System and method for generating seeded grafts

Abstract

A closed disposable seeding system is provided with an improved seeding chamber that allows for uniform seeding of patient cells into a scaffold or graft. A seeding chamber with variable width along the length of the chamber or minimal gap between the scaffold and the chamber wall improves upon prior closed disposable seeding chambers by providing faster, more efficient, and uniform seeding of grafts and scaffolds. A scaffold is also described that has biomechanical and structural properties that allow spontaneous reversal of stenosis and neotissue formation as the graft degrades, resulting in scaffold-free neovascularization.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims the benefits and priority of U.S. Provisional Application No. 62 / 936,225, filed on November 15, 2019, which is incorporated herein by reference in its entirety.

[0002] Description of research and development funded by the federal government. This invention was made with government support under authorization numbers HL098228, HL128602, HL128847, HL139796, GM068412 from the National Institutes of Health, and W81XWH-18-1-0518 from the Department of Defense. The government has certain rights in this invention.

[0003] Field of Invention The present invention generally relates to tissue engineering grafts, particularly those designed to be seeded with cells and absorbed and replaced by the patient's own tissue, and methods for producing them. [Background technology]

[0004] Background of the Invention Surgical treatment of many complex congenital cardiac malformations involves the implantation of synthetic scaffolds made of materials such as GORE-TEX® and DACRON®. A common example of such application is double vena cava-pulmonary anastomosis (TCPC) for single ventricle malformations. In some cases, the use of these synthetic grafts as scaffolds is complicated by the risk of progressive occlusion, susceptibility to infection, and thromboembolic complications. In all cases, a significant limitation to cardiovascular reconstruction is the lack of growth potential of the synthetic implant. In the case of TCPC, this can lead to suboptimal management strategies, including either delayed completion of Fontan circulation due to patient size or oversized scaffolds, resulting in suboptimal fluid properties such as expiratory regurgitation and areas of flow stagnation.

[0005] Tissue-engineered vascular grafts (TEVGs) offer the potential to overcome these problems by providing a biodegradable scaffold in which autologous cells proliferate and mature into physiologically functional blood vessels as the scaffold polymer degrades (Shin'oka, et al. N. Engl. J. Med. 344(7), 532-533 (2001); Hibino, et al. J Thorac Cardiovasc Surg. 139(2), 431-436.e432 (2010 Roh JD, et al. Biomaterials. 29(10), 1454-1463 (2008); Hibino, et al. FASEB J. 25(8), 2731-2739 (2011); Hibino, et al. FASEB J. 25(12), 4253-4263 (2011); Kurobe, et al. Tissue Eng Part C Methods 21(1), 88-93 (2015)).

[0006] Human clinical trials confirmed the growth potential of TEVG and demonstrated that no grafts were associated with death or graft failure (Shin'oka, et al. J Thorac Cardiovasc Surg. 129(6), 1330-1338 (2005)). However, the results of this study also demonstrated that stenosis was the main graft-related complication, affecting nearly 25% of graft recipients, and that 16% of recipients developed fatal stenosis (a >75% reduction in lumen diameter) (Hibino, et al. J Thorac Cardiovasc Surg. 139(2), 431-436.e432 (2010)). Despite the promising outcomes of using TEVG for the treatment of patients with congenital heart disease, the high incidence of graft stenosis in clinical applications hinders the widespread use of this technology (Fernandez, et al. Current opinion in chemical engineering 3, 83-90 (2014); Mcallister, et al. Lancet 373(9673), 1440-1446 (2009); Wystrychowski, et al. J Vasc Surg 60(5), 1353-1357 (2014)).

[0007] Furthermore, before we can recommend the routine clinical use of TEVG, we must optimize TEVG assembly to personalize the graft, minimize the time required to fabricate the graft, and improve its overall usefulness (Patterson, et al. Regenerative Medicine 7(3), 409-419 (2012)).

[0008] U.S. Patent No. 9,090,863 and U.S. Patent Application Publication No. 2018 / 0353649 describe a closed-type disposable seeding system (CDSS) for seeding cells into scaffolds and grafts. The CDSS includes seeding chambers for containing and seeding cells into scaffolds and grafts. Seeding into scaffolds and grafts using the CDSS of U.S. Patent No. 9,090,863 and U.S. Patent Application Publication No. 2018 / 0353649 results in a non-uniform cell seeding density along the length of the scaffold and graft. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] U.S. Patent No. 9,090,863 [Patent Document 2] U.S. Patent Application Publication No. 2018 / 0353649 [Patent Document 3] U.S. Patent No. 9,090,863 [Patent Document 4] U.S. Patent Application Publication No. 2018 / 0353649 [Non-patent literature]

[0010] [Non-Patent Document 1] Shin'oka, et al. N. Engl. J. Med. 344(7), 532-533 (2001) [Non-Patent Document 2] Hibino, et al. J Thorac Cardiovasc Surg. 139(2), 431-436.e432 (2010 Roh JD, et al. Biomaterials. 29(10), 1454-1463 (2008) [Non-Patent Document 3] Hibino, et al. FASEB J. 25(8), 2731-2739 (2011) [Non-Patent Document 4] Hibino, et al. FASEB J. 25(12), 4253-4263 (2011) [Non-Patent Document 5] Kurobe, et al. Tissue Eng Part C Methods 21(1), 88-93 (2015) [Non-Patent Document 6] Shin'oka, et al. J Thorac Cardiovasc Surg. 129(6), 1330-1338 (2005) [Non-Patent Document 7] Hibino, et al. J Thorac Cardiovasc Surg. 139(2), 431-436.e432 (2010) [Non-Patent Document 8] Fernandez, et al. Current opinion in chemical engineering 3, 83-90 (2014) [Non-Patent Document 9] Mcallister, et al. Lancet 373(9673), 1440-1446 (2009) [Non-Patent Document 10] Wystrychowski, et al. J Vasc Surg 60(5), 1353-1357 (2014) [Non-Patent Document 11] Patterson, et al. Regenerative Medicine 7(3), 409-419 (2012) [Overview of the Initiative] [Problems that the invention aims to solve]

[0011] There is still a need for improved seeded scaffolds and grafts that have a uniform cell density along the length of the scaffold and graft. There is still a need for scaffolds and grafts that induce reversible stenosis after implantation.

[0012] Therefore, an object of the present invention is to provide an improved seeding chamber for use with CDSS for uniform cell seeding along the length of the scaffold and graft, so as to reduce or prevent the rate of stenosis after implantation.

[0013] Another object of the present invention is to provide a system with an improved seeding chamber for seeding cells onto scaffolds and grafts to reduce, prevent, or reverse the rate of stenosis after implantation.

[0014] Another object of the present invention is to provide scaffolds and grafts having structural parameters that induce reversible stenosis after implantation.

[0015] A further object of the present invention is to provide a method for seeding cells onto scaffolds and grafts to reduce, prevent, or reverse the rate of stenosis after implantation. [Means for solving the problem]

[0016] Summary of the Invention It has been found in clinical trials that scaffolds and grafts with non-uniform cell seeding density along the length of the scaffold or graft result in an increased incidence of graft stenosis after implantation. Improved seeding chambers have been developed for use in closed-loop, disposable seeding systems that provide a uniform density of seeded cells along the length of the scaffold and graft. Cell seeding chambers, called "flip" chambers and "capacitor" chambers, have caps with lateral ports. Flip chambers typically have a variable width along their length. Flip chambers may be operated by inverting the seeding chamber by approximately half (180°) during seeding. Capacitor chambers typically have a uniform width along their length, as well as a narrow gap between the scaffold and the chamber wall.

[0017] A cell seeding chamber generally comprises a housing, a cap with one or more lateral ports, and a base. The cap typically has an opening for a suction rod that can be inserted into the housing, and a mandrel positioned on the suction rod.

[0018] The cap may include one or more lateral tubes, each connected to a lateral port. The lateral tubes typically include a lateral inlet tube connected to a lateral inlet port, a lateral outlet tube connected to a lateral outlet port, and a lateral vent tube connected to a lateral vent port. The cap typically has an upper smooth surface and a lower threaded surface. The threaded surface secures the cap to the housing. The upper portion of the cap is generally flat, and the cap may act as the base when the chamber is inverted.

[0019] A mandrel is typically a perforated porous mandrel. Alternatively, or additionally, a mandrel may have projections of any suitable arrangement configuration, such as projections arranged in an axial direction, arranged in a diamond pattern, circularly, zigzagly, and / or corrugated manner, running parallel to each other, spirally descending, in a diamond pattern.

[0020] The housing of the cell seeding chamber may have a uniform or variable width along its length. The cell seeding chamber may have a gap between the suction rod or mandrel and the housing of the flip chamber, between about 1 mm and about 30 mm, more preferably between about 3 mm and 20 mm. The cell seeding chamber may have a gap between the suction rod or mandrel and the housing of the capacitor, between about 1 mm and about 10 mm, more preferably between about 1 mm and 5 mm. The housing may have a region having a maximum width positioned between about 30% and 60% of the length of the housing. For example, the region with the maximum width may be positioned at about 50% (intermediate) along the length of the housing. The housing typically has a threaded portion for receiving the threaded surface of the cap. The "flip" seeding chamber eliminates variability in the distribution of MNCs on the scaffold. The central premise behind the device is to vary the cross-section of the seeding chamber along its length. The chamber's shape resembles an inverted hourglass, with the bulge (maximum cross-section) located approximately 40% below the top of the device's height. Both the top and bottom of the device narrow to accommodate the scaffolded mandrel, along with very minimal volume for partial clearance and excess fluid. The effect of the cross-sectional change is to effectively slow down the rate at which the MNC is drawn into the least seeded portion of the scaffold.

[0021] Methods for uniformly seeding cells onto a graft or scaffold are also provided. In some embodiments, the method includes: a) connecting a seeding chamber to a closed, disposable seeding system containing a tank with blood or a fluid enriched with cells; b) inserting the graft or scaffold onto a mandrel of the seeding chamber; c) filling the seeding chamber with fluid by gravity flow and extraction to about half the height of the graft or scaffold; d) filling the seeding chamber with fluid to cover the graft or scaffold; and e) introducing negative pressure to draw all the fluid into the graft or scaffold.

[0022] In other embodiments, the method includes: a) connecting a seeding chamber to a closed, disposable seeding system containing a tank with a fluid having blood or enriched cells; b) inserting a graft or scaffold onto the mandrel of the seeding chamber; c) filling the seeding chamber with fluid; d) applying negative pressure to the lower mandrel outlet to empty the chamber to about half its capacity; e) inverting the chamber; and f) applying negative pressure to empty the chamber through the upper outlet port.

[0023] Typically, the seeding chamber is connected to a closed, disposable seeding system via a lateral inlet port and a lateral outlet port. Negative pressure may be provided via a syringe, pump, or vacuum source. Typically, the graft or scaffold is uniformly seeded using a minimal volume of cell-containing fluid, such as blood, bone marrow aspirate, or mononuclear cell (MNC)-rich fluid, in a volume between approximately 10 ml and 200 ml, preferably between 10 ml and 150 ml. [Brief explanation of the drawing]

[0024] [Figure 1-1]Figure 1A shows an exemplary closed system (1000) for seeding and / or testing cells and / or grafts, for example, tissue grafts as described in U.S. Patent No. 9,090,863 and U.S. Patent Publication No. 2018 / 0353649. Figure 1B is a perspective view of an advanced seeding chamber (180) that can be used in place of the seeding chamber (18) shown in Figure 1A. Figure 1C is a perspective view of a seeding chamber (180) that can be used in place of the seeding chamber (18) shown in Figure 1A. Figure 1D is a perspective view of a suction rod (230) containing an integrated threaded top (170) that can be used in place of the seeding chamber (18) shown in Figure 1A. Figure 1E is a perspective view of a suction rod (230) containing a one-piece threaded top (170) and a base portion (232) configured to receive a correspondingly molded clip (600), which also contains a recess. A similar recess is provided on the top of the suction rod. Figure 1F is a perspective view of a clip (600) that can be used in place of an O-ring (22) in the seeding chamber (100) shown in Figure 1A. The clip (600) is typically hollow and has a solid wall (620) surrounding a hollow center (610). If necessary, the wall (620) contains a hinge to facilitate opening and closing the clip (600). Figure 1G is a perspective view of a clip (600) in the seeding chamber (100) shown in Figure 1A. Figure 1H is an exploded view showing the assembly of various components of a seeding chamber assembly (200), which can be used in place of the seeding chamber assembly (100) shown in Figure 1A, and includes a suction rod (230), scaffolding (20), scaffolding clips (600), and seeding chamber (180). [Figure 1-2] Same as above. [Figure 1-3] Same as above.

[0025] [Figure 2]Figures 2A and 2B are bar graphs showing the cell seeding gradient (cells / mm3; values ​​are presented as cells / mm3, as the scaffold is 1 mm thick) along the length (cm) of a scaffold using 50 mL or 100 mL of bone marrow with a standard closed-type disposable seeding system of the prior art (U.S. Patent No. 9,090,863B2 and U.S. Patent Publication No. 2018 / 0353649).

[0026] [Figure 3-1] Figures 3A-3E show the assembly and use of a flip-design seeding chamber – a "flip" seeding chamber. [Figure 3-2] Same as above.

[0027] [Figure 4-1] Figures 4A and 4B show the assembly of a capacitor-designed seeding chamber – a "capacitor" seeding chamber. Figure 4C shows the assembled seeding chamber connected to a seeding system including an IV bag, vacuum source, drain, and extraction line. [Figure 4-2] Same as above. [Figure 4-3] Same as above.

[0028] [Figure 5] Figure 5 is a bar graph showing the uniform seeding density (cells / mm3) along the length (cm) of the scaffold sown in a flip seeding chamber ("Flip", 1) or a capacitor seeding chamber ("Capacitor", 2).

[0029] [Figure 6] Figure 6 shows various mandrel structures for use with a closed-type disposable seeding system. The dimensions shown are: total length 130 mm, seeding area length 120 mm, width 15 mm.

[0030] [Figure 7]Figures 7A–7H are line graphs showing the changes in diameter (Figures 7A–7D) or thickness (Figures 7E–7H) over time (weekly) as four key parameters: δ, β, Ki h, and Ki max change. Parametric studies on these four key parameters are presented: δ modulates the onset and duration of the inflammatory response; β determines the shape and distortion of changes over time; Ki h controls the peak rate of generation and degradation of inflammatory neoplastic tissue; and Ki max scales the inflammatory effect on the degradation of neoplastic tissue.

[0031] [Figure 8] Figures 8A and 8B are line graphs (indicated by ±SD) showing plotted model simulations (solid lines) for the luminal diameter (Figure 8A) or wall thickness (Figure 8B) over time (weeks) of TEVG, which closely match actual intravascular ultrasound experimental measurements of hydraulic diameter obtained over a one-year study period.

[0032] [Figure 9-1] Figures 9A-9F are bar graphs showing stenosis resulting from excessive inflammation-driven neovascularization. Quantitative histological analysis demonstrates that wall thickness (Figure 9A) is maximum at 6 weeks post-implantation, which corresponds to a model simulation of TEVG wall thickness over time (Figure 9B) (one-way ANOVA with Tukey's multiple comparison test: a=0.05, *p<0.05). Figure 9C shows that quantitative analysis of aSMA+ area within neovascular tissue over time is consistent with a model prediction of increased smooth muscle cell density over 6 weeks (Figure 9D) (one-way ANOVA with Tukey's multiple comparison test: a=0.05, *p<0.05). Similarly, CD68+ macrophage staining and quantification (Figure 9E) confirm that significant inflammation corresponds to the development of TEVG stenosis (one-way ANOVA with Tukey's multiple comparison test: a=0.05, *p<0.05). This observation closely matches the computational model prediction of inflammatory cell density (Figure 9F). The plotted data represent mean ± SEM. [Figure 9-2] Same as above. [Figure 9-3] Same as above.

