Methods and compositions for endothelializing grafts

Abstract

This disclosure describes methods and compositions for initiating rapid endothelialization of a graft within days using autologous adipose-derived microvessel fragments. This disclosure also describes methods and compositions for endothelializing a graft.

Description

[0001] F&RRefNo.: 09531-0499WO1

[0002] METHODS AND COMPOSITIONS FOR ENDOTHELIALIZING GRAFTS

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Application Serial No. 63 / 746,766, filed on January 17, 2025. The disclosure of the prior application is considered part of the disclosure of this application, and is incorporated in its entirety into this application.

[0004] TECHNICAL FIELD

[0005] This disclosure generally relates to methods and compositions for endothelializing cardiovascular grafts.

[0006] BACKGROUND

[0007] One major hurdle to successful cardiovascular grafts is vascular patency, since a sufficient coverage of the graft by endothelial cells (ECs) is necessary to prevent thrombosis and provide proper vascular function after in vivo transplantation. Therefore, methods of improving endothelialization would significantly benefit the development of graft organs and tissues, particularly methods based on an immediate and easily accessible source of autologous ECs in a number that is sufficient to cover the luminal surface of a coronary bypass graft for urgent / emergent cases. Autologous ECs are essential at present since allogeneic ECs are among the most immunogenic cell types and lead to rapid rejection if from a donor.

[0008] SUMMARY

[0009] Methods and compositions are described for initiating rapid endothelialization of a graft within days using autologous adipose-derived microvessel fragments. This disclosure also describes methods and compositions for endothelializing a graft.

[0010] In one aspect, a method of initiating endothelialization of a cardiovascular graft with patient-derived endothelial cells is provided. Such a method typically includes contacting a cardiovascular graft ex vivo with isolated microvessel fragments under conditions appropriate for growth of endothelial cells, or singularized cells derived from the isolated microvessel fragments that includes endothelial cells and perivascular cells,F&RRefNo.: 09531-0499WO1

[0011] wherein the microvessel fragments are isolated from tissue obtained from the patient. In some embodiments, such a method takes 2-7 days.

[0012] In some embodiments, the tissue is adipose tissue. In some embodiments, the adipose tissue is obtained from the patient using liposuction. In some embodiments, the microvessel fragments are isolated from the adipose tissue using sequential enzy matic digestion-mechanical filtration steps.

[0013] In some embodiments, the cardiovascular graft is selected from a vascular graft and a peripheral bypass graft. In some embodiments, the cardiovascular graft is an AV graft, a heart valve, a vein valve, or another blood-contacting implant.

[0014] In another aspect, a method is provided. Such a method typically includes obtaining tissue from a subject; isolating microvessel fragments from the adipose tissue; and seeding a cardiovascular graft with the isolated microvessel fragments under conditions conducive for outgrowth of endothelial cells, thereby producing an endothelialized cardiovascular graft. In some embodiments, such a method further comprises transplanting the endothelialized cardiovascular graft into the subject.

[0015] In some embodiments, the method takes place over 2-7 days.

[0016] In some embodiments, the microvessel fragments are intact, or partially or completely singularized using enzymatic digestion.

[0017] In some embodiments, the tissue is adipose tissue. In some embodiments, the adipose tissue is obtained from the patient using liposuction or other surgical procedure. In some embodiments, the isolating step comprises enzy matic digestion-mechanical filtration steps. In some embodiments, the method optionally includes additional cycles of filtration of the microvessel fragments to remove associated singular cells. In some embodiments, the isolating step further comprises an optional final enzymatic treatment. In some embodiments, the method includes one or more optional steps of recycling undigested adipose for additional cycles of enzymatic digestion-mechanical filtration.

[0018] In still another aspect, a method of isolating microvessel fragments from adipose tissue is provided. Such a method typically includes providing the adipose tissue; digesting the adipose tissue with collagenase to produce a cell suspension; filtering the cell suspension through a sieve having a pore size as needed for the filtrate to include the microvessel fragments and exclude undigested adipose tissue, typically greater than 80 pm and less than 500 pm, thereby producing a cellular pellet from the filtrate; resuspending the cellular pellet and filtering the resuspended cellular pellet through aF&RRefNo.: 09531-0499WO1

[0019] sieve to filter out the microvessel fragments, typically having a pore size greater than 25 pm and less than 35 pm to produce a filtrate and a retentate; and recovering the retentate, wherein the retentate comprises microvessel fragments and associated singular cells, thereby isolating microvessel fragments and associated singular cells from adipose tissue.

[0020] In some embodiments, the undigested adipose tissue and filtrate are recycled for another such filtration, and the microvessel fragments and associated singular cells from each cycle are combined. In other embodiments, the microvessel fragments are subjected to one or more filtrations to remove associated singular cells prior to seeding the cardiovascular graft.

[0021] In some embodiments, the providing step comprises harvesting the adipose tissue. In some embodiments, the graft is pretreated with selected cell adhesion- and growthpromoting proteins such as fibronectin and / or other biomolecules. In some embodiments, the vasculogenic grow th medium further comprises D-valine or other factors to suppress grow th of any contaminating cells other than the endothelial cells and mural cells from the microvessel fragments.

[0022] In some embodiments, the method further comprises mincing the adipose tissue. In some embodiments, the method further comprises removing fat from the cell suspension. In some embodiments, further comprising contacting the treated retentate with an enzyme. In some embodiments, the method further comprises resuspending the retentate in vasculogenic growth medium and applying the treated retentate to a cardiovascular graft.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods and compositions of matter belong. Although methods and matenals similar or equivalent to those described herein can be used in the practice or testing of the methods and compositions of matter, suitable methods and materials are described below . In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0024] DESCRIPTION OF DRAWINGS FIG. 1 show s a schematic of the process for isolating the microvascular niche (MVN) described herein.F&RRefNo.: 09531-0499WO1

[0025] FIGs. 2A-2L are photographs showing the outgrow th of MVN on tissue-engineered CABG.

