Decellularized scaffold of cryopreserved rat kidney retains its recellularization potential (original) (raw)

Freezing/thawing without cryoprotectant damages native but not decellularized porcine renal tissue

Organogenesis, 2015

Whole organ decellularization of porcine renal tissue and recellularization with a patient's own cells would potentially overcome immunorejection, which is one of the most significant problems with allogeneic kidney transplantation. However, there are obstacles to achieving this goal, including preservation of the decellularized extracellular matrix (ECM), identifying the proper cell types, and repopulating the ECM before transplantation. Freezing biological tissue is the best option to avoid spoilage; however, it may damage the structure of the tissue or disrupt cellular membranes through ice crystal formation. Cryoprotectants have been used to repress ice formation during freezing, although cell toxicity can still occur. The effect of freezing/thawing on native (n=10) and decellularized (n=10) whole porcine kidneys was studied without using cryoprotectants. Results showed that the elastic modulus of native kidneys was reduced by a factor of 22 (p<0.0001) by freezing/thawing...

Cryopreservation of Complex Systems: The Missing Link in the Regenerative Medicine Supply Chain

Rejuvenation Research, 2006

Transplantation can be regarded as one form of "antiaging medicine" that is widely accepted as being effective in extending human life. The current number of organ transplants in the United States is on the order of 20,000 per year, but the need may be closer to 900,000 per year. Cadaveric and living-related donor sources are unlikely to be able to provide all of the transplants required, but the gap between supply and demand can be eliminated in principle by the field of regenerative medicine, including the present field of tissue engineering through which cell, tissue, and even organ replacements are being created in the laboratory. If so, it could allow over 30% of all deaths in the United States to be substantially postponed, raising the probability of living to the age of 80 by a factor of two and the odds of living to 90 by more than a factor of 10. This promise, however, depends on the ability to physically distribute the products of regenerative medicine to patients in need and to produce these products in a way that allows for adequate inventory control and quality assurance. For this purpose, the ability to cryogenically preserve (cryopreserve) cells, tissues, and even whole laboratory-produced organs may be indispensable. Until recently, the cryopreservation of organs has seemed a remote prospect to most observers, but developments over the past few years are rapidly changing the scientific basis for preserving even the most difficult and delicate organs for unlimited periods of time. Animal intestines and ovaries have been frozen, thawed, and shown to function after transplantation, but the preservation of vital organs will most likely require vitrification. With vitrification, all ice formation is prevented and the organ is preserved in the glassy state below the glass transition temperature (T G ). Vitrification has been successful for many tissues such as veins, arteries, cartilage, and heart valves, and success has even been claimed for whole ovaries. For vital organs, a significant recent milestone for vitrification has been the ability to routinely recover rabbit kidneys after cooling to a mean intrarenal temperature of about Ϫ45°C, as verified by life support function after transplantation. This temperature is not low enough for long-term banking, but research continues on preservation below Ϫ45°C, and some encouraging preliminary evidence has been obtained indicating that kidneys can support life after vitrification. Full development of tissue engineering and organ generation from stem cells, when combined with the ability to bank these laboratory-produced products, in theory could dramatically increase median life expectancy even in the absence of any improvements in mitigating aging processes on a fundamental level. 279

Cryopreservation of the Mammalian Kidney

Cryobiology, 1997

The objective of the present study was to determine whether rabbit kidneys could be perfused with a 7.5 M vitrification solution (VS4, which vitrifies under applied pressure) without loss of function. To answer this question, kidneys were perfused with VS4 using a computer-based machine to gradually raise and lower concentration and then attached to the aorta and vena cava of a perfusor rabbit using an apparatus that permitted renal blood flow and renal function to be measured. About half (6/13) of the kidneys so evaluated resumed substantial immediate function after a transient period of severely reduced blood flow. Loss of function did not occur if cryoprotectant concentration was limited to 3.8 M. The loss of function produced by VS4 could be partially reproduced by artificially limiting blood reflow in control kidneys to simulate the transiently low flows caused by VS4 exposure. These results provide the first evidence that both the parenchyma and the vascular system of a sensitive mammalian organ can survive exposure to a vitrifiable concentration of cryoprotectant. ᭧ 1997 Academic Press

Rat hindlimb cryopreservation and transplantation - A step towards “organ banking”