[0033] [Figure 10-1] Figures 10A-10D are graphs showing changes in the biomechanical properties of the embedded graft, which accurately follow the predictions of the computational model. Figure 10A is a bar graph showing the quantitative determination of the total collagen area fraction (%) from Picro-Sirius Red staining over time, compared with untreated IVC (last bar) (one-way ANOVA by Tukey's multiple comparison test: a=0.05, **p<0.005, ***p<0.0005, ****p<0.0001, plots represent mean ± SEM). Figure 10B is a bar graph showing the ratio (%) of thick collagen fibers to thin collagen fibers in TEVG over time, compared with untreated IVC (last bar) (mean ± SEM). Figure 10C is a graph showing that experimental measurements (symbol ± SD) of the mechanical behavior of TEVG (normalized diameter) in response to in vitro pressurization 1.5 years after in vivo onset agreed with the values ​​predicted by the computational model (solid line). Figure 10D shows the predicted graft compliance of 2–3 mmHg over two years from simulated neovascularization (solid line), with reasonable agreement at 18 months compared to experimentally measured compliance (symbols ± SD). [Figure 10-2] Same as above.

[0034] [Figure 11] Figures 11A and 11B illustrate that scaffolding implantation induces foreign body reactions and mechanically mediated necrotizing tissue remodeling. Figure 11C is a graph of a computational model showing the interaction of scaffolding breakdown in inflammation-driven and mechanically mediated necrotizing tissue formation. Factors driving the changing mass of components in the computational model of TEVG development include polymer scaffolding loss (·······) and inflammation-driven (-·-) and mechanically-mediated (---) necrotizing tissue acquisition. The sum (-) of each of these components determines the mass density of the necrotizing tissue.

[0035] [Figure 12]Figure 12 is a flowchart illustrating a pilot study investigating the clinical use of tissue-engineered vascular grafts in congenital cardiac surgery.

[0036] [Figure 13-1] Figure 13A shows a flowchart illustrating the study design and the number of animals analyzed at each time point in the sheep study. Figure 13B is a survival curve for the sheep study, showing survival (%) over the postoperative days. [Figure 13-2] Same as above.

[0037] [Figure 14-1] Figures 14A–14D are graphs showing changes in various parameters of implanted TEVGs in a sheep study. Angiography and intravascular ultrasound were used for TEVGs in the sheep model. Data were obtained from representative serial angiography and IVUS images of a single sheep's TEVG at 1 week, 6 weeks, 6 months, and 1 year post-implantation, demonstrating the development of fatal stenosis at 6 weeks and subsequent remodeling to neovascularization at 6 months. Plots of relative lumen diameter (magnification change, diameter, Figure 14A) and area change (magnification change, area, Figure 14B) as magnification changes of TEVG over one year of continuous angiography and intravascular ultrasound studies are presented, demonstrating that TEVG narrowing spontaneously reverses by 6 months (one-way ANOVA with Tukey's multiple comparison test: a=0.05, p<0.0001, mean ± SD). Weekly mean values ​​are identified by horizontal dashed lines. Figures 14C and 14D are graphs showing 6-week diameter (Figure 14C) and pressure (Figure 14D) gradient measurements from each animal in the study cohort. Two animals experienced asymptomatic ascites, and two animals experienced symptomatic ascites requiring euthanasia before the end of the study (squares). [Figure 14-2] Same as above. [Modes for carrying out the invention]

[0038] Detailed explanation I. Definition The term "tissue engineering vascular graft (TEVG)" refers to a vascular graft or scaffold designed for insertion into the body for use in repairing or enhancing one or more blood vessels, such as arteries and veins.

[0039] The term "biocompatibility" refers to materials that the body generally accepts without a major immune response and that can be implanted in biological systems, such as tissue implants, without causing excessive fibrosis or rejection. The term "biodegradable" refers to the ability of a substance or material to break down when exposed to water, enzymes, or an in vivo environment.

[0040] As used herein, "stenosis" refers to a reduction in lumen diameter of 25% or more relative to the lumen diameter of the scaffold during implantation.

[0041] The term "mandrel" refers to a cylindrical device or tube, such as a metal rod, that acts as a core, around which a material, such as a matrix scaffold for seeding and growing cells, may be cast, molded, forged, bent, or otherwise formed. Mandrels are typically open at least one end. Mandrels may also contain holes or perforations along their long axis (i.e., along their length).

[0042] As used herein, the term "porous" means having one or more openings, pores, perforations, or holes that can be filled with or permeated with liquid and / or gas, or that allow the flow of liquid and / or gas through its interior.

[0043] As used herein, the term “spontaneous” in the context of stenosis reversal refers to an action that occurs without any additional invasive or non-invasive procedure, such as surgical intervention or surgical correction.

[0044] As used herein, the term “reversal of stenosis” refers to a reduction of stenosis of at least approximately 70%, 75%, 80%, 85%, 90%, 95%, or 100% in a constricted, embedded graft without seeded cells.

[0045] As used herein, the term “substantially” refers to a measure of approximately 80%, approximately 85%, approximately 90%, approximately 95%, or approximately 98%.

[0046] II. System for sowing seeds in TEVG A. Closed-type disposable seeding system Systems and methods for seeding cells into TEVGs are known in the art. Exemplary systems are described in U.S. Patent No. 9,090,863 and U.S. 2018 / 0353649 (Figure 1A, Prior Art). The device shown in Figure 1A is a closed system (1000) for seeding, culturing, storing, transporting, and / or testing cells and / or grafts, such as tissue grafts. The operation, structure, and results of previous seeding systems are further discussed and shown in U.S. Patent No. 9,090,863 and Tissue Engineering Part C: Methods. (1):88-93 (2014).

[0047] Typically, a system for seeding cells into a tissue-engineered vascular bruf includes means for creating a vacuum, which is connected to the patient to extract biomaterial from the patient into a chamber or scaffold. The scaffold acts as an incubator, allowing at least a portion of the biomaterial to come into contact with and interact with the scaffold. The scaffold is fluid-connected to a filter / switch combination, which allows a selectable fluid to be transferred back from the biomaterial to the patient. Exemplary biomaterials transferred back to the patient include serum, erythrocytes, platelets, leukocytes, and combinations thereof. Extracting the selected biomaterial from the fluid in contact with the scaffold reduces the time required to seed the desired cell type onto the scaffold, thereby increasing the patient's healing rate. The components of the exemplary system described in c are shown in Figures 1A to 1H (prior art). Generally, the components include: Port (1) or Port (2) Cell isolation fluid container (3) Valve (5 、6、15、27 ) Fluid lines (51, 52, 53, 54, 55, 56, 57, 58a~58d, and 59) Pre-filter elements (4) Flow channels (7) Container(13) Entrance port (11) Exit port (14) Valve (10) Seeding container(18) Container(9) Valve (8) Threaded cap (17) Entrance port (16) Exit port (24) Sampling port (19) Vent port (26) Fluid containers 35a and 35b Port (25) Vacuum source, e.g., pump and regulator (28) Seedling assembly (100) Porous tube (20) Scaffolding (21) Includes clips (60).

[0048] The components are interconnected and used as described in U.S. Patent No. 9,090,863 and U.S. 2018 / 0353649.

[0049] For example, the system includes a vessel, e.g., container (3), for containing cell isolates, e.g., cell isolation fluid. Exemplary containers include a media bag or any soft or rigid container that is sterilizable and / or airtight sealed (e.g., Gibco-BFL 1L media bag). In certain embodiments, the cell isolation fluid container (3) is formed from a sterilizable, biocompatible rigid material, e.g., TEFLON®, polycarbonate, polyvinyl chloride (PVC), or stainless steel. Container (3) may have any suitable volume for containing the cell isolation fluid, typically less than 250 ml. In preferred embodiments, container (3) has at least one port suitable for sterile filling and / or dispensing of a fluid, e.g., bone marrow aspirate. For example, bone marrow aspirate (e.g., 5 ml / kg body weight) is collected sterile and passed through port (1) or port (2) into container (3), e.g., injected.

[0050] Typically, the vessel (3) has at least one inlet and one outlet. In some embodiments, the vessel (3) includes a port having one or more valves that allow unidirectional flow of a fluid or gas. For example, using the embodiment shown for reference in Figure 1A, port 1 may include a swabble valve, such as a needleless access port, and / or take the form of such a valve. The valve may be connected to the vessel (3) and / or associated with a fluid line that communicates with the vessel (3) using any suitable coupling or fastening means known to those skilled in the art, such as clamps, screws, or Luer connectors, pressure fits, friction fits, or couplings. According to the embodiment of the system (1000) shown in Figure 1A, valve (5) is associated with a scaffold, for example, a fluid line (51) that communicates with the vessel (3). If necessary, the vessel (3) may include a prefilter element (4) which includes a screen or woven element having, for example, an open or porous structure in the range of about 40 to about 150 microns. Such pre-filter elements can be used to remove undesirable materials, such as bone fragments, blood clots, and / or fat deposits, as the fluid flows out of the container.

[0051] Examples of fluids that may be used in the system include, but are not limited to, sterile fluids, contrast fluids, biological fluids, cell-containing fluids, blood, serum, bone marrow aspirate, or culture medium-containing fluids. It should be understood that during testing, seeding, and culturing in preferred embodiments, the fluid may be maintained at human body temperature and may consist of fluids with a viscosity approximating that of human blood. One exemplary example of a solution with a viscosity approximating that of blood is physiological saline containing glycerol.

[0052] The fluid in container (3) passes from the container through the fluid line (51) in Figure 1A. In a preferred embodiment, the fluid is directed away from container (3) by vacuum. In other embodiments, the system incorporates the use of a fluid pump. Fluid pumps are available from numerous commercial suppliers (e.g., Masterflex L / S Digital Drive peristaltic pump, manufactured by Cole-Palmer).

[0053] The fluid line (51) and all other fluid lines in the system (e.g., lines 52, 53, 54, 55, 56, 57, 58a-58d, and 59) may be made of any type of medical-grade, sterilizable, durable tubing suitable for transporting the fluid or gas in use. For example, the fluid lines may be made of soft or hard plastic.

[0054] The system also includes a flow channel (7) comprising at least one inlet, at least one outlet, and at least one filter, the filter medium (e.g., located within a filter housing) in between. In a preferred embodiment, the filter is positioned at an angle substantially perpendicular to the direction of flow through the flow channel (7), but in some embodiments, the filter may be positioned at an angle substantially parallel to the direction of flow, e.g., tangential flow filtration. In a preferred embodiment, the filter is adapted to allow flow in at least two directions, for example, the first and second directions being substantially opposite, such that the fluid can pass from the upstream to the downstream surface of the filter in the first direction, and the fluid can pass from the downstream to the upstream surface of the filter in the second direction. In an example of this embodiment, the cell isolation fluid passes through the filter having an appropriate pore size (or mesh size) in the first direction, and the filter medium is positioned at an angle substantially perpendicular to the direction of flow so that the filter holds cells and / or biomaterials that are too large to pass through the filter. The second fluid then passes through the filter in a second direction, allowing the retained cells and / or biomaterials to be washed off the filter medium. Filters that can be used in fluid channels are well known in the art, including, for example, those of Pall Corporation. In other embodiments, the filter (e.g., at least one filter medium) has a porosity suitable for retaining cells, e.g., bone marrow-derived mononuclear cells. In certain embodiments, the filter includes a matrix designed to reversibly bind and retain the cells of interest, for example, based on ligand-receptor interactions. In other embodiments, multiple filters can be assembled in series or in parallel for use in the systems described herein. The systems and methods are intended to have any number of desired flow paths and blockages. The liquid or gas can be supplied through the system by positive or negative pressure, such as via a syringe, pump, or vacuum source.

[0055] In certain embodiments, the system includes means for containing the collection fluid. In certain embodiments, the means is a container (13), for example, a sterilizable and / or airtight-sealed media bag or any flexible or rigid container (e.g., a GIBCO-BFL 1L media bag). In certain embodiments, the collection container (13) is formed from a sterilizable, biocompatible rigid material such as Teflon®, polycarbonate, acrylic, PVC, or stainless steel. The container (13) can have any suitable volume for containing the collection fluid.

[0056] In a preferred embodiment, the container (13) has at least one port suitable for sterile filling and / or dispensing or flow of a fluid, such as bone marrow aspirate filtrate. For example, bone marrow aspirate (e.g., 5 cc / kg body weight) is sterilely collected, passed through the container (3), for example, injected, and then passed through (e.g., through a pre-filter (4) as needed, and through fluid flow lines (51) and (52) through a fluid channel (7) containing a filter). The filter holds the cells and / or biomaterial of interest and allows the filtrate to flow into the container (13) through fluid lines (53) and (54) and the inlet port (11). In another preferred embodiment, the container (13) has at least one fluid port, such as a bidirectional fluid port; however typically, the container (13) has at least two ports. In a particular embodiment, the container (13) includes an inlet port and / or an outlet port.

[0057] In the embodiment shown in Figure 1A, the container (13) includes an inlet port (11) and an outlet port (14). In yet another embodiment, the container (13) includes a port having one or more valves that allow unidirectional flow of a fluid or gas. The valves can be associated with a fluid line that connects to and / or communicates with the container (13) using any suitable coupling or fastening means known to those skilled in the art, such as clamps, screws, or Luer connectors, pressure fits, friction fits, etc. According to the embodiment shown in Figure 1A, the system (1000) includes a valve (10) associated with a fluid line (54).

[0058] In a particular embodiment (for example, as will be described in more detail below, after elution the fluid passes through the fluid channel (7) and the cells enter the seeding container (18)), the collected fluid or filtrate contained in the container (13) passes through the fluid lines (59) and (57) and enters the seeding container (18) (Figure 1A).

[0059] In a preferred embodiment, the fluid is directed away from the container (13) by vacuum. In other embodiments, a fluid pump is used (for example, a Masterflex L / S Digital Drive peristaltic pump, manufactured by Cole-Palmer, but those skilled in the art can choose from a variety of commercially available pumps).

[0060] In certain embodiments, the system includes means for containing the eluting fluid. In certain embodiments, the means is a container (9), for example, a sterilizable and / or airtight-sealed media bag, syringe, or any flexible or rigid container (e.g., a Gibco-BFL 1L media bag).

[0061] According to the embodiment shown in Figure 1A, the system (1000) includes a valve (8) associated with a fluid line (55). The valve can be connected to a container (9) and / or associated with a fluid line using any suitable coupling or fastening means known to those skilled in the art, such as clamps, screws, or Luer connectors, pressure fits, friction fits, etc. The eluting or washing fluid passes through the valve (8) and fluid line (53), flow channel (7), fluid lines (52), (56), and (57) in Figure 1A and enters the seeding container (18).

[0062] In one embodiment, the container (9) is a syringe filled with a sterile elution or washing fluid. The elution or washing fluid, in Figure 1A, passes through the fluid channel 7 and enters the seeding container (18) through the valve (8) and fluid lines 53, 52, 56, and 57. In this embodiment, the fluid moves away from the container 9 due to the pressure applied to the fluid, for example, by pushing down the syringe plunger by hand or via a mechanical and / or electrical device. Alternatively, for example, the container (9) can be a flexible container that can be compressed. However, as will be easily understood by those skilled in the art, a fluid pump can also be used (for example, the MASTERFLEX L / S DIGITAL DRIVE peristaltic pump, manufactured by COLE-PALMER, but those skilled in the art can choose from a variety of commercially available pumps).

[0063] In certain embodiments, the system includes means for containing a cell seeding assembly. In certain embodiments, the means is a seeding container (18), for example, a sterilizable and / or airtight-sealed medium bag or any soft or rigid container. For example, a GIBCO-BFL 1L medium bag can be used. In certain embodiments, the container 18 may be made of any biocompatible rigid material that can be sterilized, such as Teflon®, polycarbonate, acrylic, PVC, or stainless steel. The seeding container (18) can have any suitable volume.

[0064] As shown in Figure 1A, the seeding container (18) comprises a rigid material including a body section with threads and a threaded cap (17). The container (18) includes at least an inlet and an outlet port (in the embodiment shown in Figure 1A, the seeding container (18) includes an inlet port (16), an outlet port (24), a sampling port (19) (for sterile acquisition of a fluid sample from the seeding container, e.g., to determine microbial contamination and / or stem cell enumeration, e.g., and the cap 17 (Figure 1A) includes the ports).

[0065] The seeding container 18 includes an inlet port 16 and an outlet port 24, allowing for the perfusion and / or circulation of fluid into and through the container. The inlet port 16 and outlet port 24 are also used to attach the container 18 to fluid lines 57 and 58a, respectively. Fluid line 58a connects the seeding container 18 to one or more residual seeding cell fluid containers 35a and 35b while maintaining a closed system. Although only one seeding container 18 is shown in Figure 1A, it should be understood that the fluid lines, for example, fluid lines 57 or 58a, may branch to connect more than one seeding container in parallel to the system.