[0026] FIGs. 3A-3N are plots showing flow cytometry characterization of MVN and SVF. As described in more detail below, “MVN” refers to digested adipose tissue, fdtered to remove large and small debris to generate a microvessel-rich population of cells, while “SVF” refers to digested adipose tissue with a single fdlration to remove large debris (e.g., 400 pm).

[0027] FIGs. 4A-4B are images showing cell coverage of static and sheared graft samples with MVFO.

[0028] FIGs. 5A-5C shows vWF expression in MVN outgrowth replicates.

[0029] FIGs. 6A-6C shows the analysis of percent coverage of the tissue-engineered vascular graft surface in areas of cell colony coverage vs. elsewhere when seeded with ovine blood outgrowth endothelial cells (oBOECs), microvascular niche (MVN), MVN seeded at double the MVFs / cm2, or accutase-singularized MVN. FIG. 6A indicates that the presence of MVN cell colonies, which are CD31+ (data not shown), reduces platelet binding in such areas as compared to non-covered graft, yet not to the extent of oBOECs. FIGs. 6B - 6C are representative images from samples seeded with MVN (6B) and singularized MVN (6C) after exposure to platelets following three days of outgrowth on the tissue-engineered vascular graft surface.

[0030] FIGs. 7A-7J are images showing cell coverage based on vWF+ expression.

[0031] FIG. 8 A shows that CD31+ coverage is indicative of functional endothelial cells (i.e. platelet resistant, relevant to an anti-thrombogenic cardiovascular implant as shown in FIG. 6).

[0032] FIG. 8B shows that the coverage of a tissue-engineered vascular graft surface increases for dfMVN (double-filtered MVF) relative to MVN and singularized MVN.

[0033] FIG. 9 shows electron micrographs of MVFs (black arrows) in MVN (top) and purified MVFs (black arrow s) following double filtration of the MVN (dfMVN) (bottom).

[0034] DETAILED DESCRIPTION

[0035] This disclosure describes novel compositions and methods to initiate endothelialization and to endothelialize cardiovascular grafts. This disclosure describes compositions and methods used to process a patient’s adipose into a population referred to as a microvascular niche (MVN) that can be used to coat cardiovascular grafts within days of a diagnosis. The compositions and methods described herein are especiallyF&RRefNo.: 09531-0499WO1

[0036] relevant to emergent coronary artery bypass graft (CABG) cases. Endothelialization of a biologically-engineered graft is described, with the graft possessing resistance to cell detachment under physiological shear flow, in multiple adipose isolations.

[0037] The methods described herein allow for the rapid (e.g., within days) creation of potentially hemocompatible, small-diameter vascular grafts using autologous cells obtained from a minimally-invasive liposuction procedure. One feature of the method is the novel use of microvessel fragments (MVFs). linear sections of microvessels isolated from a patient’s adipose using the methods described herein, which allows for the natural outgrowth of EC and support cells of the perivascular niche, such as ASC (aka pericytes), following MVN seeding on the luminal surface of a vascular graft and subsequent cell migration outward from the MVFs concurrent with their proliferation and that of the MVF-associated single cells in the MVN. The method described herein is in contrast to adipose digestion and ‘'sodding” of the entire stromal vascular fraction onto a graft, which has no EC purification (see, e.g., Williams et al., 2017, Tissue Eng. Part C Methods, 23(8):497-504) or ECs completely purified from the stromal vascular fraction (using, e.g., immunobead isolation, which excludes support cells and may not be practical in the clinic).

[0038] Also described herein is an optional digestion of the MVN prior to seeding and an optional short-term conditioning of the outgrowth cells on the seeded graft using a fluid shear stress regimen to further promote a shear-resistant anti-thrombogenic phenotype prior to the implantation step.

[0039] The method described herein allows for the rapid and efficient creation of prosthetic vascular grafts for coronary and peripheral artery bypass that can otherwise fail from occlusive clotting, even with chronic anti-coagulation therapy. It has been estimated that 60% of bypass patients lack available autologous vessels (mammary and radial arteries, saphenous veins) for grafting due to prior use or unsuitability due to atherosclerotic lesions (Schmedlen et al., 2003, Clin. Plast. Surg., 30(4):507-17). While no synthetic material has succeeded as a small-diameter (< 4 mm diameter) prosthetic bypass graft for these patients, several tissue-engineered grafts (e.g., Humacyte and Vascudyne) are showing clinical promise to fill this unmet need. However, neither offers an alternative to chronic anti-coagulation therapy if hemocompatibility does not occur via spontaneous endothelialization. The method described herein would solve these problems. Also, the failure of synthetic materials as vascular grafts can be remedied withF&RRefNo.: 09531-0499WO1

[0040] the endothelialization compositions and methods described herein, and the endothelialization compositions and methods described herein also may prove necessary for success of decellularized animal vessels or cadaver vessels used as vascular grafts.

[0041] The clinical distinction and potential impacts of this disclosure are dramatic: an endothelialized graft within days rather than months. For example, endothelial progenitor cells in blood require months to expand to the required cell number for a bypass graft and, similarly, for endothelial cells differentiated from induced pluripotent stem cells (iPSCs) obtained from a patient biopsy.

[0042] While spontaneous endothelialization has been observed in preclinical studies, they were conducted with young, healthy animals under sustained anti-coagulation to ensure occlusive clotting would not be a failure mode. It is unknown when or if spontaneous endothelialization will occur, for example, in elderly patients with atherosclerosis and co-morbidities, especially in the low-flow-rate settings of bypass grafts and in the absence of anti-coagulation therapy. If spontaneous endothelialization does not occur, lifelong anti-coagulation could be required, which has substantial associated morbidity.

