agent of the present disclosure to a polymeric material (e.g., ePTFE) by any suitable process, such that the deposited agent covers some or all of the surface of the material. In some cases, the coating includes at least partially covering the inner luminal surface area of ​​the polymeric material. The coating of the polymeric material need not be complete. In particular, in some cases, it is preferred to provide the composition only on a portion or some portion of the polymeric material to be coated, resulting in a polymeric material that is at least partially coated with one or more endothelial adhesion agents. In some cases, the coating comprises one or more coating layers. The coating can have a substantially constant or varying thickness.

[0052] Optionally, coating at least a portion of the polymeric substrate is performed at room temperature or at a temperature physiologically relevant to arterial endothelial cells, such as 37° C. Optionally, coating includes contacting at least a portion of the polymeric substrate with one or more endothelial adhesion agents for any suitable length of time, such as, without limitation, for a few minutes, a few hours, or from about 12 hours to about 24 hours, thereby resulting in a partially or completely coated substrate. Optionally, the method may include degassing the polymeric substrate prior to coating with one or more endothelial cell adhesion agents.

[0053] In another embodiment, the present invention provides a method for cryopreserving AEC-seeded polymeric vascular grafts. Cryopreservation is a process of preserving biological materials, such as cells, tissues, extracellular matrices, organs, or any other biological constructs susceptible to damage caused by unregulated chemical dynamics, by cooling to very low temperatures (usually -40°C or -80°C). The method can comprise or consist essentially of contacting the AEC-seeded polymeric vascular graft with a cryoprotectant (also referred to as cryoprotectant, cryoprotectant, and cryopreservation agent) and then exposing the contacted material to freezing temperatures. The cryoprotectant protects the biological material on the vascular graft from the damaging effects of freezing (e.g., ice crystal formation and increased solute concentration due to freezing of water molecules in the biological material). In some cases, the cryopreserved vascular graft retains at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of AEC cell viability after freezing and thawing as determined by cell counting of the graft tissue before processing and cell counting of the graft after freezing and thawing.

[0054] In another aspect, the invention provides a method for delivering a vascular graft seeded with arterial endothelial cells, the method comprising: upon receiving a request for a vascular graft seeded with arterial endothelial cells, selecting human arterial endothelial cells; seeding the selected human AECs onto a polymeric substrate at least partially coated with an endothelial cell adhesion agent; culturing the seeded polymeric substrate for about 2 to about 10 days to produce an AEC-seeded polymeric substrate suitable for implantation as a vascular graft; and delivering the AEC-seeded vascular graft within about 10 days of receiving the request. The polymeric substrate can be selected from expanded polytetrafluoroethylene (ePTFE), polyvinyl chloride (PVC), PGA (polyglycolic acid), PLA (polylactic acid), PCL (polycaprolactone), PGLA (polylactic-co-glycolic acid), polyurethane, polydioxanone, polyethylene, polyethylene terephthalate (Dacron®), tetrafluoroethylene (TFE), polytetrafluoroethylene (PTFE), silk, decellularized scaffolds, extracellular matrix protein-based scaffolds, hyaluronic acid, chitosan, and polyhydroxyalkanoates. Endothelial cell attachment agents can include one or more of dopamine, fibrin glue, RGD peptide, vitronectin, and laminin. Optionally, the vascular graft exhibits reduced leukocyte adhesion compared to a polymeric substrate seeded with venous endothelial cells. Optionally, the vascular graft exhibits reduced thrombus formation compared to a polymeric substrate seeded with venous endothelial cells or a bare uncoated polymeric substrate. Optionally, the vascular graft exhibits increased long-term patency compared to a polymeric substrate not coated with an endothelial cell adhesion agent. Optionally, the method further comprises degassing the polymeric substrate prior to coating with the endothelial cell adhesion agent. Optionally, degassing comprises washing the polymeric substrate in acetone and ethanol, washing the polymeric substrate in an organic solvent, or applying a vacuum. Preferably, the human arterial endothelial cells are non-immunogenic to the recipient of the vascular graft. Optionally, the human arterial endothelial cells comprise one or more genetic modifications such that they do not express a beta2-microglobulin gene. Optionally, the human arterial endothelial cells comprise one or more genetic modifications such that they do not express one or more proteins encoded by class I major histocompatibility complex (MHC) genes. In some cases, the human arterial endothelial cells include one or more genetic modifications so as to not express one or more proteins encoded by class II major histocompatibility complex (MHC) genes. In some cases, the human arterial endothelial cells include one or more genetic modifications so as to not express a CD58 polypeptide. In some cases, the human arterial endothelial cells include one or more genetic modifications so as to overexpress one or both of HLA-E (EDIMER) and CD47.

