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NORTH CHARLESTON, SC, United States

Patent
Cell And Tissue Systems, Inc. and Lifeline Scientific Inc. | Date: 2012-06-07

Methods for ex vivo perfusion of organs (and/or tissues) with a perfusate designed to condition the organ with the desired effect being that upon transplant, said organ, having been administered said perfusate, is less likely to experience delayed graft function, deleterious effects of ischemia/reperfusion injury, including inflammatory reactions, and/or other detrimental responses that can injure the organ or recipient including precipitating or enhancing an immunological reaction from the recipient with the potential of compromising the grafts and/or recipients short teen and/or long term health and proper functionality while monitoring, sustaining and/or restoring the viability of the organ and preserving the organ for storage and/or transport.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 268.56K | Year: 2012

DESCRIPTION (provided by applicant): In modern day medicine, cellular therapies, regenerative medicine and tissue engineering all involve technologies for harvesting, expanding, modifying and re-implanting live viable cells and tissues. Processes for preparing the therapeutic products that incorporate living cells are critical for the stability and potency of th products but may be inherently injurious to the component cells. For example, the widely practiced technique of collagenase digestion of tissues toobtain isolated cells such as pancreatic islets from pancreata, or hepatocytes from liver is fraught with detrimental side-effects and other associated problems. This widely practiced procedure has recognized pitfalls due principally to the difficulty ofcontrolling the digestive process to yield an optimum quantityof viable cells. Moreover, the process is harsh and even toxic, causing some inevitable loss of valuable cells. Furthermore, the process relying upon the purest forms of the enzymes are very expensive and may be subject to batch variations that have led to frustrating variability and inconsistency in attempts to optimize and standardize these processes. A totally new approach is proposed here that minimizes and potentially eliminates the need forenzymatic digestion of the tissue. Instead, the proposed process relies upon known susceptibilities of cells to freezing injury, to affect the separation of different cell types by virtue of a facilitated differential frezing and cryopreservation techniques. Feasibility for this novel approach has been demonstrated for isolating porcine pancreatic islets, which is a widely accepted model for research into the treatment of type I diabetes by islet transplantation. To obtain islets for cell-based therapies,te field of islet transplantation relies totally upon enzymatic digestion processes that destroy the extracellular matrix of the donor tissue releasing the entrapped islets for further processing and purification. In contrast, we propose to pre-treat the pancreas by differential perfusion of the endocrine and exocrine tissue in a way designed to maximize the destruction of the exocrine tissue at the same time as preserving the islets. More specifically, this new cryo-isolation approach involves an initial perfusion of the endocrine tissue (islets) with cryoprotective agents via a vascular access and after adequate equilibration of the islets only, the exocrine component (acini) is infused with a purely aqueous solution (distilled water or saline) via the ductal system The entire pancreas is then cooled under conditions that promote ice formation and destruction of the acinar tissue while preserving the endocrine portion by virtue of the cryoprotectant infiltration. The solid frozen pancreas is then amenable to indefinite storage and biobanking and subsequent processing to pulverize and fracture the gland into tiny fragments containing the cryopreserved islets. Finally, the freeze-disrupted tissue is thawed to release functional islets and destroyed acinar tissue. Having completed the initial proof-of-concept of this innovative new approach, this Phase I study proposes to develop a device prototype for cryo-isolation and evaluate its performance to establish baseline protocols. The approach combines basic research tools with recent advances in cryobiology science to systematically optimize the baseline technique, while developing a method to promote tissue fracturing by means of thermo-mechanical stresses, thereby increasing the effectiveness of differential freeze disruption and viable islet isolation. The study brings together a unique combination of expertise in cryobiology and thermo-mechanical engineering necessary to take this novel concept from feasibility to routine practice and subsequently validation inhuman tissue in a Phase II study. PUBLIC HEALTH RELEVANCE: Cell-based therapies in regenerative medicine and tissue engineering, which all involve processes for procurement and re-implantation of living cells, currently rely upon expensive, inconsistent and even toxic enzyme-digestion processes. A prime example is the preparation of isolated pancreatic islets for the potential treatment of Type I diabetes by transplantation. This research is focused on the development of a new and novel alternativetechnique to enzymatic digestion by relying instead on differential freeze destruction of the pancreas to release islets that are selectively cryopreserved in situ.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 262.00K | Year: 2012