[0043] Adipose is composed of an abundance of MVFs as well as fat cells and can be readily obtained using minimally-invasive methods in sufficient quantities.

[0044] As used herein, microvessels refer to intact capillary -like vessels found in adipose tissue and MVFs refer to segments of those vessels resulting from adipose processing as described herein, respectively. Representative MVFs generally include multicellular structures, predominantly interconnecting endothelial cells that form a tube and support cells like pericytes that wrap around the endothelial tubes. MVFs contained within the MVN average about 40 pm to about 100 pm in width (with occasional MVFs up to about 200 pm) and about 100 pm to about 600 pm in length (with occasional MVFs up to about 3 mm). Notably, the average size of MVFs in the double-fdtered MVN (dfMVN) described herein does not change significantly.

[0045] As used herein, “endothelialization” refers to the formation of adherent endothelial cells by natural processes in vivo or by technological means in vitro.

[0046] Endothelialization can refer to the production of a complete monolayer of endothelial cells, potentially in conjunction with perivascular cells in a multi-layer, and endothelialization also can refer to the production of a partial or incomplete monolayer of endothelial cells. In addition, initiating endothelialization. as used herein, refers to theF&RRefNo.: 09531-0499WO1

[0047] early presence of endothelial cells, even prior to formation of a monolayer. Further, endothelialization that yields an incomplete monolayer in vitro can result in a complete monolayer in vivo following implantation into the vascular system.

[0048] As used herein, “graft” typically refers to a cardiovascular graft. Cardiovascular grafts include, without limitation, vascular grafts (e.g., a coronary or peripheral bypass graft, or an arterio-venous (AV) graft), a heart valve, a vein valve, or any other bloodcontacting implant. Grafts as described herein can be biologically-engineered (e.g.. the Humacyte and Vascudyne vascular grafts grown from cells in vitro and then decellularized), synthetic (e.g., GoreTex vascular grafts), or cadaver or animal tissue that is chemically treated (e.g., decellularized cadaver arteries or glutaraldehyde-crosslinked porcine arteries).

[0049] As used herein, “conditions appropriate for growth of endothelial cells” includes a vasculogenic grow th medium, which typically includes one or more endothelial-specific growth factors, such as Endothelial Growth Factor (EGF) and basic Fibroblast Growth Factor (bFGF), in addition to a basal medium. In some instances, the conditions appropriate for providing a growth advantage of endothelial cells includes the presence of D-valine, which inhibits the growth of fibroblasts that contaminate the isolated MVFs.

[0050] Exemplary Description of Protocol

[0051] Briefly, one end of the tube is connected to the barbed Luer fitting and secured using a suture. The required length is measured, the remaining material is cut off, and the other end is connected to the Luer fitting and tightened using a suture. A square rod is used to hold the tube in place, and the tube is secured using small cable ties on both ends. The required concentration of coating solution is injected carefully without creating bubbles, the tube is capped, inverted and the syringe slowly removed. The other end is capped and the assembled tube is placed in a 50 mL tube filled with lx DPBS. After injecting the cell suspension, the tube is placed in a cell roller for 4 hrs at 37C, after which both caps are removed and the tube is immersed in Vasculife medium in the incubator for the remainder of the culture.

[0052] Specifically, the day before the MVN isolation, a tube of the biologically-engineered matrix or a punch biopsy therefrom is prepared as follows.

[0053] Autoclave one sharp scissor, one dental pick, one thin forceps, one thick forceps, one long forceps, and one surgical blade handle (#4).F&RRefNo.: 09531-0499WO1

[0054] • Wipe down a hood with ethanol and place a sterile drape in the hood.

[0055] Place the following items on the drape:

[0056] - autoclaved tools;

[0057] - petri dish with sterile PBS;

[0058] - sterile syringe to add / remove PBS;

[0059] - sterile biopsy punch (6 mm);

[0060] - 96 well plate.

[0061] • Using long forceps, remove the tube from the container and place it on the petri dish with PBS.

[0062] • Open the tube carefully (e.g., using scissors).

[0063] • Lay the tube flat with the lumen (inner side) facing upwards.

[0064] • Make a 6 mm circular sample by pressing a punch into the flattened tube wall for ~ 10 seconds, ensuring that all sides are cut.

[0065] • Carefully remove the punch biopsy with the lumen side still facing up and place it in a 96-well plate well bottom using the dental pick, ensuring that the lumen side is still facing up.

[0066] • Coat the punch biopsies with fibronectin (see below). Alternatively, gelatin or laminin can be used as a coating.

[0067] Fibronectin Coating:

[0068] • Prepare 10 pg / mL fibronectin in IX PBS (i.e., with a stock solution concentration of 1 mg / mL).

[0069] • Add 150 pl per well and incubate overnight at 4°C. Prior to plating, remove the fibronectin solution.

[0070] Autoclave Pack

[0071] • Two surgical blade handles #4

[0072] • Two scoopulas

[0073] • One blunt forceps

[0074] • One Jeweler’s forceps

[0075] • One Tissue Adson-Brown (aka Adson’s)

[0076] • One Adson's with teeth

[0077] • One long forceps (10 inch)

[0078] • One pair of scissorsF&RRefNo.: 09531-0499WO1

[0079] • Dental picks if desired (if coating tissue, keep these sterile or autoclave separately to use in case tissue floats)

[0080] Media and Solutions

[0081] • VascuLife VEG-Mv Endothelial Growth Media (Complete Kit, Lifeline Cell Technology, Cat. No. LL-0005)

[0082] o Supplemented with 1% Anti-Anti + 0.5% Amphotericin B.