American Journal of Transplantation, 2017

In 2016, over 5 million reconstructive procedures were performed in the United States. The recent successes of clinical vascularized composite allotransplantations, hand and face transplantations included, established the tremendous potential of these life-enhancing reconstructions. Nevertheless, due to limited availability and lifelong immunosuppression, application is limited. Longterm banking of composite transplants may increase the availability of esthetically compatible parts with partial or complete HLA matching, reducing the risk of rejection and the immunosuppressive burden. The study purpose was to develop efficient protocols for the cryopreservation and transplantation of a complete rodent limb. Directional freezing is a method in which a sample is cooled at a constant-velocity linear temperature gradient, enabling precise control of the process and ice crystal formation. Vitrification is an alternative cryopreservation method in which the sample solidifies without the formation of ice crystals. Testing both methods on a rat hindlimb composite tissue transplantation model, we found reliable, reproducible, and stable ways to preserve composite tissue. We believe that with further research and development, cryopreservation may lead to composite tissue "banks." This may lead to a paradigm shift from few and far apart emergent surgeries to wide-scale, well-planned, and better-controlled elective surgeries.

Charting the course of renal cryoinjury

Physiological Reports, 2015

We sought to characterize a minor renal cryoinjury that allows investigation into renal damage processes and subsequent endogenous repair mechanisms. To achieve this, we induced a small cryoinjury to mice, in which the transient superficial application of a liquid nitrogen-cooled cryoprobe to the exposed kidney induces a localized lesion that did not impair renal function. The resulting cryoinjury was examined by immunohistochemistry and Laser-Doppler flowmetry. Within hours of cryoinjury induction, tubular and vascular necrotic damage was observed, while blood flow in the directly injured area was reduced by 65%. The injured area demonstrated a peak in tubular and perivascular cell proliferation at 4 days postinjury, while apoptosis and fibrosis peaked at day 7. Infiltration of macrophages into the injury was first observed at day 4, and peaked at day 7. Vascular density in the direct injured area was lowest at day 7. As compared to the direct injured area, the (peripheral) penumbral region surrounding the directly injured area demonstrated enhanced cellular proliferation (2.5-6-fold greater), vascular density (1.6-2.9 fold greater) and blood perfusion (twofold greater). After 4 weeks, the area of damage was reduced by 73%, fibrosis decreased by 50% and blood flow in the direct injured area was reestablished by 63% with almost complete perfusion restoration in the injury's penumbral region. In conclusion, kidney cryoinjury provides a flexible facile model for the study of renal damage and associated endogenous repair processes.

Current topics in kidney cryopreservation: Cooling injury and biochemical injury

Cryobiology, 2018

The ability to cryopreserve precision-cut brain slices with full functionality is of pragmatic importance and academic interest. The present study demonstrated that rabbit hippocampal slices can be vitrified with subsequent full recovery of electrophysiological function. The cryoprotectant formulas studied were VEG, VM3 and M22. The CPAs were added and removed stepwise at first and later were added and removed continuously. There was no significant difference between naive slices and those vitrified and stored at-130 C for various periods of time. Extracellular, intracellular and optical photo diode array (PDA) recordings all indicated that electrical stimulus-response characteristics including passive membrane properties, field extracellular excitatory postsynaptic potential (fEPSP), network ensemble activity and long term potentiation (LTP) were preserved. These studies show for the first time that not just viability but also electrophysiological functionality can be cryopreserved by vitrification.

Validating the cryopreservation of tissue engineered constructs within cryobags

2021

The cryopreservation of cells and tissue engineered constructs is determined by multiple factors, such as possible cellular damages due to mass and heat transfer during the freezing and thawing process. It is assumed that the utilization of cryobags improves the heat transfer during freezing and thawing and is therefore superior in comparison to currently applied cryovials and multiwell plates. Therefore, we have analysed the cryopreservation of cell-seeded electrospun polycaprolactone/polylactide scaffolds within cryobags. Additionally, the performance of four different cryoprotective agents was analysed, which included 10% (v/v) dimethyl sulfoxide and 10% (v/v) ethylene glycol as separately applied agents as well as their combination with 0.3 M sucrose. The samples were frozen in in-house made cryobags with a controlled rate freezer. The cell viability was analysed by fluorescence microscopy before freezing and after thawing. The results show, that the application of cryobags in c...