[0066] Means for containing the residual seeding cell fluid include at least one residual seeding cell fluid container 35a, 35b, for example, a sterilizable and / or airtight-sealed media bag or any soft or rigid container. For example, a Gibco-BFL 1L media bag can be used. In certain embodiments, the containers 35a, 35b may be made of any biocompatible rigid material that can be sterilized, such as TEFLON®, polycarbonate, PVC, or stainless steel.

[0067] In a preferred embodiment, the fluid is drawn from the container 18 and enters the residual seeding cell fluid container 35a via port 25 and fluid line 58a using a vacuum assembly having a vacuum source, such as a pump and a regulator 28, and the negative pressure from the pump is transmitted through fluid lines connected to the residual seeding cell fluid containers 35a, 35b and the seeding container 18.

[0068] In a particular embodiment, the seeding container (18) includes a porous tube (20) and a scaffold (21 External 23 Internal The porous tube (20) contains a seeding assembly (100) which includes, for example, a cell or tissue scaffold or graft, such as a vascular graft scaffolding material. The porous tube (20) may contain any suitable rigid material that can be fluid permeable, such as TEFLON®, PVC, polycarbonate, plastic, or metal, such as stainless steel. One or more retaining elements, such as clips (60), O-rings, or grommets, may be placed on the tube, for example, at both ends of the scaffolding material (21), to hold the scaffolding material in place on the tube during seeding, culturing, storage, transport, or treatment.

[0069] In certain embodiments, the system is disposable. The closed-loop disposable system enables a procedure for constructing tissue engineering grafts, such as vascular grafts, that can be performed immediately while achieving similar seeding efficiency compared to the aforementioned method (Matsumura, et al., Biomaterials 2003; 24:2303-8; and FDA IDE 14127), which is incorporated entirely herein by reference. Furthermore, the use of the system allows tissue engineering grafts, such as vascular grafts, to be constructed in a clinical setting (i.e., in the operating room), eliminating the need for scaffold transport.

[0070] The seeding system may use a seeding chamber 700 (flip seeding chamber) or a seeding chamber 800 (capacitor seeding chamber) instead of the seeding container 18. 1. Seedling chamber

[0071] A seeding chamber generally comprises a housing having width and length, a cap having one or more lateral ports, and a base. The cap typically includes a suction rod that can be inserted into the housing and a mandrel positioned on the suction rod. Typically, the cap has a flat top surface. The cap generally does not include valves, ports, or accessories on its top surface.

[0072] In exemplary embodiments, the base of the seeding chamber has a radius between 10 and 100 mm, for example, between approximately 35 mm and 80 mm. In exemplary embodiments, the top of the seeding chamber has a diameter between 10 and 100 mm, for example, between approximately 25 mm and 60 mm, for example, 38.1 mm. In exemplary embodiments, the base of the seeding chamber has a height between 20 and 1000 mm, for example, between approximately 120 mm and 200 mm, for example, 180 mm. Typically, the inner diameter of the aperture of the seeding chamber is between 5 and 90 mm, for example, 25.86 mm. The typical thickness of the wall of the seeding chamber is between 0.5 and 10 mm, for example, approximately 2 mm. If the seeding chamber has a threaded top, the height of the threaded section is typically approximately 10% of the total length of the chamber, for example, 20.55 mm. The mandrel, seeding chamber, and one or more scaffold clips are typically sized to fit together within the seeding chamber, corresponding to the desired size of the vascular graft and seeding chamber. a. Flip seeding chamber

[0073] The "flip" seeding chamber has a variable cross-sectional diameter along its length.

[0074] The chamber's shape resembles an inverted hourglass, with the maximum cross-section being approximately 30%–60%, preferably about 40%, of the device's height from top to bottom. Both the top and bottom of the device narrow to accommodate the mandrel with the scaffold attached, along with very minimal volume for partial clearance and excess fluid. Typically, the cross-sectional diameter of the flip chamber at its narrowest point is between approximately 22–25 mm, including both ends. Typically, the cross-sectional diameter of the flip chamber at its widest point is between approximately 60–80 mm, including both ends.

[0075] The effect of changing the cross-sectional diameter is to effectively slow down the rate at which the MNC is drawn into the least seeded part of the scaffold.

[0076] Furthermore, the mandrel of this design has two vacuum nubs—one for drawing negative pressure upwards and one for drawing negative pressure in the opposite direction.

[0077] An exemplary flip seeding chamber is shown in Figures 3A–3E. The flip seeding chamber 700 includes a cap 710, a housing 740, and a base 730. Base section 726 This includes lateral ports, such as an inlet port 722a connected to an inlet tube 722, and an outlet port 724a connected to an outlet tube 724. The cap 710 has a flat top surface. The cap 710 may have a top surface of any shape. Typically, the top surface of the cap does not have ports, tubes, or vents.

[0078] The housing 740 has a variable cross-sectional diameter along its length. The upper 10% of the housing includes threads to receive the threaded portion of the cap 710. The bottom section of the housing 740 connects to the base portion 730. The housing 740 may include lower mandrel outlets 732 and 734. An O-ring groove 760 may also be present to position the O-ring to seal the scaffold to the mandrel.

[0079] The cap 710 is connected to the suction rod 720. The porous mandrel 750 is attached to the suction rod 720. The attachment may be by any means including clamps, screws, or Luer connectors, pressure fits, friction fits, or couplings.

[0080] Typically, the flip seeding chamber is configured to be held stably in a vertical position when positioned on its base or on its cap. This is shown in Figures 3D and 3E. b. Capacitor seeding chamber

[0081] A capacitor seeding chamber is typically a hollow tube with a consistent cross-sectional diameter along its length. Typically, the cross-sectional diameter of a capacitor chamber is between approximately 10 and 20 mm, including the values ​​at both ends. A capacitor seeding chamber is very narrow compared to an assembled mandrel / scaffolding assembly. It is designed to hold a minimal volume of fluid. The minimum and maximum gaps between the scaffolding and the chamber walls may be approximately 1 mm and 5 mm, respectively.

[0082] An exemplary capacitor seeding chamber is shown in Figures 4A–4C. The capacitor seeding chamber 800 includes a cap 810, a housing 840, and a base 830. The cap includes lateral ports, such as an inlet port 822a connected to an inlet tube 822, and an outlet port 824a connected to an outlet tube 824. The cap 810 has a flat top surface. The cap 810 may have a top surface of any shape. Typically, the top surface of the cap is free of ports, tubes, or vents.

[0083] The housing 840 has a constant cross-sectional diameter along its length. The upper 10% of the housing includes threads to receive the threaded portion of the cap 810. The O-ring 860 may be positioned between the cap 810 and the housing 840. The bottom section of the housing 840 is connected to the base portion 830.

[0084] The cap 810 is connected to the suction rod 820. The porous mandrel 850 is attached to the suction rod 820. The accessories may be attached by any means, including clamps, screws, or Luer connectors, pressure fits, friction fits, couplings, etc. 2. Mandrel

[0085] The mandrel is typically sized to fit the suction rod. The mandrel may be secured to the suction rod or cap with any suitable attachments. Exemplary attachments include clips, hooks, and slots that accommodate the width of the mandrel. The mandrel may be attached to the suction rod by molding or adhesive.

[0086] The mandrel may have any shape suitable for receiving a vascular graft or scaffold. The mandrel is typically a perforated porous mandrel. Alternatively, the mandrel may have projections arranged in an axial direction, arranged in a diamond pattern, circularly, zigzagly, and / or corrugated manner, running parallel to each other and descending spirally.

[0087] Exemplary dimensions for the mandrel are shown in Figure 6. In the exemplary embodiment, the length of the mandrel that fits into a seeding chamber with a length of 180 mm is between approximately 120 mm and 140 mm, for example, approximately 128 mm. B. Fluid volume, cell density, and seeding time

[0088] Graft or scaffold seeding typically involves bringing the graft or scaffold into contact with a cell-containing fluid. The fluid may be blood, bone marrow aspirate, or cell extracts from blood or bone marrow. The fluid may be in volumes between approximately 10 ml and 200 ml, for example, approximately 25 ml, approximately 50 ml, approximately 75 ml, approximately 100 ml, approximately 125 ml, approximately 150 ml, approximately 175 ml, and approximately 200 ml. The fluid is typically about 10 5 cells / ml~10 8Between cells / ml, preferably about 10 6 ~10 8 Between cells / ml, more preferably about 10 6 ~10 7 Has a white blood cell (WBC) concentration between cells / ml.

[0089] After seeding, the graft or scaffold typically has, along the length of the scaffold, including the values at both ends, about 0.1×10 3 cells / mm 2 ~10 5 cells / mm 2 Between, preferably along the length of the scaffold, including the values at both ends, about 0.1×10 3 cells / mm 2 ~10 4 cells / mm 2 Between, most preferably along the length of the scaffold, including the values at both ends, 1×10 3 cells / mm 2 ~10 4 cells / mm 2 Has a cell density between.

[0090] Typically, the graft is in contact with the fluid for a period of about 1 minute to 15 minutes, preferably about 1 minute to 10 minutes, most preferably about 1 minute to 7 minutes. In the most preferred embodiment, seeding ends at about 15 minutes, about 7 minutes, or about 1 minute. C. Graft or scaffold with reversible stenosis

[0091] Typically, the graft or scaffold is positioned between the mandrel and the housing. The graft or scaffold is generally a polymer and porous material made from a biodegradable polymer. The graft or scaffold may have an inner and outer structure to induce spontaneous reversal of the constriction. The graft or scaffold may have an average pore diameter on its inner surface, which is different from the average pore diameter on its outer surface. For example, the average pore diameter on the inner surface may be between about 35 μm and 50 μm, preferably between about 38 μm and 50 μm, and most preferably between about 38 μm and 45 μm. The average pore diameter on the outer surface may be between about 25 μm and 45 μm, preferably between about 27 μm and 43 μm, and most preferably between about 30 μm and 43 μm. The surface porosity may be between about 0.6 and 0.95 of the surface area of ​​the inner and / or outer surface, preferably between about 0.7 and 0.9, and most preferably between about 0.8 and 0.9.

[0092] Typically, the graft or scaffold is formed from woven polymer fibers. In some embodiments, the fibers may be woven into fiber bundles. The fiber bundles may be woven, for example, into a weft pattern. The diameter of the fibers may be between about 5 nm and 30 nm. Typically, the woven pattern forms polymer fiber layers arranged axially and polymer fiber peaks arranged circumferentially. The distance between layers may be between about 0.5 mm and 2 mm, preferably between about 0.5 mm and 1.5 mm, and most preferably between about 1 mm. The distance between peaks may be between about 0.5 mm and 2 mm, preferably between about 0.5 mm and 1.5 mm, and most preferably between about 1 mm.

[0093] The graft or scaffold may include a fibrous polymer coating. Typically, the TEVG or scaffold has an inner diameter between approximately 14 mm and 22 mm, a thickness between 0.1 mm and 3 mm, and a length between approximately 5 cm and 15 cm. The TEVG or scaffold is typically biodegradable and decomposes substantially within approximately 6 months after implantation. For example, within 6 months after implantation, the graft has been reduced by more than approximately 80% in weight, more than approximately 80% in surface area, or more than approximately 80% in thickness.

[0094] Polymer vascular grafts or scaffolds typically contain an effective number of viable cells to reduce or prevent postoperative stenosis of the graft, compared to grafts with no or few cells. Typically, a graft contains a certain number of viable cells along the length of the scaffold, including values ​​at both ends, approximately 0.1 × 10⁻⁶. 3 cells / mm 2 ~10×10 4 cells / mm 2 Between these points, preferably along the length of the scaffolding, including the values ​​at both ends, approximately 0.1 × 10 3 cells / mm 2 ~10×10 3 cells / mm 2 Between these values, most preferably along the length of the scaffolding, including the values ​​at both ends, 1 × 10 3 cells / mm 2 ~10×10 3 cells / mm 2 They are attached at a cell density between them.

[0095] Preferred cells are obtained from the patient's bone marrow. In a preferred embodiment, the vascular graft is seeded with autologous viable cells. In a specific embodiment, the cells are human bone marrow mononuclear cells. The polymer vascular graft or scaffold may contain one or more additional agents selected from the group consisting of anti-neointimal agents, chemokines, steroidal and non-steroidal anti-inflammatory drugs, conventional immunotherapeutic agents, immunosuppressants, cytokines, chemokines, and growth factors. 1. Polymer

[0096] TEGV scaffolds can be formed from one or more polymers. In some embodiments, the polymers are biodegradable. In other embodiments, the polymers are non-biodegradable. In some embodiments, the TEVG is formed from a mixture of more polymers than a single polymer. When biodegradable polymers are used, mixtures of biodegradable and non-biodegradable polymers can be used, for example, to provide long-lasting TEVG implants as desired.

[0097] In certain embodiments, the TEVG is a three-dimensional matrix formed of polymer (homopolymer and / or copolymer) fibers assembled in a random or aligned configuration onto a woven or nonwoven mesh. Preferably, the nanofiber material is an FDA-approved biodegradable nanofiber material.

[0098] The fibers of the scaffolding base material can be of any desired size, but are generally between approximately 1.5 mm and 1 nm. In certain embodiments, the fibers are nanoscale (i.e., approximately 1 nm to approximately 1000 nm) and / or microscale (approximately 1 μm to approximately 1000 μm).

[0099] Polymers useful for creating scaffolds for use in the formation of TEVGs may be inorganic (e.g., siloxanes, sulfur chains, black phosphorus, boron-nitrogen, silicones) or organic (meaning carbon-containing). Organic polymers may be natural (e.g., polysaccharides, e.g., starch, cellulose, pectin, seaweed gum, plant gum; polypeptides, e.g., casein, albumin, globulin, keratin, collagen, nucleic acids, and hydrocarbons), synthetic (thermoplastics, non-vulcanized elastomers, nylon, polyvinyl chloride, linear polyethylene, polystyrene, polypropylene, polyurethane, acrylic resins, etc.); thermosetting (e.g., vulcanized elastomers, crosslinked polyethylene, phenol, alkyds, polyesters); and semi-synthetic (e.g., cellulose, e.g., rayon, methylcellulose, cellulose acetate; and modified starch)). Furthermore, useful scaffolds may include hydrogels formed from water-soluble or water-insoluble cellulose compounds. As will be readily apparent to those skilled in the art, the specific type and composition of the scaffolds will vary depending on the desired application. However, it is generally preferable that the polymer material in the scaffold be biocompatible (i.e., does not induce an undesirable immune response). In certain embodiments, the scaffold is biodegradable. Exemplary biodegradable polymers include poly(lactic acid-glycolic acid), poly(lactic acid), poly(glycolic acid), poly(orthoester), poly(phosphazene), polycaprolactone, or polyamide. In preferred embodiments, the polymer is poly(lactic acid-glycolic acid). In one embodiment, the TEVG is formed from a biodegradable tubular scaffold made from polyglycolic acid fiber tubes. The tubes can be coated with copolymers such as a 50:50 polylactic acid (PLA) and poly-caprolactone copolymer.

[0100] In one embodiment, the graft is formed from a polymer felt or sheet-like material that can be formed into a tubular scaffold. For example, the device can be fabricated as a nonwoven, woven, or knitted structure from extruded polymer fibers. Typically, the polymer sheet is formed using any textile construction, including but not limited to weaving, knitting, braiding, or winding of filaments. Any suitable method, such as electrospinning, can be used to produce nonwoven or woven polymer textiles.