[0083] • HBSS(-)(-)(-) (no Ca, no Mg)

[0084] • HBSS(-)(-)(-) + 4% BSA (BSA = Sigma A1470)

[0085] • Cosmic Calf Serum for neutralizing the collagenase

[0086] • EIBSS(-)(-)(-) + 6 mg / mL BSA + 6 mg / mL collagenase.

[0087] o Sterile fdter 0.22 pm

[0088] • HBSS(+)(+) (+Ca, +Mg)

[0089] • lx PBS + 1% Anti-Anti + 0.5% Amphotericin B

[0090] • DMEM + 10% Cosmic Calf Serum + 1% Anti -Anti + 0.5% Amphotericin B

[0091] • Accutase

[0092] Tissue Collection

[0093] • Prepare sterile sample collection cups approximately half full with lx PBS + 1% Anti -Anti + 0.5 Amphotericin B. Bring to adipose supplier prior to sac date and have them store at 4°C.

[0094] • Bring container for transport.

[0095] Same Dav Preparations

[0096] • Warm collagenase to room temperature. Do not open until completely warmed to avoid vapor condensing from air, which will damage the enzyme.

[0097] • Warm media to room temperature.

[0098] Tissue Processing

[0099] All steps are done in tissue culture hood separated from other samples until verified mycoplasma negative.

[0100] • Rinse tissue with lx PBS + 1% Anti-Anti + 0.5% Amphotericin B until tissue is white and rinse is colorless.

[0101] • Transfer tissue to petri dish, add small amount of HBSS- + 4% BSA (HBSS4) and mince.F&RRefNo.: 09531-0499WO1

[0102] • Transfer minced tissue to a digestion vessel and weigh (usually add 360 g).

[0103] • Add equal volume HBSS(+)(+)

[0104] • Add equal volume 6 mg / ml collagenase

[0105] • Digest in incubator using rotating mixer plus more vigorous mix about every 30 min. Digestion is stopped after 4 hours

[0106] • Tissue will be mostly digested, leaving white fat globs.

[0107] • Pass sample through 400 pm cell strainer

[0108] • Add 10% Cosmic Calf Serum to the filtrate to neutralize the collagenase • Rinse the remaining adipose tissue with DMEM + 10% Cosmic Calf Serum + 1% Anti-Anti + 0.5% Amphotericin B. Filter and collect through a 400 pm cell strainer

[0109] • Aliquot all filtrate into 50 mL conicals, centrifuge each at 200xg for 10 min, remove liquid layer to about 5 ml above pellet, resuspend pellets and combine. Usually results in four 50 mL conicals of concentrated filtrate • Centrifuge each conical at 200xg for 10 min. Remove liquid layer to about 5 ml above pellet and resuspend.

[0110] • MVN Enrichment:

[0111] i. Place a 30 pm filter (pluriSelect, Cat. No. 43-50030-01) on a 50 cc conical tube

[0112] ii. Place filter on a 50 cc conical with the narrower port down. Run resuspended pellet through the 30 pm filter, discard flow through = Filtrate

[0113] iii. Flip filter into a clean 50 cc conical

[0114] iv. Run supplemented Vasculife medium through filter and rinse the surface of the filter (25 mL total) to wash MVN into conical = Retentate

[0115] v. Centrifuge Retentate at 200xg for 10 min to concentrate. Check for MVFs under the microscope, counting for MVFs / mL. vi. For Double Filtered MVN (dfMVN). repeat steps i - v

[0116] vii. Resuspend retentate in 1 mL of supplemented Vasculife medium.

[0117] Check for MVFs under the microscope, counting for MVFs / mL.F&RRefNo.: 09531-0499WO1

[0118] viii. Partial Digestion of MVFs: to concentrated pellet of MVN with all supernatant removed, add 10 mL of 37°C warmed Accutase and incubate at 37°C for 25 mins.

[0119] ix. Plate in fibronectin coated multi-wells. Remove fibronectin solution just prior to use, then add cells. Use 0.20 ml per 96 well at the desired density of MVF s / cm2.

[0120] Tube Seeding Protocol

[0121] • Tubes were washed for a week in lx PBS. The day before cell seeding, the tube is assembled.

[0122] Tube Assembly

[0123] The following items are autoclaved:

[0124] • Two barbed Luer fitting

[0125] • Two caps

[0126] • One adapter for syringe

[0127] • One square rod

[0128] • Cable ties

[0129] • Suture

[0130] • Ruler / scissors / forceps

[0131] Embodiments

[0132] Embodiment 1 is a method of initiating endothelialization of a cardiovascular graft with patient-derived endothelial cells, comprising: contacting a cardiovascular graft ex vivo with isolated microvessel fragments or singularized cells derived from the isolated microvessel fragments that includes endothelial cells and perivascular cells under conditions appropriate for growth of endothelial cells, wherein the microvessel fragments and associated singular cells are isolated from tissue obtained from the patient.

[0133] Embodiment 2 is the method of Embodiment 1, wherein the method takes between about 1 and about 7 days.

[0134] Embodiment 3 is the method of Embodiment 1 or 2, wherein the microvessel fragments are intact, or partially or completely singularized using enzymatic digestion.

[0135] Embodiment 4 is the method of any one of Embodiments 1-3, wherein the tissue is adipose tissue.F&RRefNo.: 09531-0499WO1

[0136] Embodiment 5 is the method of Embodiment 4, wherein the adipose tissue is obtained from the patient using liposuction, adipose resection, or other surgical procedure.

[0137] Embodiment 6 is the method of Embodiment 4 or 5, wherein the isolated microvessel fragments and associated singular cells are obtained from the adipose tissue using enzymatic digestion-mechanical filtration steps, with optional recycling of undigested adipose for additional cycles of enzymatic digestion-mechanical filtration steps and with optional additional cycles of fdtration of the microvessel fragments to remove associated singular cells.