[0055] In another aspect, the present invention provides a method for delivering an arterial endothelial cell-seeded vascular graft, the method comprising: upon receiving a request for an arterial endothelial cell-seeded vascular graft, selecting an appropriate cryopreserved arterial endothelial cell (AEC)-seeded vascular graft for a subject in need thereof; thawing the selected cryopreserved AEC-seeded vascular graft; removing cryopreservation solution, if present, from the thawed AEC-seeded vascular graft; and delivering the AEC-seeded vascular graft within about 1-2 days (e.g., within about 24 to about 48 hours) of receiving the request. Importantly, these methods provide a solution to a critical patient care need, specifically the ability to provide a ready AEC-seeded vascular graft to a patient within 1-2 days (e.g., within about 24 to about 48 hours) of receiving a request for graft material. As such, these methods provide a significant improvement over conventional methods that require about 30 days to provide a vascular graft seeded with a patient's autologous venous endothelial cells. As used herein, the term "patient-ready" means that the implant is pre-configured and ready for use in a patient with minimal delay or additional preparation.

[0056] In some cases, the vascular graft seeded with suitable cryopreserved AECs comprises human arterial endothelial cells that are non-immunogenic to the subject. The human arterial endothelial cells can comprise one or more genetic modifications so as not to express one or more proteins encoded by class II major histocompatibility complex (MHC) genes. In some cases, the human arterial endothelial cells comprise one or more genetic modifications so as not to express CD58 polypeptide. In some cases, the human arterial endothelial cells comprise one or more genetic modifications so as to overexpress one or both of HLA-E (EDIMER) and CD47.

[0057] manufactured goods In another aspect, the present invention provides an article of manufacture. For example, the present invention provides a container comprising a cryopreserved combination product and a cryopreservation solution, wherein the cryopreserved combination product comprises a human arterial endothelial cell population seeded on an implantable polymeric substrate at least partially coated with an endothelial cell adhesion agent. Optionally, the endothelial cell adhesion agent comprises dopamine. As described herein, the human arterial endothelial cells are preferably non-immunogenic such that the polymeric graft is "universal" and suitable for use in any human subject in need thereof. For example, the human arterial endothelial cells may comprise one or more genetic modifications such that they do not express one or more proteins encoded by the beta2-microglobulin gene and / or class I or class II major histocompatibility complex (MHC) genes. The container may be a vial, a cryotube, a bag, or any other vessel suitable for containing the polymeric vascular graft and the cryopreservation solution. Preferably, the containers can be stored at freezing temperatures, for example, without limitation, temperatures from 1° C. to about −196° C. or less (e.g., 1°, 0°, −1°, −5, −10°, −20°, −30°, −40°, −50°, −60°, −70°, −80°, −90°, −100°, −110°, −120°, −130°, −140°, −150°, −160°, −170°, −180°, −190°, −196° C., or less).

[0058] In some cases, the human arterial endothelial cell population is contacted with a cryopreservation solution prior to seeding the implantable polymeric substrate. In other cases, the implantable polymeric substrate is contacted with a cryopreservation solution after seeding with human arterial endothelial cells. Examples of suitable cryopreservation solutions include, without limitation, dimethylsulfoxide (DMSO). In some cases, a 10% DMSO solution is used for cryopreservation. In some cases, the cryopreservation solution is removed from the seeded implantable polymeric substrate prior to implantation. The solution contacting and removal steps are generally performed under aseptic, preferably sterile, conditions.