[0101] The polymers and fabrication methods selected for fabricating polymer vascular grafts are suitable for producing grafts with biomechanical properties suitable for use as vascular scaffolds. Biomechanical properties important for vascular graft function include initial burst pressure, suture retention strength, and elasticity. In one embodiment, the initial burst pressure of the polymer vascular graft is between about 1,500 mmHg and about 50,000 mmHg, preferably between about 2,000 mmHg and about 10,000 mmHg. In another embodiment, the polymer vascular graft has a suture retention strength between about 1 N and about 5 N, preferably between about 2 N and about 4 N. In another embodiment, the intrinsic elasticity of the vascular graft is between about 10 MPa and about 50 MPa, preferably between about 15 MPa and about 40 MPa. In another embodiment, the initial tensile strength of the vascular graft is between about 1 MPa and about 10 MPa, preferably between about 3 MPa and about 6 MPa. 2.Cells

[0102] In certain embodiments, the TEVG scaffold contains one or more types of cells. In some embodiments, one or more types of cells are included in the lumen of the TEVG, in the porous space throughout the wall of the TEVG, on the outside and surface of the TEVG, or in combination. Typically, the cells are attached directly to the surface of the TEVG or through one or more auxiliary materials. In some embodiments, the cells are autologous cells derived from one or more tissues of the intended recipient of the cell-seeded TEVG. In other embodiments, the cells are exogenous to the intended recipient of the graft. The cells can be undifferentiated or differentiated cells, such as pluripotent stem cells. In preferred embodiments, the cells are viable human cells. Exemplary cell types to be included in the TEVG include leukocytes (WBCs), e.g., monocytes, lymphocytes, neutrophils, basophils, eosinophils; fibroblasts, myofibroblasts, fibroblasts; smooth muscle cells; bone marrow progenitor cells; erythrocytes; embryonic stem cells; and combinations. Leukocytes are produced in the bone marrow. In certain embodiments, autologous bone marrow mononuclear cells (BM-MNCs) are contained within the TEVG.

[0103] Cells for use with TEVG can be obtained from numerous sources. Methods for isolating one or more cell types from a mixture of cells and manipulating them as needed are known in the art. In exemplary embodiments, bone marrow is collected from one or more long bones (e.g., femur and / or tibia) of the subject (e.g., the intended recipient of TEVG), and mononuclear cells are isolated using density centrifugation (Lee, et al. J Vis Exp. (88), (2014); Udelsman, et al. Tissue Eng Part C Methods. 17(7), 731-736 (2011)). a. Seeding dose

[0104] Typically, a seeding chamber provides a maximum uniform seeding density along the length of the graft or scaffold with a minimal volume of cell-containing fluid (blood, bone marrow aspirate, or mononuclear cell (MNC)-rich fraction).

[0105] Typically, the amount of cells seeded in a TEVG is directly proportional to postoperative graft patency. Therefore, in a preferred embodiment, a sufficient amount of cells is seeded in the TEVG to enhance patency or reduce the rate of postoperative stenosis of the TEVG. Methods for manually seeding cells in a TEVG are known in the art (Udelsman, et al. Tissue Eng Part C Methods. 17(7), 731-736 (2011)).

[0106] The optimal number of cells to seed can vary depending on the cell type, as well as the size and shape of the TEVG and its intended use. A typical TEVG, including both values, is 1.0 × 10⁶. 6 cells ~500×10 6 Between cells, preferably 3.0 × 10 6 cells ~250×10 6 Allow the amount of cells to come into contact with the cells.

[0107] When sown, the resulting seeding density is uniform along the length of the scaffold. The seeding density is approximately 0.1 × 10⁻⁶ along the length of the scaffold. 3 cells / mm 2 ~10×10 4 cells / mm 2 It may be between. In a preferred embodiment, the TEVG is about 0.1 × 10 along the length of the scaffolding. 3 cells / mm 2 ~10×10 3 cells / mm 2 The range is between the values ​​at both ends, preferably 1 × 10 along the length of the scaffolding. 3 cells / mm 2 ~10×10 3 cells / mm 2 The seeds are sown at a density that includes the values ​​at both ends of the spectrum.

[0108] In some embodiments, TEVG, including the values ​​at both ends, is approximately 0.1 × 10⁻⁶. 4 cells / ml~10×10 7Cells are seeded in a solution having a concentration between cells / ml. Typical volumes of cell solution are between 1ml and 100ml, including both extreme values, preferably in the range between 10ml and 100ml, for example, 10ml, 50ml, 75ml, or 100ml.

[0109] Preferably, TEVG contains 0.1 × 10 3 cells / mm 2 ~10.0×10 4 cells / mm 2 Preferably 1.0 × 10 3 cells / mm 2 ~10.0×10 3 cells / mm 2 Cells are seeded in an amount sufficient to produce a cell density between these two levels. Therefore, the seeding chamber allows for the seeding of a graft or scaffold using the minimum amount of bone marrow harvested from the patient, enabling a uniform cell density along the length of the graft or scaffold.

[0110] When autologous bone marrow mononuclear cells (BM-MNCs) are prepared for seeding, 5 ml / kg of cells from bone marrow typically provides a sufficient seeding dose. From a clinical standpoint, up to 20 ml / kg of bone marrow can be harvested from an individual without causing significant adverse effects and is routinely used for harvesting bone marrow for transplantation. 3. Additional activators

[0111] It has been established that TEVGs seeded with bone marrow-derived mononuclear cells reduce and prevent the development of postoperative stenosis through paracrine effects. The advantages of cell seeding include release signals that respond to the body's feedback mechanisms, unlike drug-eluting scaffolds that release drugs regardless of feedback. Therefore, in some embodiments, cell-seeded TEVGs are used in combination with one or more non-cell-based synthetic or non-synthetic compounds that replicate the paracrine effects of the seeded cells.

[0112] TEVG may include additional activators, such as those that enhance cell adhesion to the vascular graft or reduce the development of postoperative stenosis of the graft after insertion. Activators may include, but are not limited to, anti-intimal agents, chemokines, steroidal and non-steroidal anti-inflammatory drugs, conventional immunotherapies, immunosuppressants, cytokines, chemokines, and growth factors.

[0113] The use of growth factors to stimulate bone marrow growth is an additional strategy to increase the yield of BM-MNCs. Therefore, in some embodiments, TEVGs contain growth factors. Exemplary growth factors for incorporation into TEVGs include growth factors released in the physiological response to tissue injury, which stimulate the deposition of extracellular matrix such as platelet-derived growth factor (PDGF), potent chemotacticants, and transforming growth factor beta (TGF-β). III. Preparation Method A. Seedling chamber

[0114] One or more components of the seeding system are custom-designed so that the assembly is optimally sized to conform to the dimensions of the TEVG. Preferably, the system or components of the system are manufactured using 3D printing of a suitable material. In exemplary embodiments, one or more components of the seeding chamber of the system shown in Figures 2A–4C are manufactured by 3D printing of a suitable material. In certain embodiments, the 3D-printed components include seeding chambers (e.g., seeding chambers 700 and 800), suction rods, and one or more clips, caps, and mandrels. The components can be assembled into a seeding chamber assembly together with the manufactured scaffold. The dimensions of the components of the seeding system can be varied according to what is desired, for example, by the dimensions of the vascular graft.

[0115] Suitable materials for forming the various components of a seeding system include polymers, as well as metals, including, but not limited to, stainless steel, iridium, platinum, gold, tungsten, tantalum, palladium, silver, niobium, zirconium, aluminum, copper, indium, ruthenium, molybdenum, niobium, tin, cobalt, nickel, zinc, iron, gallium, manganese, chromium, titanium, aluminum, vanadium, and carbon, as well as combinations, alloys, and / or laminates thereof. B. Mandrel

[0116] Mandrels for use in the formation of TEVGs can be fabricated using means known in the art. Exemplary methods for fabricating mandrels include 3D printing. In some embodiments, the final mandrel design is converted into a computer-readable format suitable for 3D fabrication. An exemplary computer-readable format is the STL format. Suitable materials for 3D printing of mandrel models include polymers, metals, and carbon, as well as combinations thereof, alloys, and / or laminates. In some embodiments, the mandrel is fabricated from a liquefiable material, thereby enabling the mandrel to be easily released from the graft. The use of liquefiable mandrels also allows for the formation of complex shapes in the graft. How to create a C.TEVG

[0117] Methods for manufacturing TEVGs based on mold structures, such as custom-designed mandrels, are provided. TEVGs can be manufactured by electrospinning, stamping, mold forming, molding, weaving, and combination using any suitable method, such as melting, solvent processing, leaching, foaming, extrusion, injection molding, compression molding, blow molding, spray drying, extrusion coating, and fiber spinning, followed by processing into woven, nonwoven, or knitted structures.

[0118] In some embodiments, the TEVG is fabricated to include pores within the graft. Pores can be induced by any suitable method, including salt leaching, sublimation, solvent evaporation, spray drying, foaming, processing of the material into fibers, and subsequent processing into a woven or nonwoven device. In preferred embodiments, the fibrous matrix of the scaffold contains pores of appropriate size, allowing cells to adhere, grow, and / or differentiate. Since the diameter of cells is approximately 10 μm to 20 μm, pore diameters within this range are desired in certain embodiments. Preferably, the pores of the device are between 5 and 500 μm in diameter, more preferably between 5 and 250 μm, and more preferably between 5 and 100 μm. In certain embodiments, the polymer scaffold is generated or fabricated to more closely mimic the structure and composition of the natural extracellular matrix in order to promote the growth and differentiation of seeded cells and to facilitate the transplantation and / or embedding of the scaffold or cells grown thereon.

[0119] In preferred embodiments, TEVGs are produced by electrospinning a stock solution containing one or more polymers. Typically, the one or more polymers used to produce the TEVGs are biodegradable polymers. How to sow seeds in D.TEVG

[0120] Typically, seeding chambers, such as Chamber 700 or 800, are connected to a closed, disposable seeding system containing a fluid with cells.

[0121] Typically, the number of cells used to contact a graft or scaffold may be proportional to the surface area of ​​the graft, and the number of cells, including the values ​​at both ends, is approximately 1.0 × 10⁻⁶. 4 cells / mm 2 Graft ~1.0 × 10 6 cells / mm 2 Between the grafts, preferably including the values ​​at both ends, 0.7 × 10 5 cells / mm 2 Graft ~7.0×10 5 cells / mm 2This is between the grafts. In some embodiments, the polymer vascular graft or scaffold has a value of 0.5 × 10 including the values ​​at both ends. 6 cells ~500×10 6 Between cells, preferably 1.0 × 10 6 cells ~100×10 6 Contact is made with a quantity of cells between the cells. In a preferred embodiment, the graft is in contact with the cells for less than 3 hours, preferably less than 2 hours, for example, about 30 minutes, about 20 minutes, about 15 minutes, about 7 minutes, about 5 minutes, or about 1 minute. The contact is typically carried out in a sterile, closed seeding chamber.

[0122] Methods are also provided for increasing the patency of polymer vascular grafts or scaffolds, comprising the step of administering an effective amount of viable cells onto the graft or scaffold to reduce macrophage infiltration into the graft, promote host cell replenishment into the graft, or reduce or prevent platelet activation.

[0123] Methods have been developed to reduce or prevent postoperative stenosis in subjects. Subjects may be those at or at risk of restenosis or other vascular proliferative disorders. For example, in some embodiments, subjects may have, have, or will have undergone vascular trauma, angioplasty, vascular surgery, or graft artery disease. Methods may reduce neointima, stenosis, or restenosis, reduce or prevent thrombosis, or any combination thereof, in subjects compared to untreated control subjects.

[0124] Restenosis refers to the recurrence of treated coronary artery stenosis over time. Restenosis is most commonly defined as a reduction of more than 50% of the lumen (bidifferentiated angiographic restenosis) within the stent (intrastent restenosis) or within the stent, including 5 mm proximal or distal to the stent end (intrasegmental restenosis), on follow-up angiography (typically 6 or 9 months later). Restenosis can clinically appear over a period of 1 to 6 months after PCI (percutaneous coronary intervention).

[0125] Customizable systems and compositions for seeding cells into vascular grafts or scaffolds are described in U.S. Patent No. 9,090,863 and U.S. Patent Publication No. 2018 / 0353649. IV.How to use

[0126] Typically, seeding chambers are used for the rapid and efficient seeding of grafts and scaffolds. The seeded scaffolds are then used in cardiovascular surgery to repair or replace damaged blood vessels. A. Method using a seeding chamber 1. Flip seeding chamber

[0127] The method using the flip seeding chamber involves the following steps: fill the chamber, apply negative pressure, allow the chamber to expel 50% of its volume (at which point approximately 50% of the bottom of the scaffold is saturated with mononuclear cells (MNCs)), interrupt the negative pressure, invert the device, reintroduce negative pressure, and draw the remaining 50% of the volume into the "upper" half of the scaffold, which is then also saturated with MNCs. Thus, the bottom and upper halves of the scaffold are successively saturated, resulting in a fully saturated scaffold with minimal MNC loss (high seeding efficiency).

[0128] Negative pressure may be provided by a syringe, pump, or vacuum source. 2. Capacitor seeding chamber

[0129] The method of using a capacitor seeding chamber includes the following steps: The mandrel and scaffold assembly are carefully placed inside the seeding chamber. The seeding chamber is very narrow compared to the assembled mandrel / scaffold assembly. It is designed to hold a minimum volume of MNC solution. The cell seeding chamber may have a gap between the suction rod or mandrel and the housing between approximately 1 mm and 10 mm, more preferably between approximately 1 mm and 5 mm.

[0130] After the mandrel and chamber are secured together, the MNC is introduced into the device. The fluid quickly fills the chamber and "overflows" into an IV bag positioned above the chamber. After the entire volume of the MNC has been introduced into both the seeding chamber and the overflow (capacitor) IV bag, negative pressure is applied. The MNC fluid begins to pass through the scaffold, and the entire scaffold remains immersed in the MNC as the fluid level drops in the IV bag. The fluid is not completely drained from the IV bag until near the end of the seeding process, and the fluid level eventually falls below the top of the seeding device. The time during which the bottom of the scaffold remains immersed while the fluid is drained from the top of the scaffold and the scaffold is exposed to filtered air is very short. Thus, the top and bottom of the scaffold remained immersed throughout most of the seeding process. Only with respect to a small portion of the MNC solution is the top exposed while the bottom remains immersed. Therefore, the seeding gradient is minimized along the length of the scaffold.

[0131] Negative pressure can be provided by a syringe, pump, or vacuum source. B. Methods using grafts or scaffolding

[0132] The cell seeding dose for tissue-engineered vascular grafts (TEVGs) has been established as an effect-dependent variable for improving graft performance and utility, independent of cell incubation time. Typically, cells are seeded in TEVGs before implantation into the target. Typically, the cells are autologous cells from the intended recipient, and the seeding method may include the step of harvesting the cells from the recipient. One or more cell types can be isolated from the cell mixture using any technique known in the art. Thus, the method may also include the step of isolating or purifying the cells before application (i.e., seeding).

[0133] Cell seeding into TEVGs is performed using a kit or device, such as a closed-loop disposable seeding system. The closed-loop disposable seeding system may include either a flip seeding chamber or a capacitor seeding chamber for seeding cells onto a graft or scaffold. Preferably, the kit or device allows for the sterile and efficient seeding of a controllable quantity of cells into the TEVG. 1. How to use TEVG with seeded cells

[0134] Cell-seeded TEVGs can reduce or prevent the rate of postoperative stenosis in TEVGs compared to equivalent TEVGs without cells. Therefore, TEVGs can be seeded with an effective amount of cells to reduce or prevent one or more immune processes associated with the development of postoperative stenosis, including inflammation.

[0135] Tissue repair has four distinct phases, including a) coagulation; b) inflammation; c) fibroblast migration / proliferation; and d) the final remodeling phase in which the architecture of normal tissue is restored. In the earliest stages after tissue injury, epithelial and / or endothelial cells release inflammatory mediators, initiating an antifibrinolytic-coagulation cascade and inducing the development of coagulation and pseudoextracellular matrix (ECM). Platelet aggregation and subsequent degranulation promote vasodilation and increased permeability, allowing for efficient replenishment of inflammatory cells such as neutrophils, macrophages, lymphocytes, and eosinophils to the damaged tissue. Neutrophils are the most abundant inflammatory cells in the earliest stages of wound healing, but are rapidly replaced by macrophages after neutrophil degranulation. Activated macrophages and neutrophils induce fibroblast proliferation and replenishment, as well as generating various cytokines and chemokines that clear the wound, eliminate any invading organisms, and amplify the inflammatory response. Upon activation, adenoblasts are converted into muscle fiber cells, which secrete α-smooth muscle actin and ECM components. Finally, in the remodeling phase, epithelial / endothelial cells divide and migrate onto the temporary matrix to regenerate the damaged tissue. Thus, healing and neovascularization are finely regulated processes that balance the need to regenerate tissue and thicken the blood vessel walls without excessive thickening and stenosis or fibrosis. a. Macrophages

[0136] The presence of circulating monocytes and infiltrating macrophages has been shown to be crucial for wound healing and neotissue formation (Arras, et al., J Clin Invest, 101(1): 40-50 (1998)). However, the degree of macrophage infiltration at the site of tissue injury was also correlated with proliferation dysregulation and neointima formation (Hibino, et al., FASEB J. 25(12):4253-63 (2011)). Furthermore, numerous studies have shown that macrophages and fibroblasts are key effector cells involved in the pathogenesis of fibrosis (re-examined in Wynn, Nat Rev Immunol. 4(8):583-94 (2004)).