[0138] Embodiment 7 is the method of any one of the preceding Embodiments, wherein the cardiovascular graft is selected from vascular grafts (e.g., a coronary or peripheral bypass graft, or an arterio-venous (AV) graft), a heart valve, a vein valve, or any other blood-contacting implant.

[0139] Embodiment 8 is the method of any one of the preceding Embodiments, w herein the cardiovascular graft is biologically-engineered, non-biologically engineered, or cadaver or animal tissue that is chemically treated.

[0140] Embodiment 9 is the method of any of the preceding Embodiments, wherein, after the contacting step, the cardiovascular graft is resistant to platelet adhesion.

[0141] Embodiment 10 is a method, comprising: obtaining tissue from a subject; isolating microvessel fragments and associated singular cells from the adipose tissue; and seeding a cardiovascular graft with the isolated microvessel fragments and associated singular cells under conditions conducive for outgrowth of endothelial cells, thereby initiating an endothelialized cardiovascular graft.

[0142] Embodiment 11 is the method of Embodiment 10, further comprising transplanting the endothelialized cardiovascular graft into the subject.

[0143] Embodiment 12 is the method of Embodiment 10 or 11, wherein the method takes place over 1 -7 days.

[0144] Embodiment 13 is the method of any one of Embodiments 10-12, wherein the tissue is adipose tissue.

[0145] Embodiment 14 is the method of Embodiment 13, wherein the adipose tissue is obtained from the patient using liposuction or other surgical procedure.

[0146] Embodiment 15 is the method of any one of Embodiments 10-14, wherein the isolating step comprises sequential enzymatic digestion-mechanical filtration steps.F&RRefNo.: 09531-0499WO1

[0147] Embodiment 16 is the method of any one of Embodiments 10-15, wherein the isolating step further comprises an optional final enzymatic treatment.

[0148] Embodiment 17 is the method of any one of Embodiments 10-16, wherein, after the seeding step, the cardiovascular graft is resistant to platelet adhesion.

[0149] Embodiment 18 is a method of isolating micro vessel fragments from adipose tissue, comprising: providing the adipose tissue; digesting the adipose tissue with collagenase or similar enzyme to produce a cell suspension; filtering the cell suspension through a sieve having a pore size as needed for the filtrate to include the microvessel fragments and exclude undigested adipose tissue, typically greater than 20 pm and less than 120 pm, thereby producing a cellular pellet from the filtrate; resuspending the cellular pellet and filtering the resuspended cellular pellet through a sieve to filter out the microvessel fragments, typically having a pore size greater than 25 pm and less than 35 pm to produce a filtrate and a retentate; and recovering the retentate, wherein the retentate comprises microvessel fragments, thereby isolating microvessel fragments from adipose tissue.

[0150] Embodiment 19 is the method of Embodiment 18, wherein the providing step comprises harvesting the adipose tissue.

[0151] Embodiment 20 is the method of Embodiment 18 or 19, further comprising mincing the adipose tissue.

[0152] Embodiment 21 is the method of any one of Embodiments 18-20, further comprising removing fat from the cell suspension.

[0153] Embodiment 22 is the method of any one of Embodiments 18-21, further comprising resuspending the retentate in vasculogenic growth medium and applying the treated retentate to a cardiovascular graft.

[0154] Embodiment 23 is the method of Embodiment 22, wherein the cardiovascular graft is pretreated with selected cell adhesion- and growth-promoting proteins such as fibronectin and / or other biomolecules.

[0155] Embodiment 24 is the method of Embodiment 22, wherein the vasculogenic growth medium further comprises D-valine or other factors to suppress growth of any contaminating fibroblasts among the endothelial cells and mural / support cells from the microvessel fragments.F&RRefNo.: 09531-0499WO1

[0156] Embodiment 25 is the method of any one of Embodiments 22-24, further comprising conditioning the endothelialized cardiovascular graft prior to implantation using fluid shear flow.

[0157] Embodiment 26 is the method of any one of Embodiments 22-25, wherein the endothelialized cardiovascular graft is resistant to platelet adhesion.

[0158] In accordance with the present invention, there may be employed conventional molecular biology, microbiology, biochemical, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. The invention will be further described in the following examples, which do not limit the scope of the methods and compositions of matter described in the claims.

[0159] EXAMPLES

[0160]

[0161] Isolation

[0162] FIG. 1 is a schematic of the MVN isolation process. The adipose-derived microvascular niche (MVN) seeded on a tissue-engineered coronary artery bypass graft (CABG) material is isolated via the method described herein. Subcutaneous adipose is resected from the midsection of a recently sacrificed sheep, rinsed w ith PBS (+ 1% Anti / Anti, + 0.5% Amphotericin B), minced manually into ~1 cm2pieces in a solution of 4% BSA in HBSS(-)(-)(-), and added to a solution of collagenase (6 mg / mL collagenase + 6 mg / mL BSA in HBSS(-)(-)(-)) and HBSS(+)(+) at a volume ratio of 1 : 1 : 1, tissue : collagenase : HBSS(+)(+). This digestion solution is incubated at 37°C for an initial 60 minutes before starting a cycle of intermediate filtrations. A single intermediate filtration includes the sequential use of 400 pm and 30 pm reusable filters to retain all microvessels dissociated from the adipose at a size between 400 pm and 30 pm. All retentate is rinse-removed from the 30 pm filter with a solution of DMEM-C (+ 10% Cosmic Calf Serum (CCS), + 1% Anti / Anti, + 0.5% Amphotericin B) and stored on ice. Intermediate filtrations take place every 30 minutes after the initial, incubating at 37°C in-between, until either the reusable 30 pm filter is impermissibly clogged with fatty particulate or a total of 3.5 h of incubation time is reached. If the filter clogs before the total incubation is reached, the adipose is incubated without intermediate filtration for the remaining time until 3.5 h is reached.F&RRefNo.: 09531-0499WO1

[0163] After the total incubation time, all digestion solution is filtered through the 400 pm filter to exclude all larger material and collected in 50 mL coni cals (a sample of this solution is considered the SVF) with 10% CCS to quench enzymatic activity. Each SVF conical is centrifuged, combined to one, and filtered through a final 30 pm filter, again collecting all material >30 pm (MVN). The retentate is again rinse-removed from the filter with supplemented DMEM-C and then combined with the retentate from the intermediate filtration steps.