[0059] In another embodiment, the present invention provides a container containing a cryopreserved combination product and a cryopreservation solution, wherein the cryopreserved combination product comprises an implantable polymeric substrate seeded with human arterial endothelial cells, and the implantable polymeric substrate is at least partially coated with one or more endothelial cell attachment agents. The polymeric substrate can be selected from expanded polytetrafluoroethylene (ePTFE), polyvinyl chloride (PVC), PGA (polyglycolic acid), PLA (polylactic acid), PCL (polycaprolactone), PGLA (polylactic-co-glycolic acid), polyurethane, polydioxanone, polyethylene, polyethylene terephthalate (Dacron®), tetrafluoroethylene (TFE), polytetrafluoroethylene (PTFE), silk, decellularized scaffolds, extracellular matrix protein-based scaffolds, hyaluronic acid, chitosan, and polyhydroxyalkanoates. Optionally, the human arterial endothelial cells are non-immunogenic to the recipient of the implantable polymeric substrate. In some cases, the human arterial endothelial cells include one or more genetic modifications so as not to express the beta2-microglobulin gene. The human arterial endothelial cells can include one or more genetic modifications so as not to express one or more proteins encoded by class I and / or class II major histocompatibility complex (MHC) genes. In other cases, the human arterial endothelial cells include one or more genetic modifications so as not to express the CD58 polypeptide. Alternatively, or in addition, the human arterial endothelial cells can include one or more genetic modifications so as to overexpress one or both of HLA-E (EDIMER) and CD47. The container can be a vial, a cryotube, or a bag. The cryopreservation solution can include about 10% dimethyl sulfoxide (DMSO). In some cases, the human arterial endothelial cell population is contacted with the cryopreservation solution before seeding on the implantable polymeric substrate. Preferably, the cryopreservation solution is removed from the seeded implantable polymeric substrate before implantation.Preferably, the combination product is configured to be stored at a temperature between 37°C and about -196°C (e.g., about 37°, 30°, 25°, 15°, 10°, 4°, 1°, 0°, -1°, -5, -10°, -20°, -30°, -40°, -50°, -60°, -70°, -80°, -90°, -100°, -110°, -120°, -130°, -140°, -150°, -160°, -170°, -180°, -190°, -196°C, or lower) without significant loss of cell viability compared to a control not stored under such conditions.

[0060] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In case of conflict, the present application, including definitions, will govern. Unless otherwise required by context, singular terms include the plural and plural terms include the singular. All publications, patents and other references mentioned herein are incorporated by reference in their entirety for all purposes as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference, unless only a specific portion of a patent or patent publication is indicated to be incorporated by reference. To further clarify this disclosure, the following terms, abbreviations and definitions are provided. The indefinite articles "a" and "an," as used in the specification and claims, unless clearly indicated to the contrary, should be understood to mean "at least one." Any reference to "or" herein is intended to include "and / or" unless specifically stated otherwise.

[0061] When used in the present specification and claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" is interpreted as being inclusive, i.e., including at least one of a number or list of elements, but also including more than one, and may include additional unlisted items. Only terms clearly indicated to the contrary, such as "only one" or "exactly one" or "consisting of" when used in the claims, refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein should be interpreted as indicating exclusive alternatives (i.e., "one or the other, but not both") only when preceded by a term of exclusivity, such as "either," "one," "only one," or "exactly one." "Consisting essentially of" when used in the claims has its ordinary meaning as used in the field of patent law.

[0062] The terms "comprise" and "consist of" as used herein are synonymous with "comprise" or "contain" and are inclusive or open-ended and do not exclude additional, unlisted members, elements, or method steps. The terms and terminology used herein are for purposes of description and should not be considered limiting. The use of "comprise", "comprising", "having", "containing", "involving" and variations thereof means to include the items listed thereafter as well as additional items. In other words, these terms are intended to be non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or device that includes listed elements is not necessarily limited to only those elements, but may include other elements that are not expressly listed or inherent to such composition, mixture, process, method, article, or device. The use of ordinal terms such as "first," "second," "third," etc. in the claims to modify claim elements does not, in itself, imply any priority, superordinate, or ordering of one claim element relative to another, or the temporal order in which acts of a method are performed. Ordinal terms are used merely as markers to distinguish claim elements from one claim element having a certain name from another element having the same name (except for the use of the ordinal term).