[0137] Following vascular injury, inflammatory monocytes (CD16-hi, CD64-hi, and CD14-hi in humans; CD115+, CD11b+, and Ly6c-hi in mice) are replenished in the injured tissue and differentiate into activated macrophages (Emr1-hi in humans; F4 / 80-hi in mice) after exposure to local growth factors, pro-inflammatory cytokines, and microbial compounds (Geissmann et al., Science 327: 656-661 (2010)). Excessive macrophage infiltration leads to stenosis, while complete inhibition of macrophage infiltration prevents neovascularization (Hibino, et al., FASEB J. 25(12):4253-63 (2011)).

[0138] Two distinct states of macrophage polarization activation have been defined: the classically activated (M1) macrophage phenotype and the surrogate-activated (M2) macrophage phenotype (Gordon and Taylor, Nat. Rev. Immunol. 5: 953-964 (2005); Mantovani et al., Trends Immunol. 23: 549-555 (2002)). The role of classically activated (M1) macrophages is that of effector cells in the TH1 cell immune response, while surrogate-activated (M2) macrophages appear to be involved in immunosuppression and wound healing / tissue repair. M1 and M2 macrophages have entirely different chemokine and chemokine receptor profiles; M1 secretes the TH1 cell-attracting chemokines CXCL9 and CXCL10, while M2 macrophages express the chemokines CCL17, CCL22, and CCL24.

[0139] The presence of M2 macrophages is associated with neointima-genesis and stenosis (Hibino, et al., FASEB J. 25(12):4253-63 (2011)). The correlation between macrophage infiltration, neotissue formation, and the degree of stenosis at specific time points after tissue graft implantation provides a means of preventing stenosis through modulation of macrophage activity.

[0140] Furthermore, macrophages are typically located near collagen-producing muscle fibers, and monocyte-derived macrophages have been shown to significantly prolong the post-injury inflammatory response as a prerequisite for fibrosis (Wynn and Barron, Semin Liver Dis., 30(3):245-257 (2010)). Macrophages produce pro-fibrosis mediators that activate fibroblasts, including platelet-derived growth factor (PDGF), potent chemotacticians, and transforming growth factor beta (TGF-B). In particular, a marked increase in the non-classical M2 (CD14+, CD16+) subset of macrophages correlated with pro-inflammatory cytokines and clinical progression in patients with chronic liver disease. During the progression of fibrosis, monocyte-derived macrophages release cytokines that perpetuate chronic inflammation and directly activate hepatic stellate cells (HSCs), resulting in their proliferation and conversion to collagen-producing myofibroblasts (Zimmermann, et al., PLOS One, 5(6):e11049 (2010)). b. Platelets

[0141] Aggregated platelets assist in vascular repair by secreting chemicals that attract fibroblasts into the wound area from surrounding connective tissue or, in the case of a dysregulated inflammatory response, from scar tissue, in order to heal the wound. In response to tissue injury, platelets become activated and release numerous growth factors that stimulate the deposition of extracellular matrix, such as platelet-derived growth factor (PDGF), a potent chemotactic, and transformed growth factor beta (TGF-β). Both of these growth factors have been shown to play a crucial role in the repair and regeneration of connective tissue. PDGF acts as a primary mitogen and chemotactic, significantly increasing the influx of fibroblasts and inflammatory cells, while also stimulating cell proliferation and gene expression. PDGF firmly adheres leukocytes to the vessel wall, eventually allowing them to migrate into the subendothelial tissue. However, platelet-derived chemokines are also known to induce smooth muscle cell (SMC) proliferation and play a role in neointimal hyperplasia and organofibrosis (Chandrasekar, et al., J Am College Cardiology, Vol 35, No. 3, pp. 555-562 (2000)). Increased expression of PDGF and its receptor is associated with the lung and skin tissues of scleroderma. In particular, there is evidence of an autocrine PDGF receptor-mediated signaling loop in the lung and cutaneous fibroblasts of scleroderma, indicating both TGF-β and PDGF pathways in chronic fibrosis of scleroderma (Trojanowska, Rheumatology;47:v2-v4 (2008)). Furthermore, deregulation of PDGF signaling is associated with cardiovascular signs such as pulmonary hypertension and atherosclerosis.

[0142] Medium layer smooth muscle cell (SMC) proliferation and migration in response to injury-induced PDGF are essential events involved in the thickening of the neointima (Fingerle, et al., Proc Natl Acad Sci., 86:8412 (1989); Clowes, et al., Circ. Res., 56:139-145 (1985)), ultimately leading to vascular narrowing and stenosis.

[0143] Other healing-related growth factors released by platelets include basic fibroblast growth factor, insulin-like growth factor 1, platelet-derived epidermal growth factor, and vascular endothelial growth factor.

[0144] TEVG can be seeded with an effective number of cells to create a regenerative immune environment that enhances wound healing and prevents stricture. TEVG can also be seeded with an effective number of cells to modulate platelet activity and function. Thus, TEVG can also be seeded with an effective number of cells to reduce or prevent platelet biological functions, such as platelet aggregation and platelet-derived growth factor (PDGF) production / expression.

[0145] A method using cell-seeded tissue-engineered vascular grafts to reduce postoperative graft stenosis includes the step of surgically implanting the cell-seeded graft into the patient's body or administering it by other means. Typically, the implantation method includes the step of attaching the graft to the arterial section to be replaced or augmented. Methods for attaching vascular grafts are known in the art. The method typically reduces or inhibits macrophage cell infiltration or conversion of macrophage cells from M1 to M2 phenotype or both, compared to a control such as an equivalent graft with no or fewer cell seeding. In some embodiments, the method reduces or inhibits macrophage cell proliferation without reducing or inhibiting the development of angiogenic tissue. Subjects may be identified as having, having, or being at risk of restenosis or other vascular proliferative disorders, such as vascular trauma, angioplasty, surgery, or graft arteropathy. Any of the methods described may include a step of identifying the subject that requires treatment.

[0146] The present invention will be further understood by reference to the following non-limiting embodiments. [Examples]

[0147] (Example 1) Seeding chamber for uniform cell seeding in grafts. material and method

[0148] U.S. Patent No. 9,090,863 describes how a closed, disposable tissue engineering vascular graft seeding device is used to seed mononuclear cells (MNCs) onto an implantable scaffold.

[0149] All scaffolds seeded using this method exhibited a gradient (distribution) of MNCs along the longitudinal axis of the graft (meaning fewer cells were seeded at the top of the graft than at the bottom). With the new concentration and seeding volume, the seeded scaffolds reached a saturation point towards the bottom 3 cm of the graft (Figures 2A and 2B), which did not allow for further cell seeding, resulting in bone marrow waste and potential risks to the patient.

[0150] The entire graft (13 cm in length) met the release criteria by saving 5 mm from the top and sowing sufficient MNCs in the bottom graft, but many more MNCs were found in the lower section of the graft than in the upper section. This embodiment presents two seeding chambers—flip and capacitor—regarding seeding devices to eliminate this imbalance. Flip seeding chamber

[0151] The "flip" seeding chamber eliminates variability in the distribution of MNCs on the scaffold. The central premise behind the device is to change its cross-section along the length of the seeding chamber.

[0152] Figures 3A–3E show examples of flip seeding chambers. The chamber's shape resembles an inverted hourglass, with the bulge (maximum cross-section) located approximately 40% below the top of the device's height. Both the top and bottom of the device narrow to accommodate a scaffolded mandrel, along with very minimal volume for partial clearance and excess liquid.

[0153] The effect of changing the cross-section is to effectively slow down the rate at which the MNC is drawn into the least seeded part of the scaffold.

[0154] The chamber is filled and vacuumed, the chamber having 50% of the discharged volume (at which point approximately 50% of the bottom of the scaffold is saturated with MNC), the vacuum is interrupted, the device is inverted, the vacuum is reintroduced, and the remaining 50% of the volume is drawn into the "upper" half of the scaffold, which is then also saturated with MNC. Through this method, the bottom and upper halves of the scaffold are successively saturated, resulting in a fully saturated scaffold with minimal MNC loss (high seeding efficiency).

[0155] Furthermore, the mandrel for this design has two vacuum nubs—one for drawing negative pressure upwards and the other for drawing negative pressure in the opposite direction. How to sow seeds in a graft using a flip chamber

[0156] 1. Secure the scaffolding to the mandrel (upper part of the device). 2. Secure the mandrel (upper) to the device's chamber (lower) portion by rotating it counterclockwise until the sealing O-ring is compressed. 3. Place all necessary mounting hardware, tubing, and air vents onto the device. 4. Fill the chamber with MNC. 5. Introduce negative pressure to the lower mandrel outlet. 6. 50% of the chamber's volume is drawn out through its interior. 7. Discontinue the vacuum. 8. Reverse the device 9. Reintroduce the vacuum. 10. Draw the remaining 50% of the volume into the "upper" part of the scaffolding. 11. Disassemble the device, and at this point the scaffolding will be ready for embedding.

[0157] The total sowing time was 7 minutes (7:0.1 ± 0.03 minutes).

[0158] After seeding, the seeded scaffold was cut into 1 cm wide rings along its length, forming 12 rings. The number of cells in each ring was counted, and the data is shown in Figure 5. The cells were counted using the PicoGreen dsDNA assay, and the total number of cells / mm 3 Presented as follows. Capacitor seeding chamber

[0159] The "capacitor" seeding chamber eliminates variations in the distribution of MNCs on the scaffold. The central premise behind the device is to reduce the MNC gradient by exposing the entire scaffold to the MNC seeding process for as long as possible.

[0160] Figures 4A-4C show an example of a capacitor seeding chamber. In this chamber, the mandrel has a scaffold attached to it. The scaffold and mandrel assembly is then carefully placed inside the seeding chamber. The seeding chamber is very narrow compared to the assembled mandrel / scaffold assembly. It is designed to hold a minimum volume of MNC solution. The minimum and maximum gaps between the scaffold and the chamber wall for uniform seeding may be 1 mm and 10 mm, for example, 1 mm and 5 mm.

[0161] After securing the mandrel and chamber together, the MNC is introduced into the device. The liquid quickly fills the chamber and "overflows" into the IV bag positioned above the chamber.

[0162] After the entire volume of MNC is introduced into both the seeding chamber and the overflow (capacitor) IV bag, a vacuum is applied. The MNC liquid begins to pass through the scaffold, and the entire scaffold remains immersed in the MNC as the liquid level drops within the IV bag. From the very end of the seeding process until just before the end of the process, the fluid is not completely drained from the IV bag, and the fluid level eventually falls below the top of the seeding device. The time during which the bottom of the scaffold remains immersed while the fluid is drained from the top of the scaffold and the scaffold is exposed to filtered air is very short. Thus, the top and bottom of the scaffold remained immersed throughout most of the seeding process. Only with respect to a small portion of the MNC solution is the top exposed while the bottom remains immersed. Therefore, the seeding gradient is minimized along the length of the scaffold.

[0163] Graft seeding method using a capacitor chamber 1. Fill the IV bag. 2. Fill the chamber. 3. Extract air from the chamber. 4. Fill the chamber with blood and cover the scaffolding. 5. Introduce a vacuum and use the vacuum to draw the fluid into the scaffolding. 6. Discharge from the chamber. 7. Remove the seeded scaffolding.

[0164] The total sowing time was 1 minute and 15 seconds.

[0165] After sowing, the sown scaffold was cut into 1 cm wide rings along its length, forming 12 rings. The number of cells in each ring was counted, and the data is shown in Figure 5. result

[0166] The results, shown in Figure 5, demonstrate uniform seeding of the scaffolds sown in either the flip seeding chamber (1) or the capacitor seeding chamber (2).

[0167] (Example 2) Scaffolding parameters that provide a spontaneous reversal of the constriction.

[0168] We developed a computational model to simulate neovascularization. This model was initially formulated from data collected in previous studies of TEVG development in mouse models and successfully described and predicted neovascularization over a two-year period. An initial analysis of data from US clinical trials is presented, as well as computational simulations suggesting that the early stenosis observed in previous clinical trials may have reversed spontaneously without intervention. Experiments using established large animal models are also presented to test a simulation proposal that a transitional period of TEVG stenosis may occur and then resolve spontaneously as part of the natural course of neovascularization. The changing geometry, composition, and biomechanical properties of TEVGs for up to 1.5 years after implantation in large animal models are characterized. The data validated the primary predictions of the computational model. Comparison of in vivo observations with results from the computational model allowed for further refinement of model parameter values, thereby enabling the collection of increased insights into the underlying mechanisms of the transformation of TEVGs from an autologous cell-seeded scaffold to living neovascularization capable of growth and remodeling. material and method TEVG Characterization of scaffolding

[0169] The scaffolds were characterized using scanning electron microscopy (SEM). A non-embedded scaffold sample was cut axially to create a 0.5 cm square, which was then attached to the SEM stage with carbon tape. The sample was sputter-coated with gold to a thickness of 3 nm under vacuum in argon gas, and imaged using a Hitachi S4800 SEM at 5 kV and 10 mA. The images were analyzed using FIJI image analysis software. Pore diameter was calculated from seven 100 × SEM images. Fiber diameter was calculated by averaging over at least five PGA fibers. Release standards and post-process testing

[0170] Cell samples (0.2 mL aliquots) were obtained, seeded on scaffolds (5 × 5 mm sections), and subjected to release and post-process monitoring. Cell counting and viability were performed using trypan blue-excluded hemocytometers. FACS was performed using FITC-CD45 and 7AAD to determine leukocyte counts and cell viability. Seeding efficacy was determined by quantifying the number of cells in the sample obtained from the pre-seeding and post-seeding solutions using a hemocytometer, and then calculating the difference between the number of cells in the pre-seeding and post-seeding solutions by dividing it by the number of cells in the pre-seeding solution. Clinical trials research design

[0171] The primary objective of this pilot study was to evaluate the safety of TEVG as an extracardiac modification Fontan conduit in patients with single ventricle cardiac malformations. The secondary objective was to determine the potential for TEVG growth by assessing its length changes between 6 months and 3 years after implantation. The initial design involved enrolling six patients and monitoring them with serial echocardiography and MRI over a 3-year period. growth analysis

[0172] The growth potential of the TEVG was assessed using serial MRI studies conducted at 6 months and 3 years post-implantation. TEVG growth potential was estimated by comparing the change in length over time to that of the patient's Glenn shunt SVC, measured from its first branch to the pulmonary artery anastomosis. Safety analysis

[0173] Patients were observed and evaluated by the research team during their initial hospitalization, at all planned follow-up appointments after TEVG implantation (1, 6, 12, 24, and 36 months post-surgery), and at any unplanned cardiology or cardiac surgery appointments or hospitalizations. In addition, the study nurses contacted the patients' parents or guardians monthly by telephone to re-examine their medical status using standardized surveys. All adverse events were recorded, their grade and attribution determined by the research team, and reviewed by the data safety monitoring board. The data were analyzed and compared to the incidence of graft-related complications in an initial pilot study conducted at Tokyo Women's Medical University Hospital (Japan). angioplasty

[0174] Patients who developed stenosis, defined as a reduction of >50% in the tubular diameter of the TEVG, were treated with angioplasty without stent placement. Computational model Constraint-mixed framework

[0175] The structurally significant components α=1, ..., n possess individual time rates of generation and decomposition as well as material properties, but are constrained to deform together with the graft as a whole. Therefore, the deformation gradient specific to the components at the current growth and remodeling time s with respect to the material generated at the intermediate time τ is,

number

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number

number

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[0176] First, a computational model was used to predict the changing normalized lumen diameter and wall thickness of the embedded clinical TEVG using model parameter values ​​determined by fitting biomechanical and geometric data from mouse experiments, and then the geometric (diameter / thickness ratio, D / H=16 for clinical scaffolds vs. D / H=3 for mouse scaffolds) and microstructural (scaffold pore diameter r for clinical scaffolds) of the simulated TEVG.p Appropriate modifications were made to the properties (41.9 μm vs. 11.2 μm for mouse scaffolds) to account for clinical and mouse scaffold differences. Based on mouse data, the inflammation-driving dynamics were considered, and since the immune response is known to play a crucial role in stenosis, four important inflammatory parameters (δ, β, K) were considered. i h , and K i max The effect of the parameters on the model output was investigated. Parametric studies were performed by varying the values ​​of individual inflammation parameters while keeping the other three parameters constant, and vice versa. This made it possible to isolate the effect of each parameter on the changing properties of TEVG, including normalized diameter, wall thickness, diameter compliance, and inflammation surface mass density. Diameter and thickness were normalized by their initial values ​​relative to the embedded TEVG scaffold; normalized diameter compliance was calculated as a change in diameter that increased by 50% in pressure from the homeostatic pressure normalized by the initial graft diameter. The parameters shown (see Table 1) were selected to obtain a wide range of potential physiological outcomes based on pilot simulations.