DESCRIPTION (provided by applicant): Autologous regenerative medicine tissue therapy costs are very high because when individual tissues are manufactured all the costs fall on a single patient. Allogeneic tissues are cheaper because large tissue batches can be made and the costs are shared by many patients. Allogeneic tissues are commonly treated to remove cell-associated antigens by decellularization. Decellularization employs harsh chemicals and freezing methods that may damage tissue matrices. Our preliminary data in an in vivo allogeneic sheep model and an in vitro xenogeneic model with ice-free cryopreserved porcine tissue and human responder cells indicates that an ice- free cryopreservation method developed by the Company modifies the recipient's immune reaction. The primary goal of this SBIR proposal is a feasibility study to determine whether ice-free cryopreservation of human tissue also results in little or no immune reaction when combined with allogeneic human peripheral blood mononuclear cells using a panel of in vitro assays. This goal will be pursued in two Specific Aims and associated Hypotheses. The human tissues will be a tissue engineered vascular graft being developed by our collaborators for therapeutic applications. In the first aim cellular grafts will be ice-free cryopreserved and washing procedures to optimize cryoprotectant removal will be developed. Endothelial cell attachment and proliferation on the treated human tissue engineered vascular grafts will be the criteria for assessmentof washing adequacy. It is critical that the ice-free tissues be non-toxic to recipient cells for integration in to the recipient. Nanoliter osmometry will also be employed to quantify residual cryoprotectant concentrations. In the second aim evaluation ofimmunogenicity will be performed using cell proliferation and cytokine release assays. Fresh untreated, decellularized and ice-free cryopreserved human engineered blood vessels will be compared using allogeneic human peripheral blood mononuclear cells asresponders in vitro. The anticipated outcome is that the ice-free cryopreserved tissues will be equivalent or less immunogenic compared with decellularized controls. This outcome will be followed by in vivo transplant studies in a subsequent Phase II SBIRproposal. Retention of materials properties will also be confirmed in Phase II. This research will have a far-reaching clinical impact on surgical repairs by providing unprecedented access to low cost non- immunogenic tissue allografts for a variety of surgical applications and diseased artery replacement in particular. Our commercialization strategy involves exclusive and non-exclusive licensing of ice-free cryopreservation methods to companies developing specific human allogeneic tissue-based therapies.PUBLIC HEALTH RELEVANCE: Natural and engineered allogeneic tissues potentially impact huge orthopedic, urinary, cardiac and vascular surgery applications. The potential worldwide market for vascular grafts alone is predicted to be 2,588M in 2013. The technology development in this proposal will minimize costs by reducing the manufacturing steps required for engineered human tissue-derived products to be non-immunogenic. This technology simultaneously provides a long-term tissue storage method, whichhas proven retention of extracellular matrix components and biomaterial properties compared with alternative preservation strategies, with the potential for little if any immune response in vivo after implantation in patients.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 224.81K | Year: 2011