[0063] As used herein, the terms "approximately" or "about" in connection with numbers are generally intended to include numbers within a range of 5% in either direction of that number (larger or smaller) unless otherwise stated or apparent from the context (except where such number exceeds 100% of possible values). Where a range is given, the endpoints are included within that range unless otherwise stated or apparent from the context. In this disclosure, any numerical value, range, or end point of either range that is preceded by the words "about," "substantially," or "approximately" (including, for example, "about none," "almost none," "almost all," etc.) is understood to also describe or disclose the same numerical value, range, or end point of either range that is not preceded by the words "about," "substantially," or "approximately." The following examples are provided to better illustrate various embodiments and should not be construed as limiting the scope of the present disclosure. EXAMPLES

[0064] Improving cell adhesion by surface modification of ePTFE Meinhart et al. (ASAIO J 43:M515-521, 1997) reported the construction of ePTFE (expanded polytetrafluoroethylene) vascular grafts coated with venous endothelium, which showed improved in vivo patency compared to ePTFE without venous endothelial cells. Because arterial grafts are preferred over venous grafts for arterial bypass surgery, we seeded ePTFE with arterial endothelial cells to further improve patency. ePTFE is hydrophobic and therefore exhibits low cell adhesion. To improve cell adhesion, we performed plasma treatment to make ePTFE hydrophilic. The results showed that plasma treatment promoted cell density but only to about 20% of confluence (Figure 1A), suggesting that cell adhesion molecules are required to further improve cell attachment. Since RGD peptide, collagen, fibronectin (FN), laminin, vitronectin (VTN), and Matrigel® are widely used for cell attachment, we coated ePTFE with these cell attachment agents. The cells were then seeded onto the ePTFE using a cell seeding device. After seeding, the cells were cultured for an additional 2-20 days in culture medium containing basal medium supplemented with FGF, VEGF, TGF-beta inhibitors (e.g., SB431542), and resveratrol (RESV) to form a chemically defined FVIR medium (see Table 1). Surprisingly, only Matrigel® and VTN were able to achieve cell confluence greater than 95% (Figure 1B).

[0065] Next, we coated ePTFE with dopamine (Figure 2A), which could be deposited on the surface of ePTFE via self-polymerization. The results showed that dopamine coating improved cell density and reduced cell death on the synthetic substrate (Figure 2B-2C). Fibrin glue was used to improve endothelial cell seeding on ePTFE (Zilla et al., 1989). However, this method involves multiple steps and is therefore difficult to scale up for large-scale clinical applications. Therefore, we investigated whether other endothelial cell glues, such as dopamine, could be used for cell seeding instead of fibrin glue. We compared cell seeding on ePTFE coated with dopamine. As shown in Figure 2D, immunostaining revealed that AEC density was comparable on ePTFE coated with dopamine and fibrin glue. These data indicate that various endothelial cell glues can be used to seed human AECs on polymeric substrates.

[0066] [Table 1] Comparison of cell seeding efficiency of ePTFE coated with dopamine and fibrin glue Fibrin glue was used to improve endothelial cell seeding on ePTFE (Zilla et al., 1989). However, this method involves multiple steps and is therefore difficult to scale up for large-scale clinical applications. Therefore, we investigated whether other endothelial cell adhesives, such as dopamine and extracellular matrix peptides and proteins, could be used for cell seeding instead of fibrin glue. We compared cell seeding on ePTFE coated with dopamine, fibrin glue, RGD (Arg-Gly-Asp) peptide, VTN (vitronectin), and laminin. As shown in Figure 2A, immunostaining revealed that AEC density was comparable on ePTFE coated with dopamine and fibrin glue. As shown in Figure 2B, AECs adhered well to ePTFE coated with RGD peptide, vitronectin, and laminin. These data indicate that various endothelial cell adhesives can be used to seed human AECs on polymeric substrates.