Cryopreservation of rat precision-cut liver and kidney slices by rapid freezing and vitrification

Cryobiology, 2007

Precision-cut tissue slices of both hepatic and extra-hepatic origin are extensively used as an in vitro model to predict in vivo drug metabolism and toxicity. Cryopreservation would greatly facilitate their use. In the present study, we aimed to improve (1) rapid freezing and warming (200°C/min) using 18% Me 2 SO as cryoprotectant and (2) vitrification with high molarity mixtures of cryoprotectants, VM3 and VS4, as methods to cryopreserve precision-cut rat liver and kidney slices. Viability after cryopreservation and subsequent 3-4 h of incubation at 37°C was determined by measuring ATP content and by microscopical evaluation of histological integrity. Confirming earlier studies, viability of rat liver slices was maintained at high levels by rapid freezing and thawing with 18% Me 2 SO. However, vitrification of liver slices with VS4 resulted in cryopreservation damage despite the fact that cryoprotectant toxicity was low, no ice was formed during cooling and devitrification was prevented. Viability of liver slices was not improved by using VM3 for vitrification. Kidney slices were found not to survive cryopreservation by rapid freezing. In contrast, viability of renal medullary slices was almost completely maintained after vitrification with VS4, however vitrification of renal cortex slices with VS4 was not successful, partly due to cryoprotectant toxicity. Both kidney cortex and medullary slices were vitrified successfully with VM3 (maintaining viability at 50-80% of fresh slice levels), using an optimised pre-incubation protocol and cooling and warming rates that prevented both visible ice-formation and cracking of the formed glass. In conclusion, vitrification is a promising approach to cryopreserve precision-cut (kidney) slices.

Discarded human kidneys as a source of ECM scaffold for kidney regeneration technologies

Biomaterials, 2013

In the United States, more than 2600 kidneys are discarded annually, from the total number of kidneys procured for transplant. We hypothesized that this organ pool may be used as a platform for renal bioengineering and regeneration research. We previously showed that decellularization of porcine kidneys yields renal extracellular matrix (ECM) scaffolds that maintain their basic components, support cell growth and welfare in vitro and in vivo, and show an intact vasculature that, when such scaffolds are implanted in vivo, is able to sustain physiological blood pressure. The purpose of the current study was to test if the same strategy can be applied to discarded human kidneys in order to obtain human renal ECM scaffolds. The results show that the sodium dodecylsulfate-based decellularization protocol completely cleared the cellular compartment in these kidneys, while the innate ECM framework retained its architecture and biochemical properties. Samples of human renal ECM scaffolds stimulated angiogenesis in a chick chorioallantoic membrane assay. Importantly, the innate vascular network in the human renal ECM scaffolds retained its compliance. Collectively, these results indicate that discarded human kidneys are a suitable source of renal scaffolds and their use for tissue engineering applications may be more clinically applicable than kidneys derived from animals. (G. Orlando). 1 CB and ZW are equally second authors.

Recellularization of Well-Preserved Acellular Kidney Scaffold Using Embryonic Stem Cells

Tissue Engineering Part A, 2014

For chronic kidney diseases, there is little chance that the vast majority of world's population will have access to renal replacement therapy with dialysis or transplantation. Tissue engineering would help to address this shortcoming by regeneration of damaged kidney using naturally occurring scaffolds seeded with precursor renal cells. The aims of the present study were to optimize the production of three-dimensional (3D) rat whole-kidney scaffolds by shortening the duration of organ decellularization process using detergents that avoid nonionic compounds, to investigate integrity of extracellular matrix (ECM) structure and to enhance the efficacy of scaffold cellularization using physiological perfusion method. Intact rat kidneys were successfully decellularized after 17 h perfusion with sodium dodecyl sulfate. The whole-kidney scaffolds preserved the 3D architecture of blood vessels, glomeruli, and tubuli as shown by transmission and scanning electron microscopy. Microcomputerized tomography (micro-CT) scan confirmed integrity, patency, and connection of the vascular network. Collagen IV, laminin, and fibronectin staining of decellularized scaffolds were similar to those of native kidney tissues. After infusion of whole-kidney scaffolds with murine embryonic stem (mES) cells through the renal artery, and pressure-controlled perfusion with recirculating cell medium for 24 and 72 h, seeded cells were almost completely retained into the organ and uniformly distributed in the vascular network and glomerular capillaries without major signs of apoptosis. Occasionally, mES cells reached peritubular capillary and tubular compartment. We observed the loss of cell pluripotency and the start of differentiation toward meso-endodermal lineage. Our findings indicate that, with the proposed optimized protocol, rat kidneys can be efficiently decellularized to produce renal ECM scaffolds in a relatively short time, and rapid recellularization of vascular structures and glomeruli. This experimental setup may open the possibility to obtain differentiation of stem cells with long lasting in vitro perfusion.