DESCRIPTION (provided by applicant): Twenty four million Americans are affected with end-stage lung disease which is the 4th leading cause of death in the US, and there are currently 2,122 candidates on the lung transplant waiting list. Emphysema and chronic obstructive pulmonary disease are the most common diagnoses leading to lung transplantation. The limited availability of suitable lung donors has become an increasing concern in the transplant community. The primary impact of this SBIR proposal will beincreased numbers of lungs being considered acceptable for transplantation and decreased patient waiting times on the transplant list. This will be a consequence of technology focused on development of a lung hypothermic perfusion preservation device and methods that will maintain lungs ex vivo for up to 24 hours. Furthermore, the device should permit evaluation of organ quality; and it will also result in an important research tool being available to the lung research community. The objective of this PhaseI SBIR proposal is to determine the feasibility of hypothermic machine perfusion preservation of the lung by ex vivo normothermic testing of lung function after 12 hours in a portable hypothermic lung perfusion prototype. The portable lung perfusion prototype will be continuously optimized during the course of the experiments for optimal performance. Swine lungs preserved by hypothermic (4-8 C) perfusion for 12 hours will be compared with lungs submitted to cold static preservation on ice. Organ quality will be assessed ex vivo where the lungs will be perfused and ventilated at 37 C while biochemical and physiological tools are used to determine lung function. At the conclusion of this feasibility study the portable preclinical lung perfusion prototype design will be reviewed and subjected to failure mode and effects analysis to insure the safety and ease of use that will permit its clinical application. The proposed Phase I study will progress to Phase II if feasibility is demonstrated in Phase I. Feasibility will be verified by demonstration of statistically better lung function after perfusion preservation than current practice static storage controls. In Phase II porcine lung transplant model studies as well as ex vivo human lung evaluations will be performed. PUBLIC HEALTH RELEVANCE: This proposal aims at the technical breakthrough with the potential to address critical national needs for transplantable lungs. The device described in this proposal has the potential to increase the lung donor pool to levels that would satisfy current demand by evaluation, reconditioning and approval of marginal organs and controlled non-heart beating donors that are presently not considered for transplantation.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 278.18K | Year: 2011

DESCRIPTION (provided by applicant): Vascular grafting is performed clinically to repair or replace diseased coronary artery and peripheral vessels to restore normal blood flow patterns. Synthetic grafts composed of polymers such as Dacron and expanded polytetrafluoroethylene do not work well in small diameter (lt 6 mm) vessels. Such grafts exhibit low patency rates and fail, in large part, due to compliance mismatch. Compliance describes how the mechanical properties of a vascular graft change as a function of the internal hemodynamic pressure. Natural blood vessels display a complex non-linear 'J-shaped' stress-strain biomechanical behavior which is a function of extracellular matrix elastin and collagen nanofibers. Elastic fibers with straight conformation dominate the low elastic modulus at low levels of vessel distention. While collagen nanofibers with a wavy or helical orientation, with little resistance to expansion at lower values of vessel distention, dominate the high elastic modulus at higher levels of vessel distention as the nanofibers straighten. In addition to compliance, possession of a non-thrombogenic inner lining, biocompatibility and, after recipient cell ingrowth, vasoactivity is important for long term function of vascular grafts. The innovation in this proposal is design and manufacturing of composite nanofiber-based tissue-engineered vascular grafts (TEVGs) which mimic the potential implant site's arterial extracellular matrix microstructure and mechanical properties. In other words thegrafts will be designed to match the compliance of each type of artery that requires replacement. Our preliminary data has demonstrated our ability to fabricate synthetic nanofibrous composite materials with overall mechanical properties matching those ofa natural blood vessel (aorta) by employing a non-degradable elastin-like nanofiber and degradable collagen-like nanofibers. In this proposal these materials will be used in the construction of TEVGs mimicking the rabbit's carotid artery followed by evaluation in three specific aims. These aims include biomechanics and graft seeding with cells and in vitro assessment of remodeling profiles and retention of mechanical properties including compliance, burst strength and suture pull strength over time. Finally, cell-free TEVG designs will be assessed by vascular grafting in vivo. Patency, quantitative histology, mechanical properties and development of vasoactivity will be determined after one month post-implantation. Feasibility for progression to Phase II SBIR studies will be demonstrated by retention of biomaterial properties with e80% patency, the development of significantly better carotid-like vasoactivity after ingrowth of recipient cells and less anastomotic hyperplasia than controls (TEVGs without collagen-like microstructures) at explant. In Phase II we will propose large animal preclinical studies and other testing required for federal regulatory clearance for human trials. PUBLIC HEALTH RELEVANCE: Cardiovascular disease is a leading cause of patient morbidity and mortality. Effective small diameter vascular grafts are an unmet clinical need. The potential impact of this project is design and production of effective composite nanofiber-based tissue-engineered vascular grafts for patients requiring small diameter artery repair or replacement. The potential world-wide market for vascular grafts is predicted to be 1,657,000 units valued at 2,588M by the year 2013. The simplicity, versatility, and scalability of our proposed approach will allow rapidclinical translation and market penetration.

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