[0164] The remaining adipose slurry is rinsed again with DMEM-C to dislodge more cells and microvessels still available for harvest from the adipose. The solution is filtered through a 400 pm filter into 50 mL conicals to collect all rinsing liquid and cellular material. Each conical is centrifuged, combined to one, and filtered through a 30 pm filter, again collecting all material >30 pm via rinse-removal (MVN). This retentate material is combined with the other retentates to obtain the complete MVN which will be centrifuged and seeded at a desired concentration on a tissue-engineered CABG material.

[0165] This process may be improved via digestion times longer than 3.5 hrs as complete digestion of the adipose had not been achieved. Another improvement would be the use of larger conicals for centrifugation to minimize time constraints associated with the current volume of conicals to combine. The process is ultimately aimed for use on a liposuction sample, therefore future experimentation should utilize such samples for similar efficacy.

[0166] MVN was isolated from a digestate of subcutaneous ovine adipose via sequential 400 pm and 30 pm filtration steps.

[0167] Example 2 — Growth of Microvascular Niche (MVN) on Engineered Tissue

[0168] FIG. 2 shows MVN outgrowth on tissue-engineered CABG. Adipose-derived MVN was seeded onto fibronectin-coated TCP (FIGs. 2A-2C) and 6 mm tissue-engineered CABG punchouts (FIGs. 2D-2L) at a concentration of -100 MVFs / cm2, culturing for 7 days in Vasculife endothelial growth media. The images depict the fixed and immunostained samples on D7 from the third replicate isolation conducted.

[0169] Immunocytochemistry analysis includes a secondary antibody control (first column; FIGs. 2A, 2D, 2G, and 2J) as well as stains for vWF+aSMA (second column; FIGs. 2B, 2E, 2H. and 2K) and vWF+CD31 (third column; FIGs. 2C, 2F, 21, and 2L). TCP images were performed at 20x magnification (FIGs. 2A-2C). Tile scan images are presented forF&RRefNo.: 09531-0499WO1

[0170] each staining condition on CABG punchouts, performed at 4x magnification (FIGs. 2D-2F), with higher magnification images provided at 20x (FIGs. 2G-2I) and 63x (FIGs. 2J-2L). All color and brightness thresholding is identical across staining conditions at each respective magnification (within each row). Blue = Hoechst 33342, Green= aSMA (second column) or CD31 (third column), Red = vWF. *Note: graft samples in the first and third columns are from Lot 2 while samples in the second column are from Lot 3.

[0171] Adipose-derived MVN adheres to tissue-engineered CABG punchouts and forms a population of vWF+ / aSMA+ / CD31- endothelial cells when cultured in EGM for 7 days

[0172] 3 — Characterization of Microvascular Niche

[0173]

[0174] The adipose-derived SVF and MVN were characterized for endothelial identity via flow cytometry. (FIGs. 3A-3C) Representative gates to narrow analysis to only include (FIG. 3A) singlet (FIG. 3C) cells while excluding dead matter (FIG. 3B) for all samples. Representative panels are given characterizing a secondary' only control (FIGs.

[0175] 3D. 3G, 3J), SVF (FIGs. 3E, 3H. 3K), and MVN (FIGs. 3F, 31, 3L) within a single isolation for CD31+ identity (FIGs. 3D-3F), vWF+identity (FIGs. 3G-3I), and for CD31 and vWF+dual staining signatures (FIGS. 3J-3L). (FIGs. 3M-3N) Graphical representation comparing CD31+and vWF+cell proportions (respectively) between SVF and MVN. A one-tailed, ratio paired t-test was conducted for each plot (p < 0.05*).

[0176] The simple filtration method presented here increases the proportion of endothelial cells (vWF+and CD31+) in the seeded MVN in comparison to SVF, a population used previously for vascular graft endothelialization, acting as an endothelial purification of the seeded population.

[0177] Example 4 — Comparison of SVF and MVF

[0178] Table 1 describes the results of a comparison of cell identity in SVF and MVF. Data obtained from flow cytometry' is shown, denoting the proportion of endothelial cells present in the SVF and MVF fractions as characterized by the markers vWF and CD31. Cells were characterized across three isolations (n=3). gated based on a single live / dead experiment. Average percent difference in cell proportions was calculated as an average of the individual percent differences between fractions (MVF vs. SVF) within each isolation.F&RRefNo.: 09531-0499WO1

[0179] Table 1

[0180]

[0181] In comparison to SVF, MVF isolated here doubles the proportion of vWF+ cells in the seeded cell population and increases the CD31+ proportion by almost seven-fold.

[0182] Example 5 — Shear-Conditioning Protocol Summary

[0183] Short term (12-36 hr) physiological fluid shear flow exposure of an endothelialized graft (e.g., 15 dyne / cm2laminar shear stress) is known to induce an anti- thrombogenic phenotype of endothelial cells as well as demonstrate “shear resistance” of the cells to detachment from the surface.

[0184] To prepare surfaces for shearing, a graft prepared with MVN outgrowth (MVNO), cut the 6 mm-diameter graft tubes into flat -6 mm squares. Embed the squares in 4% agarose in the custom stainless-steel slides so that the luminal surface of graft is fully exposed. Using clip-on wells, coat the graft surfaces with 10 pg / mL fibronectin overnight at 4°C.