[0067] AEC-ePTFE exhibits lower leukocyte adhesion Increased leukocyte adhesion is a hallmark of the initiation of atherosclerosis (De Caterina et al., 1995; Legein et al., 2013). Arterial endothelial cells (AECs) showed lower leukocyte adhesion when compared to venous endothelial cells in static culture (Zhang et al., 2017), suggesting that AECs are more resistant to atherosclerosis. To address whether ePTFE materials seeded with human AECs ("AEC-ePTFE grafts") maintain arterial specific functions with flow, we compared leukocyte adhesion on ePTFE seeded with AECs and HUVECs (human umbilical vein endothelial cells; "HUVEC-ePTFE grafts"), respectively. Leukocytes were stained by exposure to 2 μM calcein AM for approximately 15 min. Calcein AM-labeled leukocytes were then transferred to AEC- and HUVEC-seeded ePTFE at approximately 1 × 10 6The cells were added at a cell density of 1000 cells / ml. Both the calcein AM-labeled leukocyte cell suspension and the ePTFE were placed in a 0.5 ml tube, and the tube was rotated at 60 rpm for 1 h. After 1 h, the cell-seeded ePTFE was gently washed three times with fresh medium and then fixed and stained with DAPI for imaging. Leukocyte adhesion assay was performed under shear stress to mimic fluid flow through blood vessels.

[0068] Before TNFα treatment, few leukocytes were observed adhering to the AEC-seeded and HUVEC-seeded substrates (Figure 3A). After TNFα treatment, many leukocytes adhered to the HUVEC-ePTFE grafts, but much fewer adhered to the AEC-ePTFE grafts (Figures 3A-3B). The results suggested that AEC-ePTFE may be more resistant to vascular injury compared to ePTFE coated with venous endothelium.

[0069] Degassing of ePTFE prevents the formation of cell aggregates and improves CD31 expression and cell density ePTFE vascular grafts contain 70% air by volume (Bensen et al., 1991). When fluid flows, bubbles or gas nuclei form on the surface of ePTFE (Bensen et al., 1991), which may damage the dopamine coating and therefore the endothelial covering. We performed degassing using acetone and ethanol. ePTFE was submerged in acetone for 10–60 min, followed by rinsing with 100% EtOH, 90% EtOH, and 70% EtOH for 30 min each. Degassed ePTFE was kept in H2O before use. After degassing, ePTFE was coated with dopamine and then seeded with AECs. We observed that the mean fluorescence intensity of CD31 expression (red staining) increased after degassing (Figures 4A, 4C). Degassing also increased the cell density on the seeded substrates, as determined by comparing the number of nuclei before and after 8 days of degassing (Figures 4A, 4B). In addition, CD31 expression was increased as measured by CD31 fluorescence intensity (Figures 4A, 4C). It was observed that cell aggregates formed in ePTFE sample 2 seeded with AECs (Figure 4A), but degassing treatment reduced the number of cell aggregates (Figure 4A). Overall, these data indicated that degassing improved the endothelial coverage of ePTFE.

[0070] To develop a "ready-to-go" product, we tested various cryopreservation solutions to be used with AEC-ePTFE vascular grafts. Glycerol was used for cryopreservation of clinically used human skin substitutes (US Patent Publication No. 20140271583). However, our results showed that glycerol was not suitable for cryopreservation of AEC-ePTFE vascular grafts (Figure 5A). Although Recovery™ cell culture freezing medium (Thermofisher) improved cell viability in cryopreservation of five different adherent and suspension cell lines, most cells died when AEC-ePTFE vascular grafts were frozen using Recovery™ (Figure 5B). DMSO combined with FBS (fetal bovine serum) is commonly used for cryopreservation (Ha et al., 2005). Interestingly, our results showed that FBS reduced cell survival on frozen AEC-ePTFE vascular grafts (Figure 5B). In contrast, serum-free medium containing 10% DMSO showed the highest cell viability, which was comparable to that of the non-frozen control samples (Figure 5B). Lowering or increasing the DMSO concentration had a negative effect on cell viability (Figure 5C), indicating that 10% DMSO is well suited for cryopreservation of AEC-ePTFE vascular grafts.