[0177] [Table 1]

[0178] Production rate m of various constituent components α α and removal rate q α The key equations within the overall computational model developed for TEVG, which explain the mass change through the changes in t, are the inflammatory burden (superscript i) resulting from the foreign body response and the circumferential wall stress t from the homeostatic target value. θ and the shear stress τ of the inner wall w The deviation Δ may be dependent on the biomechanical response (superscript m). Four key model parameters and their possible ranges were identified from previous experiments in immunocompetent and immunodeficient mice and linked to the inflammatory process potentially involved in neoangiogenesis. Metabolic turnover of varying mass (production m α (τ) and removal q α (due to s, τ)) ρ(s) (graft mass) = ρ p (s) (polymer mass) + ρ i (s) (immunologically mediated mass) + ρ m (s) (mechanically mediated mass) is,

Number

Number

Number

[0179] The aim of this study was to test the computationally generated output, i.e., to quantify the 1-year natural course of neotissue formation and thus neovascularization in an established IVC-interposed TEVG model. Seeded TEVGs were implanted into 24 lambs, and in vivo data were collected at 1 week, 6 weeks, 6 months, and 1 year via serial angiography and intravascular ultrasound. The 1-week time point was used for baseline anatomical information; providing data equivalent to the immediate postoperative period, but the animals were able to recover from the initial surgical insult and the risks associated with the prolonged anesthesia required to perform the implantation surgery and initial catheter placement during the same period were reduced. Pilot data demonstrated significant stenosis at 6 weeks, consistent with computational predictions, and later time points were selected to monitor graft performance over the long term. The primary endpoint was the narrowest cross-sectional area of the graft at each intravascular ultrasound imaging. No data were excluded from the study. Large animal scaffold

[0180] The TEVG scaffold was supplied by Ginze Ltd (Tokyo, Japan) and was identical to that used in clinical trials: a braided PGA core containing a 50:50 copolymer sealant of PCLA. Bone marrow aspiration, assembly, and TEVG implantation

[0181] Twenty-four young lambs underwent bone marrow aspiration (5 mL / kg body weight) and implantation of autologous cell-seeded TEVG as intrathoracic IVC intercalated grafts. The animals were anesthetized with propofol (5 mg / kg) for induction and isoflurane (1–4%) or propofol (20–40 mg / kg / hour) for maintenance. The lambs were placed in a lateral position, the area covering the iliac crest was shaved, and they were prepared using standard sterile techniques. A 2 mm incision was made, and an aspiration needle was inserted into the bone. 5 mL / kg of bone marrow was aspirated using a heparinized syringe (100 U / mL).

[0182] After aspiration, bone marrow was treated using Ficoll density gradient separation to isolate bone marrow-derived mononuclear cells as described above. Briefly, bone marrow was filtered through a 100 μm cell strainer to remove osteophytes and coagulations. A 1:1 dilution was achieved with phosphate-buffered saline (PBS), and the bone marrow was layered on Ficoll 1077 (Sigma-Aldrich, St. Louis, MO). The plasma and mononuclear cell layers were isolated after centrifugation. The mononuclear cell layer was washed twice with PBS to obtain a cell pellet, which was diluted with 20 mL of PBS and seeded onto a scaffold. The mononuclear cells were vacuum seeded onto the scaffold and incubated in plasma until the embedding time.

[0183] The scaffold was implanted in the intrathoracic IVC as described above. The lamb was placed in the left lateral decubitus position. Depending on the anatomical structure of each animal, a right thoracotomy was performed in the 5th or 6th intercostal space, and the thoracic IVC was dissected between the diaphragm and the right atrium. The superior vena cava atrial shunt was positioned so that perfusion was maintained during cross-clamping of the IVC. The vessels were clamped, and 2 cm segments of the seeded scaffold, matched to the diameter, were implanted, and end-to-end anastomoses were performed using continuous non-absorbable monofilament sutures. The natural vessels were not removed. Titanium vascular clips were applied to the tail of the sutures to mark the anastomoses for postoperative imaging. The chest wall, covering muscles, and skin layers were brought together again with absorbable sutures. Interventional imaging

[0184] Postoperative catheterization was performed at 1 week, 6 weeks, 6 months, and 1 year. Additional imaging was performed as needed based on the animal's clinical condition. After sedation and intubation, the lamb was placed in the left lateral decubitus position. The right internal jugular vein was cannulated, and a 9-French sheath (Terumo, Somerset, NJ) was inserted, followed by an intravenous bolus of heparin (150 U / kg). A 5-French JR 2.5 catheter (Cook Medical, Bloomington, IN) was passed through the SVC to the right internal jugular vein and into the right atrium. Then, using an angled Glidewire (Terumo), the JR catheter was passed through the TEVG and into the intraperitoneal IVC, where a Rosen exchange guidewire (Cook Medical, Bloomington, IN) was placed. Next, the JR catheter was replaced with a 5-French multitrack angiography catheter (NuMed, Hopkinton, NY), which was used to measure hemodynamic pressure in the intraperitoneal IVC, intrathoracic IVC below and above the TEVG, and within the TEVG. The mean pressure gradient was calculated by subtracting the mean pressure above the TEVG from the mean pressure below the TEVG. Digital angiography was then obtained by injecting Ioversol 68% (Mallinckrodt Pharmaceuticals, Raleigh, NC) through the multitrack angiography catheter positioned in the intraperitoneal IVC. Diameter was measured at seven points: intraperitoneal IVC, lower intrathoracic IVC (diaphragmatic side of the TEVG), proximal anastomosis (defined relative to blood flow), intermediate graft, distal anastomosis, upper intrathoracic IVC (atrial side of the TEVG), and the most severely narrowed area. The proximal and distal anastomoses were identified by the surgically placed radiopaque clips mentioned earlier. A 0.035-inch digital intravascular ultrasound catheter (Volcano, San Diego, CA) was advanced within the graft, over the Rosen guidewire. This was used to obtain images at the same seven points measured during angiography. These images were analyzed using Volcano software to obtain the cross-sectional areas described above.

[0185] Due to the requirements for size-matched grafts in implants, angiography and IVUS data are normalized to their respective 1-week measurements, and therefore graft stenosis is expressed as a magnification change relative to the baseline intermediate graft size at 1 week, i.e.

number

[0186] The development of neovascular tissue within the TEVG was measured using intravascular ultrasound and reported as a percentage of the TEVG diameter. Graph reconstruction of the IVUS imaging data was performed using Rhino 3D (Seattle, WA). Euthanasia

[0187] At the prescribed endpoint, animals were deeply sedated with ketamine (20 mg / kg) and diazepam (0.02–0.08 mg / kg), followed by induction of bilateral pneumothorax and blood loss. A complete veterinary necrosis was performed at the time of TEVG explantation. Animals were also euthanized if fatal stenosis occurred, which was defined here as graft stenosis with systemic symptoms. Animals not euthanized for fatal stenosis were euthanized at 6 months (n=5) and 12 months (n=2) post-implantation. The remaining animals were kept alive for long-term follow-up, and three were euthanized at 18 months for late mechanical testing. Characterization of the new organizational structure Histology and immunohistochemistry

[0188] TEVG explants were fixed in 4% formalin, dehydrated, and embedded in paraffin. Subsequently, 4 μm transverse sections of the intermediate grafts were prepared, placed on slides, and heat-fixed. Standard techniques were employed for hematoxylin, iosin, and Picro-Sirius Red staining. Immunohistochemistry was used to detect macrophage antigen CD68 (CD68, 1:500, Abcam) and alpha smooth muscle actin (aSMA, 1:2000, Dako). The samples were subjected to heat-induced antigen retrieval with Dako target retrieval solution (90°C, pH 6.0), followed by blocking of endogenous peroxidase activity (0.3% H2O2 in H2O) and nonspecific binding (3% normal goat serum in Background Sniper, BioCare Medical). After incubation of the primary antibody, the sections were sequentially incubated with appropriate biotinylated secondary antibody (1:1500, Vector) and streptavidin-horseradish peroxidase (Vector). DAB+ substrate chromogen (Vector) was used for color development. All samples were counterstained with Gill's hematoxylin (Vector), then dehydrated and covered with coverslips. Image quantification

[0189] Microscopic images of tissue staining were quantified using ImageJ. For Picro-Sirius Red stained specimens, the entire vascular area was obtained using tiled 25× magnification images. The area of ​​interest was quantified using the Color Threshold command. For immunohistochemically stained slides, four representative tiled images were taken at 100× around the vessels, each capturing the full width of the graft or the section of the original IVC. Images were evaluated using color deconvolution and threshold commands to quantify the positively stained area or count positive cells. Biaxial mechanical testing

[0190] The composite specimen, including the TEVG and adjacent proximal and distal thoracic IVC, was dissected from the right atrium to the diaphragm (59.3±16.2 mm) at 18 months after lamb implantation and then the perivascular tissue was removed by blunt dissection. The specimen was cannulated with a conventional acrylic cannula, mounted in a conventional computer-controlled test device, and immersed in Hank's buffered physiological solution at room temperature. The outer diameter was measured using a video camera, the length was defined via a stepping motor; the luminal pressure and axial force were measured using standard transducers. The specimen was equilibrated for 15 minutes at a low flow rate at a pressure of 5 mmHg and then pre-conditioned with 6 cycles of pressurization (1 to 30 mmHg), both performed at a fixed value of in vivo axial elongation. Passive data acquisition consisted of cyclic pressure-diameter tests (1.5 to 30 mmHg) at three different fixed values of axial elongation (95%, 100%, and 105% of the in vivo value). Data from the unloaded curves of each protocol were used for the analysis. Verification of the computational model

[0191] After collecting data on the changing sheep TEVG and noting the differences between the predicted (based on parameters from previous mouse studies) geometry and the measured changing geometry, the values of four important inflammatory parameters were identified using a non-invasive optimization technique, the Surrogate Management Framework previously detailed, to obtain the changing normalized lumen diameter and wall thickness of the lamb grafts throughout the first year of implantation as measured by intravascular ultrasound. Since intravascular ultrasound showed a non-circular graft cross-section, the hydraulic diameter of the TEVG was calculated from area measurements related to the fitting process. The boundaries for the optimization of the Surrogate Management Framework were informed by a parametric study. To validate the computational model, the compliance of the TEVG measured at 18 months was compared with that predicted from the growth and remodeling model. Statistical analysis

[0192] Statistical analysis was performed and graphs were created using GraphPad Prism version 7.03 (GraphPad Software, Inc., La Jolla, CA). Comparisons of the incidence of early stenosis in clinical trials in Japan versus the United States were performed via two-sided Fisher's exact test. Serial measurements (angiography and IVUS) from the sheep study were first normalized to paired weekly values ​​to control for the various scaffold sizes used in implantation and to ensure size agreement with the varying degrees of anatomical stenosis resulting from congenital vessels or implantation procedures, and are therefore expressed as a magnification change relative to each weekly measurement. Pressure measurements or normalized angiography and IVUS values ​​were analyzed using a standard one-way ANOVA with Tukey's post-hoc multiple comparison test. Histological morphometric and microscopic data (wall thickness, aSMA+ area fraction, CD68+ cells / mm²) were also analyzed. 2 Collagen (and collagen area fraction) was analyzed via a standard one-way ANOVA with Tukey's post-hoc multiple comparison test. In all statistical tests, α was restricted to 0.05, and p-values ​​< 0.05 were considered statistically significant. Ethical compliance Clinical trials

[0193] Institutional Review Board (IRB) approval was obtained from Yale University (HIC #0701002198) and Nationwide Children's Hospital (IRB12-00357). This clinical trial was conducted under FDA IDE 14127 in accordance with the Clinical Trial Standards Guidelines. Sheep research

[0194] The Institutional Animal Care and Use Committee of Nationwide Children's Hospital (Columbus, OH) reviewed and approved the protocol (Ar13-00079). A representative of the animal care staff monitored all animals during the intraoperative and postoperative process. Animal care was within the scope of the humanitarian guidelines published by the Public Health Service and the National Institutes of Health (Bethesda, MD) in the Care and Use of Laboratory Animals (2011), and within the scope of USDA regulations as described in the Animal Welfare Act. result TEVG design and characterization

[0195] TEVGs were assembled on a biodegradable tubular scaffold by seeding autologous bone marrow-derived mononuclear cells. The scaffold (Ginze Ltd, Kyoto, Japan) was prepared from poly(glycolic acid) fibers (PGA) and a copolymer of caprolactone and lactide (PCLA) synthesized by ring-opening polymerization in a 50:50 molar composition. The PGA fibers were knitted into tubes, the inner and outer surfaces were coated with a PCLA solution, and then freeze-dried under vacuum to create a knitted PGA fiber matrix embedded in a porous sponge.

[0196] The scaffold was designed to decompose through hydrolysis over approximately six months. The dimensions of the scaffold were measured as an inner diameter of either 16±0.5 mm or 18±0.5 mm, a length of 13±0.5 cm, and a wall thickness of 0.7±0.1 mm (Figure 1A). Quantitative scanning electron microscopy of the inner surface revealed an average pore size of 41.9±2.7 μm and a porosity of 0.87±0.01. On the outer surface, the average pore size was 36.4±6.6 μm and the porosity was 0.86±0.02. PGA fiber bundles were woven into a weft pattern, with an axial interlayer distance of 1 mm and a circumferential peak-to-peak distance of 1 mm. The average PGA fiber diameter was measured as 15.8±1.0 μm. The TEVG was assembled in accordance with Good Manufacturing Practice (GMP) regulations. Bone marrow (5 ml / kg body weight) was collected on the day of surgery, and the mononuclear cell fraction was separated using density centrifugation in Ficoll. In a US clinical trial, this procedure yielded an average of 20.6 × 10⁶ cells. 6 (Range 19.0~22.2×10 6 Mononuclear cells were obtained, with an average cell viability of 92.6% (range 86.5–96.8%). Flow cytometry demonstrated that 78.3% (range 73.2–85.3%) of these cells were CD45+. These cells were then seeded onto polymer scaffolds using a conventional vacuum system, resulting in an average seeding efficiency of 42.7% (range 23–61.4%). The seeded scaffolds were incubated for 2 hours in autologous plasma (obtained from the non-mononuclear cell fraction after density gradient centrifugation) and then implanted as vascular scaffolds on the same day the TEVGs were assembled. All TEVGs used in the clinical trial met the release and post-process monitoring criteria (Table 2).

[0197] [Table 2] Clinical performance of TEVG

[0198] FDA-approved clinical trials evaluated the safety and growth potential of TEVG when used as a vascular conduit connecting the inferior vena cava (IVC) to the pulmonary artery in children with single ventricle malformations undergoing modified Fontan surgery (Figure 12 and Table 3). [Table 3]

[0199] Growth potential was assessed using serial magnetic resonance imaging (MRI) studies performed at 6 months and 3 years post-implantation. Noting that IVCs are volumetric vessels whose diameter changes moment by moment, TEVG growth was estimated by comparing its change in length over time to an internal control: the superior vena cava (SVC) when anastomosed to the pulmonary artery (referred to as the Glenn shunt, which is a component of the Fontan procedure). Four TEVGs increased in length by 2.5 mm (range 1.1–4.2 mm) between 6 months and 3 years post-implantation, compared to the Glenn shunt which increased in length by 1.5 mm (range 0.9–2.4 mm) during the same period. The mean percentage increase in length for both TEVGs and Glenn shunts was 7%.