[0185] Isolate ovine adipose MVN (OAMV) according to the standard protocol, then seed at ~100 MV / cm2. Culture the OAMV to confluence in HAMEC grow th medium (usually -7-10 days) with a 75% medium change every 2-3 days. When the MVNO reaches confluence, load half of the samples into a Flexcell® Streamer® system modified to hold slides and ramp the shear from 0 to 15 dyn / cm2over 12 hours, then hold 15 dyn / cm2for another -12 hours. Give the other half of the samples a 100% medium change and keep them under static conditions.

[0186] When the shearing is complete, immediately fix all the samples for staining and imaging.

[0187] Example 6 — Characterization of Sheared Microvascular Niche (MVN)

[0188] FIG. 4 are images showing cell coverage of static and sheared graft samples with MVNO. (Blue = Hoechst 33342). (FIG. 4A) Static graft sample with confluent MVNO.F&RRefNo.: 09531-0499WO1

[0189] (FIG. 4B) Graft sample w ith MVNO after 24 h of shear. Graft samples used in this image are from Lot 4.

[0190] The population of cells grown from MVN on tissue-engineered CABG samples is retained under physiologic shear conditions.

[0191] Example 7 — Microvascular Niche (MVN) Outgrowth

[0192] FIG. 5 shows vWF expression in MVN outgrowth (MVNO) replicates. Three replicate adipose-derived MVF outgrowth experiments were conducted on three separate lots of tissue-engineered CABG. Each punchout was seeded with ~90 microvessels / cm2and cultured for 7 days in EGM. Three replicate experiments are given (FIGs. 5A-5C). For each experiment, a panel of images are included as follows: right=4x tile scan, top left=20x image of outgrowth on graft, middle left=63x image of outgrowth on graft, bottom left=20x image of outgrowth on TCP. Red=vWF; Blue=Hoechst 33342. All vWF brightness and thresholding is identical across experiments at each respective magnification (panel location). Graft lots for each replicate image panel given here are as follows: (FIG. 5A) Lot 1, (FIG. 5B) Lot 2. (FIG. 5C) Lot 3.

[0193] Adipose-derived MVF outgrowth is reproducible across three different lots of tissue-engineered CABG samples in forming a confluent layer of cells, characterized by punctate vWF expression.

[0194] The surface of the tissue-engineered vascular graft was analyzed to determine the percent cell colony coverage w hen seeded with ovine blood outgrowth endothelial cells (oBOECs), microvascular niche (MVN), MVN seeded at double the MVFs / cm2, or accutase-singularized MVN. Data indicated that the presence of MVN cell colonies reduces platelet binding in such areas as compared to non-covered graft, yet not to the extent of oBOECs. FIG. 6A. FIG. 6B shows representative images from samples seeded with MVN and FIG. 6C shows representative images from samples seeded with singularized MVN after exposure to platelets following three days of outgrowth on the tissue-engineered vascular graft surface.

[0195] Example 8 — Endothelial Coverage using Microvascular Niche (MVN)

[0196] FIG. 7 are images showing vWF+ cell coverage analysis. Tile scan 4x images are provided for immunostained CABG punchouts after culturing with adipose-derived MVF for seven days in EGM. Samples are included from each experiment as follows: (FIG.F&RRefNo.: 09531-0499WO1

[0197] 7 A) - isolation 1, graft Lot 1; (FIGs. 7B, 7C) - isolation 2, graft Lots 1 and 2; (FIGs. 7D, 7E, 7F) - isolation 3. graft Lots 2 and 3. Red=vWF; Blue=Hoechst 33342. All vWF brightness and thresholding is identical for images shown in FIGs. 7A-7F. ImageJ was utilized to threshold all individual 4x images for each scan to evaluate the approximate % of vWF+ cells on the CABG surface. An example of the ImageJ workflow is given (FIGs.

[0198] 6G-7J). (FIG. 7G) Images were segmented for an ROI which presents near confluent nuclei. (FIGs. 7H, 71) Images were spliced to isolate the vWF channel, thresholding to remove background and determine all area above a designated background level to be considered vWF+ cell coverage. (FIG. 7J) The binary vWF threshold was combined with ROI segmentation to determine a %vWF+ cell coverage in such ROI. All individual 4x images were combined based on area-weighted average to determine a %vWF+ coverage in a full sample.

[0199] Adipose-derived MVF outgrowth across experiments generates a confluent layer of cells which is approximated to be 72.6 ± 15.7% positive for vWF.

[0200]

[0201] Filtrations

[0202] Double-filtered MVN (dfMVN) were obtained as described herein, contacted with an antibody against CD31, and the CD31+ colony coverage across timepoints (day 3 - day 7) was plotted (FIG. 8A). CD31+ coverage is indicative of functional endothelial cells (i.e. platelet resistant, relevant to an anti-thrombogenic cardiovascular implant) (FIG. 8B). These data indicate that the coverage of a tissue-engineered vascular graft surface increases when dfMVN (purified MVF) is used relative to MVN or singularized MVN.

[0203] FIG. 9 are electron micrographs showing MVFs (black arrows) in the MVN (top) and purified MVFs (black arrows) following double filtration (dfMVN) (bottom). As can be seen from FIG. 9, one additional filtration using a 30 pm filter virtually eliminated the single cells within the MVN.