[0071] Methods and Materials Fibrin adhesive coating: The fibrinogen component (Baxter, TISSEEL) was prepared by diluting a 2 ml portion of fibrinogen with 4 ml of heated fibrinolysis inhibitor followed by the addition of 1 ml of tranexamic acid (20 mg / ml). The thrombin component was prepared by diluting 2 ml of fibrinogen in 4 ml of CaCl2, 75 ml of H2O, and 4 ml of tranexamic acid (20 mg / ml). The fibrinogen component was flowed three times through the ePTFE. The thrombin component was then flowed through the ePTFE for 5 minutes. The ePTFE was rinsed three times with distilled water. After the coating process was repeated once, the ePTFE was flushed with 5 ml of 50 U / ml heparin. Dopamine coating: Dopamine was dissolved in 10 mM Tris solution (pH = 8.5) at a concentration of 2 mg / mL. ePTFE was immediately immersed in the solution and incubated in the solution at room temperature or 37°C for 4 to 24 hours. The coated ePTFE was washed five times with distilled water.

[0072] Seed the cells onto ePTFE: Endothelial cells were seeded (1.5 × 10) in cell culture medium containing Y27632 (ROCK inhibitor). 6 The cells were suspended at a density of 1000 x 1000 cells / ml and seeded onto the ePTFE. The ePTFE was placed into a tube and then loaded onto a cell seeding device (Endostradilisator III, Biggler). The ePTFE (inside the tube) was rotated at 4 rph for 3 hours. Alternatively, the ePTFE can be incubated for 1 hour, then manually rotated 90° and incubated for another hour. The 90° rotation was repeated 4 times. Degassing of ePTFE: Degassing was performed by soaking the ePTFE in acetone for 3 hours, followed by washing the acetone-treated ePTFE with 70% ethanol for 30 minutes (repeated three times). The degassed ePTFE was rinsed in distilled water for 30 minutes (repeated three times). From this point on, the ePTFE should be soaked in distilled water or phosphate buffered saline (PBS) to avoid regassing. TIFF0007669266000002.tif189170 Yet another aspect of the present invention may be as follows. [1] (a) a polymeric substrate at least partially coated with an endothelial cell adhesion agent; and (b) Human arterial endothelial cells attached to the coated polymeric substrate. 13. A vascular graft comprising: [2] The implant according to [1], wherein the polymeric substrate is selected from expanded polytetrafluoroethylene (ePTFE), polyvinyl chloride (PVC), PGA (polyglycolic acid), PLA (polylactic acid), PCL (polycaprolactone), PGLA (polylactic-co-glycolic acid), polyurethane, polydioxanone, polyethylene, polyethylene terephthalate (Dacron®), tetrafluoroethylene (TFE), polytetrafluoroethylene (PTFE), silk, decellularized scaffolds, extracellular matrix protein-based scaffolds, hyaluronic acid, chitosan, and polyhydroxyalkanoates. [3] The graft according to [1] above, wherein the endothelial cell adhesion agent comprises one or more of dopamine, fibrin glue, RGD peptide, vitronectin, and laminin. [4] The graft described in [1] above, wherein the vascular graft exhibits reduced leukocyte adhesion compared to a polymeric substrate seeded with venous endothelial cells. [5] The vascular graft is compared to a polymeric substrate that is not coated with human arterial endothelial cells. (a) reduced thrombus formation; (b) increased long-term patency, and (c) Decreased platelet adhesion The implant according to claim 1, wherein the implant exhibits one or more of the following: [6] The graft described in [1] above, wherein the human arterial endothelial cells are produced from human pluripotent stem cells. [7] The graft described in [6] above, wherein the human pluripotent stem cells are induced pluripotent stem cells. [8] The graft described in [7] above, wherein the induced pluripotent stem cells are autologous to the patient. [9] The graft described in [7] above, wherein the induced pluripotent stem cells are at least 50% HLA-compatible with the patient.

[10] The graft described in [1] above, wherein the human arterial endothelial cells are non-immunogenic to the recipient of the vascular graft.

[11] The graft described in [1] above, wherein the human arterial endothelial cells contain one or more genetic modifications so that they do not express the beta 2-microglobulin gene.

[12] The graft described in [1] above, which contains one or more genetic modifications so that human arterial endothelial cells do not express one or more proteins encoded by class I or class II major histocompatibility complex (MHC) genes.

[13] The graft described in [1] above, wherein the human arterial endothelial cells contain one or more genetic modifications so that they do not express CD58 polypeptide.