[0200] Safety analysis demonstrated no graft-related deaths, worst-case graft failures, or complications requiring graft replacement during the 3-year study. All four patients remained healthy for 4–7 years post-implantation. However, three of the four patients developed fatal stenosis (narrowing of >50% of graft diameter), which was successfully treated with angioplasty 5–8 months post-implantation. There were no additional graft-related complications. Enrollment was limited to four patients instead of the intended six due to this unexpectedly high incidence of early TEVG stenosis: in the US trial, three of the four patients (75%) developed stenosis and were treated with angioplasty, while in the original Japanese trial, only one of 25 patients (4%) developed stenosis and required angioplasty within 3 years post-implantation (bilateral Fisher's exact test, p<0.01).

[0201] Figure 12 shows a flowchart of a pilot study investigating the clinical use of tissue-engineered vascular grafts in congenital cardiac surgery. The study objectives were as follows: The primary objective of this pilot study was to determine the safety of using tissue-engineered vascular grafts as large-diameter, high-flow, low-pressure conduits in pediatric patients requiring extracardiac double vena cava pulmonary anastomosis (EC TCPC) for palliative treatment of single-ventricle cardiac malformations. The secondary objective was to determine the growth potential of tissue-engineered vascular grafts using serial magnetic resonance angiography. The study design was as follows: This study was a promising non-randomized Phase 1 clinical trial to determine the safety of using tissue-engineered vascular grafts as conduits for EC TCPC. Eligibility criteria: All patients who were considered candidates for EC TCPC for completion of modified Fontan for palliative treatment of their congenital cardiac malformations during the course of institutional investigation were considered for enrollment in the study. Patients were not excluded on the basis of age, sex, or race. Inclusion criteria included volunteers and informed consented patients with single ventricular anomalies who were candidates for EC TCPC. Exclusion criteria: Emergency / rescue surgery status, major chromosomal anomalies, need for pacemaker, 4µm 2This included pulmonary vascular resistance greater than 2 (u = Wood's unit), abnormal venous drainage (obstructed IVC), the presence of moderate to severe atrioventricular valve regurgitation, or other significant medical problems. In the investigator's opinion, a history of any other condition or other significant medical problems excluded compliance with the procedures specified in the protocol. Study site: This single-institutional study was initiated at Yale-New Haven Hospital but subsequently moved to Nationwide Children's Hospital. Data housing facility is on-site (Nationwide Children's Hospital, Columbus, OH). Outcomes: The primary endpoints of the study included determining graft failure rates as well as graft-related morbidity and mortality. Graft failure was defined as any graft narrowing / occlusion or dilation / rupture requiring surgical or endovascular intervention. Graft-related morbidity and mortality included any postoperative complications, such as any thromboembolic or infectious events, that were deemed likely to be caused by the tissue-engineered vascular grafts requiring treatment, determined by the investigators, and confirmed by the data safety monitoring board. Sample size: The initial plan was to enroll a total of 6 patients. The study was terminated with a total of 4 patients because a higher-than-expected stenosis incidence was experienced. No additional trials were conducted in the study, as it was a pilot study to see the rate of adverse events among patients receiving these vascular grafts. Study dates: The study received FDA approval in December 2009 and implanted the first patient in August 2011 while at Yale New Haven Children's Hospital, affiliated with Yale University. The study moved to Nationwide Children's Hospital, affiliated with Ohio State University, in September 2012. Enrollment resumed in March 2014 after completion of necessary institutional testing, and all clinical follow-ups were completed in August 2017. Study concluded: A higher incidence of TEVG stenosis was experienced in the clinical trial than predicted based on the initial pilot study in Japan. Based on these findings, the study was voluntarily suspended and a closed DSMB meeting was requested.The DSMB was subsequently held and it was recommended to temporarily suspend the study and re-evaluate the data. Trial registration: NCT01034007. Construction of a computational model of angiogenesis

[0202] To describe changes in soft tissue mass and microstructure as criteria for the model, general constrained mixture theory was used. A computational model was previously developed to simulate the changing geometry, composition, and mechanical properties of TEVGs implanted in mice over time. The model considered the degradable polymer scaffold, the organization of the angiogenic tissue, and the extracellular matrix occupied by newly synthesized collagen as separate structurally significant components. Specifically, data-driven constitutive relationships defined the unique material properties for each of the α = 1, 2,... n components, as well as their individual production and removal rates: the neoHookean relationship described the mechanical behavior of the polymer, the Fung-exponential functional relationship described the relationship of the neo-tissue, the mass density production rate m)τ>0 was dependent on mechanobiological stimuli such as wall stress and immunobiological stimuli, was proportional to macrophage invasion, and removal was defined via a survival function q)s, τ∈[0,1] that tracked the percentage of material produced at time τ∈[0,s] that was retained at time s. For example, deviations in wall stress from steady state regulated mechanosensitive-mediated dynamics since vascular cells typically promote mechanobiological homeostasis, while the scaffold microstructure, as quantified by scanning electron microscopy, modulated inflammation-promoting dynamics since pore sizes sufficient for cell infiltration are extremely important regulators of macrophage activity and phenotype.

[0203] Previous studies in immunocompetent and immunodeficient mice identified four important model parameters that control the deposition and degradation of the inflammation-promoting extracellular matrix (Table 1): δ modulates the onset and duration of the inflammatory response, β controls the strain of the production function, Κ i h controls the rates of inflammatory extracellular matrix production and degradation, Κ i maxIt expands and contracts the scale of the inflammatory effect of extracellular matrix degradation, and its production is

number

number

number

number

[0204] Numerous simulations of neovascularization (Figure 7A) were obtained by varying the values ​​of individual inflammatory parameters while maintaining other fixed parameters (Table 1). This made it possible to decouple the effects of individual contributors on changes in key clinical parameters, including TEVG radius, wall thickness, diameter compliance, and neovascular tissue accumulation over desired time courses, which is not possible in in vivo studies. The parameter ranges shown yielded a wide range of potential physiological outcomes based on more extensive preliminary studies (not shown). Overall, the simulations predicted similar findings to those suggested by clinical trials, namely, early TEVG narrowing due to increased inflammation-driven neovascular tissue wall thickening and lumen narrowing. Interestingly, however, the model also predicted that such narrowing could spontaneously reverse in a wide range of cases as the immune response weakens along with polymer degradation, and subsequent mechanically mediated neovascular tissue degradation outweighs the accumulation, as wall stresses due to both narrowing and thickening fall well below normal homeostatic values ​​(Figures 7A-7H). The possibility that TEVG stenosis could spontaneously reverse had not been considered until now. Consequently, computational models produced unpredictable outcomes that were experimentally tested using large animal models.

[0205] In Figures 7A-7H, each plot represents δ, β, and Κ. i h , and K i max Focusing on the variability, the graph shows early changes in normalized lumen diameter (top row) and wall thickness (bottom row) up to 52 weeks post-implantation, with arrows indicating the direction of increasing values. The graph shows the predicted transient decrease and subsequent recovery in lumen diameter, which are partly due to wall thickening that initially penetrates the lumen within the rigid polymer scaffold and then degrades, thus increasing the mechanical control of biological and mechanical factors while reducing immunoregulation. TEVG stenosis spontaneously reverses in large animal models.

[0206] The predictions of the computational model were tested by conducting a time-course study on a sheep intrathoracic IVC intervening graft model. The sheep IVC intervening graft is a validated model used as a surrogate for the Fontan procedure because there are no large animal models with single ventricle malformations and the performance of the Fontan procedure in animals with structurally normal hearts is associated with excessive mortality (>80%). Size-matched TEVGs were implanted in 24 young lambs (Figures 13A and 13B: Sheep study design and surgical outcomes). TEVG morphology was continuously monitored in all living lambs over a period of 12–18 months using angiography and intravascular ultrasound to measure lumen diameter and cross-sectional area. Angiography revealed significant TEVG stenosis at 6 weeks (-0.48 ± 0.16 times diameter change from 1 week, one-way ANOVA, Tukey's multiple comparison test: a=0.05, p<0.0001). As predicted, the stenosis spontaneously improved within 6 months without endovascular intervention, and all TEVGs remained patent for 1 year and beyond.

[0207] Intravascular ultrasound suggested that early TEVG stenosis resulted from the additional growth of neoplastic tissue on the lumen surface of the scaffold, thus leading to wall thickening and lumen narrowing. At 6 weeks, TEVG stenosis was localized to the intermediate distal segment of the graft (i.e., the region closer to the heart), while no change was observed at the proximal anastomosis. Quantitative intravascular ultrasound evaluation showed a significant decrease in lumen area from 1 to 6 weeks (a change in area from the intermediate graft area at 1 week to -0.67 ± 0.17 times, by one-way ANOVA and Tukey's multiple comparison test: a = 0.05, p < 0.0001), the stenosis reversed at 6 months, and the graft remained patent at 1 year (Figure 8A). During the same period, the wall reached its maximum thickness in 6 weeks (range 1.5–4.6 mm, one-way ANOVA, Tukey's multiple comparison test: a=0.05, p<0.0001), and then gradually thinned over the course of one year (Figure 8B).

[0208] Hemodynamic data were also collected during each angiography. The mean pressure gradient across the graft followed the morphological changes described above. The gradient increased significantly from weeks 1 to 6 (0.5±0.5 vs. 11.8±5.5 mmHg, p<0.001), then decreased to near baseline by 6 months (1.3±2.8 mmHg, one-way ANOVA, Tukey's multiple comparison test: a=0.05, p<0.001 vs. 6 weeks, p=0.8883 vs. 1 week) (Figures 14A-14D: Analysis of angiography, IVUS, and hemodynamic data). The maximum pressure gradient tended to correspond to the most narrowed graft. Of the 22 animals that developed stenosis, 2 (9.1%) also developed symptoms including ascites, lethargy, and weight loss. Both of these lambs had a mean pressure gradient >19 mmHg at 6 weeks, and their grafts were two of the three narrowest on angiography, both measuring a lumen diameter of less than 4 mm at their narrowest point. These two symptomatic animals were euthanized. Despite the formation of significant TEVG stenosis in all animals, the stenosis resolved in the remaining lambs, who remained asymptomatic. No grafts suddenly closed, and no animals died as a result of stenosis. Validation of the computational model

[0209] The parametric study was parameterized based on a mouse IVC-mediated TEVG study consisting of a scaffold design similar to those used in sheep and clinical grafts, but we also tested whether the model could describe in vivo sheep data. These data fit well over all time (R²=0.83) using many model parameters from the mouse experiment (e.g., the mechanical properties of collagen and the baseline ratio of collagen turnover), but generated sheep-specific values ​​for four key parameters controlling transient inflammatory responses: δ=0.32 ⋅ 1, β=3.96, K i h =5.13, and K i max=72. These best-fit values ​​for the parameters were identified using the optimization method of the Surrogate Management Framework, as in previous studies of the growth and remodeling of innate blood vessels. Among the various computational measurements, particular attention should be paid to the changes in lumen diameter and wall thickness that determine the presence or absence of stenosis (Figures 8A and 8B). The changes in the cellular composition of neovascularization are consistent with the predictions of the computational model.

[0210] Changes in sheep neonatal tissue over time were characterized using histology and immunohistochemistry. Histological sections confirmed changes in the lumen and wall of the TEVG observed by in vivo imaging and verified that transient lumen narrowing was mainly due to scaffold thickening and, in part, to neonatal tissue formation on the lumen surface of the scaffold, which appeared to resolve at 6 months post-implantation. Quantitative histological morphometry demonstrated that wall thickening peaked at 6 weeks post-implantation, consistent with computational predictions (Figures 9A and 9B). Immunohistochemical staining for α-smooth muscle actin (aSMA) identified some positive cells along the lumen surface of the scaffold and within the scaffold; their number was highest at 6 weeks. The aSMA+ cells formed along the lumen surface were presumed to be smooth muscle cells that proliferated and contributed to the neonatal tissue causing TEVG narrowing. The total number of aSMA+ cells peaked early, fitting well with the early overgrowth and generation of the matrix predicted by computational models (Figures 9C and 9D). The extent of macrophage infiltration was assessed using immunohistochemical staining for CD68. Quantitative immunohistochemistry revealed the maximum macrophage density at 6 weeks. Beyond 6 months, the number of macrophages decreased, consistent with scaffold degradation, again fitting well with computational predictions (Figures 9E and 9F). In summary, these findings indicate that TEVG stenosis formation is largely driven by inflammation. The computational model accurately predicts the biomechanics of neovascularization.

[0211] The sum of the mechanical contributions of the polymer scaffold and neonatal tissue (primarily determined by the extracellular matrix components of the neonatal tissue) determines the biomechanical properties of the TEVG. Scaffold degradation and neonatal tissue formation were characterized using polarized images of Picro-Sirius Red (PSR) stained sections from sheep TEVGs obtained at 1 week, 6 weeks, 6 months, and 1 year post-implantation, revealing both PGA fiber degradation and collagen fiber deposition and maturation. PGA fibers remained highly organized within the scaffold 1 week post-implantation but began to thin at 6 weeks, showing evidence of premature fragmentation. Very few thin individual fragments of PGA fibers could be seen at or beyond 6 months post-implantation. In contrast, minimal staining was detected in fibrous collagen at 1 week. The total amount of collagen in the neonatal tissue peaked at 6 weeks post-implantation, with a significant amount of fine (green) fibers present within the scaffold and along its luminal surface. Collagen density steadily increased over the first year as it compacted and matured (Figures 10A and 10B). This collagen appeared to be the major extracellular matrix component of the neonatal tissue associated with TEVG stenosis. The collagen also appeared to remodel between 6 weeks and 6 months as the scaffold degraded and the wall thinned; at 6 months, the collagen fibers appeared orange, indicating they were of medium thickness. One year post-implantation, the wall had thinned further, and the collagen fibers appeared thicker (red) and denser, showing normal, mature vascular collagen.

[0212] In vitro biaxial mechanical testing of TEVGs excised 1.5 years after implantation revealed a structural response (pressure-diameter) of TEVGs similar to that predicted by the model (Figure 10C). The final compliance of TEVGs predicted by the computational model as a validation step, rather than based on fitting to the data, was in good agreement with the values ​​found from in vitro mechanical characterization (Figure 10D). Importantly, the relevant values ​​of material parameters in the computational model suggested that this bulk behavior was derived from inflammation-driven matrix turnover rather than mechanically mediated matrix turnover throughout much of neovascularization, as mechanical stimuli for neovascularization are attenuated by high wall thickness and low diameter. Consideration

[0213] The first FDA-approved clinical trial evaluating the use of TEVG for the repair of complex congenital heart defects confirmed that while stenosis is the most widespread graft-related complication, the formation of early TEVG stenosis occurs at a much higher incidence than previously observed.

[0214] To gain mechanical insights into the complex immunological and mechanical processes underlying the formation of early TEVG stenosis, a computational model of neovascularization was used to parameterize the relative contributions of numerous critical factors controlling neovascularization. This model predicted spontaneous resolution of stenosis, and the findings were reproducibly confirmed in a sheep IVC-mediated graft model. Asymptomatic TEVG stenosis in lambs could be safely monitored without acute graft failure or thrombosis throughout the entire study period of 1–1.5 years. Based on the available data, symptoms and the mean pressure gradient across the graft could be used as primary criteria for assessing the clinical significance of TEVG stenosis, as well as morphometric changes alone. Since most stenosis resolved spontaneously, appropriate monitoring rather than overly invasive intervention may be a positive approach.