[0204] Example 10 — Evaluating Patency of a MV-Seeded Bvpass Graft in a Sheep Carotid Arterv

[0205] Three sheep are studied with bilateral interposition grafting in the common carotid artery using 6 mm diameter tubes of biologically-engineered matrix. This is performed at APS, which has previously performed these specific implantations. Each sheep isF&RRefNo.: 09531-0499WO1

[0206] implanted with an autologous MV-seeded, shear-conditioned graft (Experimental group), with an identical, unseeded graft on the contralateral side serving as the paired control. The grafts are evaluated with ultrasound at 4 wks and explanted at 8 wks following angiography. Histology (H&E, Masson’s Trichrome) and immunohistochemical analysis of key markers for endothelium (vWF, VE-cadherin), intact basement membrane (laminin, fibronectin, collagen IV), and platelet adhesion (GpIIb / IIIa) of explanted grafts are compared between the MV-seeded and unseeded grafts. Together, these results demonstrate robust efficacy of the method proposed herein to achieve rapid endothelialization of bypass grafts.

[0207] It is to be understood that, while the methods and compositions of matter have been described herein in conjunction with a number of different aspects, the foregoing description of the various aspects is intended to illustrate and not limit the scope of the methods and compositions of matter. Other aspects, advantages, and modifications are within the scope of the following claims.

[0208] Disclosed are methods and compositions that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that combinations, subsets, interactions, groups, etc. of these methods and compositions are disclosed. That is, while specific reference to each various individual and collective combinations and permutations of these compositions and methods may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular composition of matter or a particular method is disclosed and discussed and a number of compositions or methods are discussed, each and every’ combination and permutation of the compositions and the methods are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed.

Claims

F&RRefNo.: 09531-0499WO1WHAT IS CLAIMED IS:

1. A method of initiating endothelialization of a cardiovascular graft with patient-derived endothelial cells, comprising:contacting a cardiovascular graft ex vivo with isolated microvessel fragments that includes endothelial cells and perivascular cells under conditions appropriate for growth of endothelial cells, wherein the microvessel fragments are isolated from tissue obtained from the patient.

2. The method of claim 1, wherein the method takes between about 1 and about 7 days.

3. The method of claim 1 or 2, wherein the microvessel fragments are intact, or partially or singularized using enzymatic digestion.

4. The method of any one of claims 1-3, wherein the tissue is adipose tissue.

5. The method of claim 4, wherein the adipose tissue is obtained from the patient using liposuction, adipose resection, or other surgical procedure.

6. The method of claim 4 or 5, wherein the isolated microvessel fragments are obtained from the adipose tissue using enzymatic digestion-mechanical filtration steps, with optional recycling of undigested adipose for additional cycles of enzymatic digestion-mechanical filtration steps and with optional additional cycles of filtration of the microvessel fragments to remove associated singular cells.

7. The method of any one of the preceding claims, wherein the cardiovascular graft is selected from vascular grafts (e.g., a coronary or peripheral bypass graft, or an arterio-venous (AV) graft), a heart valve, a vein valve, or any other bloodcontacting implant.F&RRefNo.: 09531-0499WO18. The method of any one of the preceding claims, wherein the cardiovascular graft is biologically-engineered, non-biologically engineered, or cadaver or animal tissue that is chemically treated.

9. The method of any of the preceding claims, wherein, after the contacting step, the cardiovascular graft is resistant to platelet adhesion.

10. A method, comprising:obtaining tissue from a subject;isolating microvessel fragments from the tissue; andseeding a cardiovascular graft with the isolated microvessel fragments under conditions conducive for outgrowth of endothelial cells, thereby initiating an endothelialized cardiovascular graft.

11. The method of claim 10, further comprising transplanting the endothelialized cardiovascular graft into the subject.

12. The method of claim 10 or 11, wherein the method takes place over 1-7 days.

13. The method of any one of claims 10-12, wherein the tissue is adipose tissue.

14. The method of claim 13, wherein the adipose tissue is obtained from the patient using liposuction or other surgical procedure.

15. The method of any one of claims 10-14, wherein the isolating step comprises sequential enzymatic digestion-mechanical filtration steps.

16. The method of any one of claims 10-15, wherein the isolating step further comprises an optional final enzymatic treatment.F&RRefNo.: 09531-0499WO117. The method of any one of claims 10-16, wherein, after the seeding step, the cardiovascular graft is resistant to platelet adhesion.

18. A method of isolating microvessel fragments from adipose tissue, comprising:providing the adipose tissue;digesting the adipose tissue with collagenase or similar enzyme to produce a cell suspension;filtering the cell suspension through a sieve having a pore size as needed for the filtrate to include the microvessel fragments and exclude undigested adipose tissue, typically greater than about 20 pm and less than about 120 pm, thereby producing a cellular pellet from the filtrate;resuspending the cellular pellet and filtering the resuspended cellular pellet through a sieve to filter out the microvessel fragments, typically having a pore size greater than 25 pm and less than 35 pm to produce a filtrate and a retentate; and recovering the retentate, wherein the retentate comprises microvessel fragments, thereby isolating microvessel fragments from adipose tissue.

19. The method of claim 18, wherein the providing step comprises harvesting the adipose tissue.

20. The method of claim 18 or 19, further comprising mincing the adipose tissue.

21. The method of any one of claims 18-20, further comprising removing fat from the cell suspension.

22. The method of any one of claims 18-21, further comprising resuspending the retentate in vasculogenic growth medium and applying the treated retentate to a cardiovascular graft.F&RRefNo.: 09531-0499WO123. The method of claim 22, wherein the cardiovascular graft is pretreated with selected cell adhesion- and growth-promoting proteins such as fibronectin and / or other biomolecules.

24. The method of claim 22, wherein the vasculogenic growth medium further comprises D-valine or other factors to suppress growth of any contaminating fibroblasts among the endothelial cells and mural / support cells from the microvessel fragments.

25. The method of any one of claims 22-24, further comprising conditioning the endothelialized cardiovascular graft prior to implantation using fluid shear flow.

26. The method of any one of claims 22-25, wherein the endothelialized cardiovascular graft is resistant to platelet adhesion.

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