[14] The graft described in [1] above, wherein the human arterial endothelial cells contain one or more modifications so as to overexpress one or both of HLA-E (EDIMER) and CD47.

[15] A method for forming a cell-seeded vascular graft, comprising: (a) coating a polymeric substrate with an endothelial cell attachment agent; (b) seeding the coated polymeric substrate with human arterial endothelial cells; and (c) culturing the coated and seeded polymeric substrate for about 2 to about 20 days, thereby obtaining a cell-seeded vascular graft. The method includes:

[16] The method according to

[15] , further comprising degassing the polymeric substrate prior to coating with one or more endothelial cell adhesion agents. 17. The method of claim 16, wherein degassing comprises washing the polymeric substrate in acetone and ethanol, washing the polymeric substrate in an organic solvent, or applying a vacuum.

[18] The method according to

[15] , wherein the polymeric substrate is selected from expanded polytetrafluoroethylene (ePTFE), polyvinyl chloride (PVC), PGA (polyglycolic acid), PLA (polylactic acid), PCL (polycaprolactone), PGLA (polylactic-co-glycolic acid), polyurethane, polydioxanone, polyethylene, polyethylene terephthalate (Dacron®), tetrafluoroethylene (TFE), polytetrafluoroethylene (PTFE), silk, decellularized scaffolds, extracellular matrix protein-based scaffolds, hyaluronic acid, chitosan, and polyhydroxyalkanoates.

[19] The method according to

[15] , further comprising contacting the cell-seeded vascular graft with a cryopreservation solution and freezing the contacted cell-seeded vascular graft.

[20] The method according to

[15] above, wherein freezing comprises storing at a temperature of 1°C to about -196°C.

[21] A method for producing a vascular graft seeded with AEC cells, comprising: coating at least a portion of the polymeric substrate with one or more endothelial adhesion agents; Contacting the coated polymeric substrate with human arterial endothelial cells forms an AEC-seeded vascular graft that is substantially non-adherent to leukocytes or cell fragments thereof. The method includes:

[22] The method according to

[21] , wherein the polymeric substrate is selected from expanded polytetrafluoroethylene (ePTFE), polyvinyl chloride (PVC), PGA (polyglycolic acid), PLA (polylactic acid), PCL (polycaprolactone), PGLA (polylactic-co-glycolic acid), polyurethane, polydioxanone, polyethylene, polyethylene terephthalate (Dacron®), tetrafluoroethylene (TFE), polytetrafluoroethylene (PTFE), silk, decellularized scaffolds, extracellular matrix protein-based scaffolds, hyaluronic acid, chitosan, and polyhydroxyalkanoates.

[23] The method according to

[21] , further comprising degassing the polymeric substrate prior to coating with one or more endothelial cell adhesion agents.

[24] The method according to

[23] , wherein degassing comprises washing the polymeric substrate in acetone and ethanol, washing the polymeric substrate in an organic solvent, or applying a vacuum.

Claims

1. (a) a degassed polymeric substrate, wherein said degassed polymeric substrate is at least partially coated with an endothelial cell attachment agent; and (b) Human arterial endothelial cells (AECs) attached to the degassed, coated polymeric substrate.

13. A vascular graft comprising:

2. 2. The implant of claim 1, wherein the polymeric substrate is selected from expanded polytetrafluoroethylene (ePTFE), polyvinyl chloride (PVC), PGA (polyglycolic acid), PLA (polylactic acid), PCL (polycaprolactone), PGLA (polylactic-co-glycolic acid), polyurethane, polydioxanone, polyethylene, polyethylene terephthalate Dacron®, tetrafluoroethylene (TFE), polytetrafluoroethylene (PTFE), silk, decellularized scaffolds, extracellular matrix protein-based scaffolds, hyaluronic acid, chitosan, and polyhydroxyalkanoates.

3. The implant of claim 1 , wherein the endothelial cell attachment agent comprises one or more of dopamine, fibrin glue, RGD peptide, vitronectin, and laminin.

4. 10. The graft of claim 1, wherein the vascular graft in the patient exhibits reduced leukocyte adhesion compared to a polymeric substrate seeded with venous endothelial cells.