[0215] In summary, results from previous studies linked with human clinical trials, computational simulations, and sheep studies suggest that the implantation of cell-seeded PGA / PCLA scaffolds as TEVGs within vascular structures initiates inflammation-driven, mechanics-mediated changes in neovascularization over time. The polymer initially provokes a strong xenobiotic response, causing host inflammatory cells to infiltrate the scaffold. Simultaneously, the scaffold partially shields the infiltrating vascular cells and the neovascular tissue they deposit from hemodynamically imposed mechanical forces until the structural integrity of the polymer is lost. Thus, the mechanicobiological processes appear to intensify as the integrity of the scaffold diminishes, and low wall stresses resulting from excessive graft thickening cause degradation that outpaces subsequent deposition, resulting in the progressive resolution of initial inflammation-driven stenosis. However, the inflammatory consequences are not entirely negative; in fact, some inflammation underlies early host cell replenishment and neovascularization. Infiltrating single-cell / macrophage orchestrates early neotissue formation, particularly by inducing endoplasty of host endothelial and smooth muscle cells from adjacent blood vessel walls. Therefore, overall TEVG functionality requires balanced degradation of the polymer scaffold (primarily by hydrolysis) and proper deposition of neotissue. It appears crucial to promote but limit inflammation while simultaneously optimizing the timing of load transfer from the initial rigid polymer scaffold to the cell-matrix complexes constituting the neotissue (Figures 11A-11C).

[0216] Figures 11A and 11B illustrate how scaffolding implantation triggers a foreign body reaction and mechanically mediated neovascular tissue remodeling. Macrophages infiltrate the scaffolding. While macrophages are essential for neovascular tissue formation, excessive macrophage infiltration leads to stenosis. Immunobiological contributions continue to build while polymers fragment and degrade, but begin to subside as the polymers disappear; simultaneously, smooth muscle cells change the amount of stress shielding to establish mechanical homeostasis in response to mechanical stimuli of the hemodynamic environment by remodeling the extracellular matrix. In situations where cells of neovascular tissue sense low mechanical stimuli, the cells disintegrate and remodel the extracellular matrix, reducing stress shielding until mechanical homeostasis is re-established.

[0217] Similarly, under conditions of high mechanical stimulation, these cells generate and remodel extracellular matrix to increase stress shielding until mechanical homeostasis is achieved. The biomechanical contribution continues to build as the stress shielding capacity of the degradable polymer scaffold diminishes. During the first six months after implantation, macrophage orchestration of neotissue formation occurs through parasecretory signaling that drives cell migration and extracellular matrix generation, while after scaffold degradation and loss, its biomechanical integrity and dynamics are mediated by the ability of vascular cells to sense and respond to their local mechanical environment through the deposition and degradation of neotissue. It should be noted that the loss of scaffold biomechanical integrity precedes its disappearance, and therefore the overlap in inflammation and mechanical contributions to neotissue turnover. Importantly, the overgrowth of neotissue during the inflammation-driven period results in neotissue wall stresses well below the normal homeostatic target, thus promoting biomechanically mediated degradation of neotissue beyond deposition and contributing to the spontaneous resolution of stenosis.

[0218] In conclusion, this study highlighted the usefulness of combining advanced computational modeling and model-driven preclinical experiments in translational studies. A framework was developed to create robust computational models that can accurately predict the geometric, compositional, and biomechanical changes in the newly formed tissue that ultimately determine graft performance. The results suggested that the early stenosis observed in clinical trials, which led to the premature termination of the study, may have resolved spontaneously without angiogenesis. Computational simulations predicted that the scaffold could be modified to reduce the degree and duration of stenosis by changing the scaffold design (including fiber diameter, fiber alignment, porosity, and pore size) to reduce associated inflammation and stress shielding. The present invention provides, for example, the following items: (Item 1) A cell seeding chamber for use in a closed-type disposable cell seeding system, Housing having width and length, A suction rod that can be inserted into the housing, and a cap including one or more lateral fluid ports, A mandrel positioned on the suction rod, and Base Includes, A cell seeding chamber in which the housing has a variable width along its length, or a gap between the suction rod or the mandrel and the housing. (Item 2) The cell seeding chamber according to item 1, wherein the mandrel is a porous mandrel. (Item 3) A cell seeding chamber according to item 1 or 2, wherein there is a gap between the suction rod or the mandrel and the housing between approximately 1 mm and 30 mm, preferably between approximately 1 mm and 20 mm, approximately 1 mm and 10 mm, or approximately 1 mm and 5 mm. (Item 4) A cell seeding chamber according to item 1 or 2, wherein the housing has a variable width along the length of the housing. (Item 5) The cell seeding chamber according to item 4, wherein the housing has a region having a maximum width positioned between approximately 30% and 60% of the length of the housing. (Item 6) A cell seeding chamber having a lateral vent port, as described in any one of items 1 to 5. (Item 7) A cell seeding chamber according to any one of items 1 to 6, configured to receive a scaffold between the mandrel and the housing. (Item 8) A polymer biodegradable vascular graft or scaffold comprising internal and external structures for inducing spontaneous reversal of stenosis, wherein the polymer biodegradable vascular graft or scaffold allows for uniform dispersion of cells within the vascular graft or scaffold. (Item 9) A polymer biodegradable vascular graft or scaffold according to item 8, comprising polymer fiber layers arranged axially and polymer fiber peaks arranged circumferentially. (Item 10) A polymer biodegradable vascular graft or scaffold according to item 8 or 9, comprising an average pore diameter on the inner surface and different average pore diameters on the outer surface. (Item 11) A polymer biodegradable vascular graft or scaffold according to any one of items 8 to 10, having an average pore diameter on the inner surface between approximately 35 μm and 50 μm, preferably between approximately 38 μm and 50 μm, and most preferably between approximately 38 μm and 45 μm. (Item 12) A polymer biodegradable vascular graft or scaffold according to any one of items 8 to 10, having an average pore diameter on its outer surface between approximately 25 μm and 45 μm, preferably between approximately 27 μm and 43 μm, and most preferably between approximately 30 μm and 43 μm. (Item 13) A polymer biodegradable vascular graft or scaffold according to any one of items 9 to 12, having a distance between layers of approximately 0.5 mm to 2 mm, preferably between approximately 0.5 mm to 1.5 mm, most preferably about 1 mm. (Item 14) A polymer biodegradable vascular graft or scaffold according to any one of items 9 to 13, having a distance between peaks of approximately 0.5 mm to 2 mm, preferably between approximately 0.5 mm to 1.5 mm, and most preferably about 1 mm. (Item 15) A polymer biodegradable vascular graft or scaffold according to any one of items 8 to 14, comprising a surface porosity of about 0.6 to 0.95, preferably about 0.7 to 0.9, most preferably about 0.8 to 0.9, of the surface area of ​​the inner and / or outer surface. (Item 16) A polymer biodegradable vascular graft or scaffold according to any one of items 8 to 15, comprising polymer fibers woven in a weft pattern. (Item 17) A polymer biodegradable vascular graft or scaffold according to any one of items 8 to 16, having a fiber diameter between approximately 1 μm and approximately 100 μm, preferably between approximately 1 μm and approximately 50 μm, and most preferably between approximately 5 μm and approximately 30 μm. (Item 18) A polymer biodegradable vascular graft or scaffold as described in any one of items 8 to 17, further comprising a polymer coating. (Item 19) A polymer biodegradable vascular graft or scaffold according to any one of items 8 to 18, having an inner diameter between approximately 14 mm and 24 mm, preferably between approximately 14 mm and 20 mm, and most preferably between approximately 15 mm and 20 mm. (Item 20) A polymer biodegradable vascular graft or scaffold according to any one of items 8 to 19, having a length between approximately 5 cm and 25 cm, preferably between approximately 5 cm and 15 cm, and most preferably between approximately 10 cm and 15 cm. (Item 21) A polymer biodegradable vascular graft or scaffold according to any one of items 8 to 20, comprising one polymer selected from the group consisting of polyester, poly(orthoester), poly(phosphazene), poly(caprolactone), polyamide, polysaccharide, and blends and copolymers thereof. (Item 22) A polymer biodegradable vascular graft or scaffold described in any one of items 8 to 21, which substantially degrades within approximately six months after implantation. (Item 23) A polymer biodegradable vascular graft or scaffold according to any one of items 8 to 22, further comprising autologous viable cells. (Item 24) The polymer biodegradable vascular graft or scaffold described in item 23, wherein the viable cells are bone marrow mononuclear cells. (Item 25) A polymer biodegradable vascular graft or scaffold according to any one of items 8 to 24, further comprising one or more additional agents selected from the group consisting of anti-neointimal agents, chemotherapeutic agents, steroidal and non-steroidal anti-inflammatory agents, conventional immunotherapeutic agents, immunosuppressants, cytokines, chemokines, and growth factors. (Item 26) A method for seeding cells onto a graft or scaffold, a) Connecting a seeding chamber described in any one of items 1 to 7 to a closed, disposable seeding system including a tank containing a fluid containing blood or enriched cells, b) The step of inserting the graft or scaffold onto the mandrel of the seeding chamber, c) Filling the seeding chamber with the fluid by gravity flow and extraction to about half the height of the graft or scaffold, d) The steps of filling the seeding chamber with the fluid and covering the graft or scaffold, e) A method comprising the step of introducing negative pressure to draw all of the fluid into the graft or scaffold. (Item 27) A method for seeding cells onto a graft or scaffold, a) Connecting a seeding chamber described in any one of items 1 to 7 to a closed, disposable seeding system including a tank containing a fluid containing blood or enriched cells, b) The step of inserting the graft or scaffold onto the mandrel of the seeding chamber, c) A step of filling the seeding chamber with the fluid, d) Applying negative pressure to the lower mandrel outlet and emptying the chamber to about half its capacity. e) The step of inverting the chamber, and f) Applying negative pressure to empty the chamber through the upper outlet port. A method that includes this. (Item 28) The method according to item 26 or 27, wherein negative pressure is provided by a syringe, pump, or vacuum source. (Item 29) The method according to any one of items 26 to 28, wherein the graft or scaffold is a polymer graft or scaffold according to any one of items 8 to 25. (Item 30) The method according to any one of items 26 to 29, wherein the fluid has a volume between approximately 10 ml and 200 ml. (Item 31) The aforementioned fluid is about 10 5 White blood cells (WBC) / ml~10 8 Preferably about 10 WBC / ml 6 ~10 8 Between WBC / ml, more preferably about 10 6 ~10 7 The method according to any one of items 26 to 30, having a WBC / ml range. (Item 32) The method according to any one of items 26 to 31, wherein the graft or scaffold has a length between 10 cm and 15 cm. (Item 33) The method according to any one of items 26 to 32, wherein sowing is completed within a period of about 1 to 15 minutes, preferably about 1 to 10 minutes, most preferably about 1 to 7 minutes. (Item 34) The method according to any one of items 26 to 33, wherein cells are seeded in the graft or scaffold at substantially the same cell density along the length of the scaffold. (Item 35) The cell density along the length of the scaffold, including the values ​​at both ends, is approximately 0.1 × 10 3 cells / mm 2 ~10 5 cells / mm 2 Between which, preferably along the length of the scaffold, including the values ​​at both ends, approximately 0.1 × 10 3 cells / mm 2 ~10 4 cells / mm 2 Between these values, most preferably along the length of the scaffold, including the values ​​at both ends, 1 × 10 3 cells / mm 2 ~10 4 cells / mm 2 The method described in any one of items 26 to 34, which is between. (Item 36) A method for reducing or preventing postoperative stenosis in a subject, comprising the step of administering a polymer vascular graft or scaffold described in any one of items 8 to 25 to the subject. (Item 37) The method according to item 36, wherein the subject is at or has a risk of restenosis or other vascular proliferative disorder. (Item 38) The methods described in item 36 or 37, wherein the subject has had, is having, or will have had vascular trauma, angioplasty, vascular surgery, or graft artery disease. (Item 39) The method according to any one of items 36 to 38, wherein the polymer vascular graft or scaffold reduces or prevents postoperative neointima formation, stenosis or restenosis in a subject, reduces or prevents thrombosis, or any combination thereof, compared to a control subject.

Claims

1. A cell seeding chamber for use in a closed-type disposable cell seeding system, wherein the cell seeding chamber is Housing with variable width and length, An opening for a suction rod that can be inserted into the housing, one or more lateral fluid ports, an open end for receiving a cap that includes a perforated or porous mandrel that can be inserted into the housing and positioned on the suction rod, and Base Includes, A cell seeding chamber having a housing with a bulge positioned between 30% and 60% of the length of the housing that forms the bulge.

2. A cap for a cell seeding chamber for use in a closed-type disposable cell seeding system, wherein the cell seeding chamber is Housing having width and length, A cap comprising an opening for a suction rod insertable into the housing and one or more lateral fluid ports, the cap being for securing a perforated or porous mandrel that can be positioned on the suction rod, and Base Includes, A cap having a housing with a variable width along its length and a bulge positioned between 30% and 60% of the length of the housing, forming a bulge.

3. A cell seeding chamber for use in a closed-type disposable cell seeding system, wherein the cell seeding chamber is Housing having width and length, A cap comprising an opening for a suction rod insertable into the housing and one or more lateral fluid ports, for securing a perforated or porous mandrel that can be positioned on the suction rod, and Base Includes, A cell seeding chamber having a housing with a bulge positioned between 30% and 60% of the length of the housing that forms the bulge.

4. The cell seeding chamber according to claim 3, wherein the mandrel is a porous mandrel and comprises a suction rod inserted therein and a graft or scaffold on the mandrel of the seeding chamber.

5. The cell seeding chamber according to claim 3 or 4, wherein the cell seeding chamber has a lateral vent port.

6. A polymer biodegradable vascular graft or scaffold comprising an internal structure and an external structure, wherein the graft or scaffold has a uniform dispersion of cells in and on it, and is formed within the cell seeding chamber according to claim 5.

7. (a) A polymer fiber layer arranged in the axial direction and polymer fiber peaks arranged in the circumferential direction, (b) The inner surface has an average pore diameter, and the outer surface has different average pore diameters, (c) Having an average pore diameter between 35 μm and 50 μm on the inner surface, (d) Having an average pore diameter between 25 μm and 45 μm on the outer surface, (e) Having a distance between layers between 0.5 mm and 2 mm, (f) Having a distance between peaks between 0.5 mm and 2 mm, (g) including a surface porosity of 0.6 to 0.95 of the surface area of ​​the inner surface and / or the outer surface, (h) containing polymer fibers woven into a weft pattern, (i) Having a fiber diameter between 1 μm and 100 μm, (j) further comprising a polymer coating, (k) Having an inner diameter between 14 mm and 24 mm, (l) Having a length between 5 cm and 25 cm, (m) comprising one or more polymers selected from the group consisting of polyester, poly(orthoester), poly(phosphazene), poly(caprolactone), polyamide, polysaccharide, and blends and copolymers thereof, (n) Substantially decomposes within 6 months after implantation. (o) further comprising autologous surviving cells, (p) further comprising one or more additional agents selected from the group consisting of anti-intimal agents, chemotherapeutic agents, steroidal and non-steroidal anti-inflammatory agents, immunotherapeutic agents, immunosuppressants, cytokines, chemokines, and growth factors, The polymer biodegradable vascular graft or scaffold according to claim 6.

8. A method for seeding cells onto a graft or scaffold, a) Connecting a seeding chamber according to any one of claims 3 to 5 to a closed, disposable seeding system including a tank containing a fluid containing blood or enriched cells; b) Inserting the graft or scaffold onto the mandrel of the seeding chamber, c) A step of filling the seeding chamber with the fluid, d) Applying negative pressure to the lower mandrel outlet and emptying the chamber to half its capacity. e) The step of inverting the chamber, and f) Applying negative pressure to empty the chamber through the upper outlet port. A method that includes this.

9. (a) Negative pressure is provided by a syringe, pump, or vacuum source. (b) The graft or scaffold is a polymer biodegradable vascular graft or scaffold according to claim 6 or 7. (c) The fluid has a volume between 10 ml and 200 ml. (d) The fluid is 10 ５ White blood cells (WBC) / ml ~ 10 ８ Having a WBC / ml range, (e) The graft or scaffolding has a length between 10 cm and 15 cm. (f) Sowing is completed within a period of 1 to 15 minutes. (g) The graft or scaffold is seeded with cells at substantially the same cell density along the length of the scaffold, and / or (h) The cell density along the length of the scaffold, including the values ​​at both ends, is 0.1 × 10 ３ cells / mm ２ ~10 ５ cells / mm ２ It is between, The method according to claim 8.

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