5. Vascular grafts in patients were compared to polymeric substrates not coated with human arterial endothelial cells. (a) reduced thrombus formation; (b) increased long-term patency, and (c) Decreased platelet adhesion The implant of claim 1 , exhibiting one or more of the following:

6. The graft of claim 1 , wherein the human arterial endothelial cells are produced from human pluripotent stem cells.

7. The graft of claim 6 , wherein the human pluripotent stem cells are induced pluripotent stem cells.

8. The graft of claim 7 , wherein the induced pluripotent stem cells are autologous to the patient.

9. The graft of claim 7 , wherein the induced pluripotent stem cells are at least 50% HLA-matched to the patient.

10. The graft of claim 1 , wherein the human arterial endothelial cells are non-immunogenic to a recipient of the vascular graft.

11. The graft of claim 1, wherein the human arterial endothelial cells contain one or more genetic modifications so that they do not express the beta2-microglobulin gene.

12. The graft of claim 1, wherein the human arterial endothelial cells contain one or more genetic modifications such that they do not express one or more proteins encoded by class I or class II major histocompatibility complex (MHC) genes.

13. The graft of claim 1 , wherein the human arterial endothelial cells contain one or more genetic modifications such that they do not express CD58 polypeptide.

14. The graft of claim 1, wherein the human arterial endothelial cells contain one or more modifications to overexpress one or both of HLA-E (Edimer) and CD47.

15. 1. A method for forming a cell-seeded vascular graft, comprising: (a) degassing a polymeric substrate; (b) coating the polymeric substrate with an endothelial cell attachment agent; (c) seeding the polymeric substrate with human arterial endothelial cells; and (d) culturing the polymeric scaffold seeded with human arterial endothelial cells for about 2 to about 20 days, thereby obtaining a cell-seeded vascular graft. The method includes:

16. 16. The method of claim 15, wherein the degassing comprises washing the polymeric substrate in acetone and ethanol, washing the polymeric substrate in an organic solvent, or applying a vacuum to the polymeric substrate.

17. 16. The method of claim 15, wherein the polymeric substrate is selected from expanded polytetrafluoroethylene (ePTFE), polyvinyl chloride (PVC), PGA (polyglycolic acid), PLA (polylactic acid), PCL (polycaprolactone), PGLA (polylactic-co-glycolic acid), polyurethane, polydioxanone, polyethylene, polyethylene terephthalate Dacron®, tetrafluoroethylene (TFE), polytetrafluoroethylene (PTFE), silk, decellularized scaffolds, extracellular matrix protein-based scaffolds, hyaluronic acid, chitosan, and polyhydroxyalkanoates.

18. 16. The method of claim 15, further comprising contacting the cell-seeded vascular graft with a cryopreservation solution and freezing the contacted cell-seeded vascular graft.

19. 20. The method of claim 18, wherein freezing comprises storing at a temperature of from 1°C to about -196°C.

20. A method for producing a vascular graft seeded with human arterial endothelial cells (AECs), comprising: (a) degassing a polymeric substrate; (b) coating at least a portion of the polymeric substrate with one or more endothelial cell attachment agents; and (c) contacting the polymeric substrate with human arterial endothelial cells to form a vascular graft seeded with human AECs that are substantially non-adherent to leukocytes or cell fragments thereof; The method includes:

21. 21. The method of claim 20, wherein the polymeric substrate is selected from expanded polytetrafluoroethylene (ePTFE), polyvinyl chloride (PVC), PGA (polyglycolic acid), PLA (polylactic acid), PCL (polycaprolactone), PGLA (polylactic-co-glycolic acid), polyurethane, polydioxanone, polyethylene, polyethylene terephthalate Dacron®, tetrafluoroethylene (TFE), polytetrafluoroethylene (PTFE), silk, decellularized scaffolds, extracellular matrix protein-based scaffolds, hyaluronic acid, chitosan, and polyhydroxyalkanoates.

22. 21. The method of claim 20, wherein the degassing comprises washing the polymeric substrate in acetone and ethanol, washing the polymeric substrate in an organic solvent, or applying a vacuum to the polymeric substrate.

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