New Approaches to Cryopreservation of Cells, Tissues, and Organs.

In this concept article, we outline a variety of new approaches that have been conceived to address some of the remaining challenges for developing improved methods of biopreservation. This recognizes a true renaissance and variety of complimentary, high-potential approaches leveraging inspiration by nature, nanotechnology, the thermodynamics of pressure, and several other key fields. Development of an organ and tissue supply chain that can meet the healthcare demands of the 21st century means overcoming twin challenges of (1) having enough of these lifesaving resources and (2) having the means to store and transport them for a variety of applications. Each has distinct but overlapping logistical limitations affecting transplantation, regenerative medicine, and drug discovery, with challenges shared among major areas of biomedicine including tissue engineering, trauma care, transfusion medicine, and biomedical research. There are several approaches to biopreservation, the optimum choice of which is dictated by the nature and complexity of the tissue and the required length of storage. Short-term hypothermic storage at temperatures a few degrees above the freezing point has provided the basis for nearly all methods of preserving tissues and solid organs that, to date, have proved refractory to cryopreservation techniques successfully developed for single-cell systems. In essence, these short-term techniques have been based on designing solutions for cellular protection against the effects of warm and cold ischemia and basically rely upon the protective effects of reduced temperatures brought about by Arrhenius kinetics of chemical reactions. However, further optimization of such preservation strategies is now seen to be restricted. Long-term preservation calls for much lower temperatures and requires the tissue to withstand the rigors of heat and mass transfer during protocols designed to optimize cooling and warming in the presence of cryoprotective agents. It is now accepted that with current methods of cryopreservation, uncontrolled ice formation in structured tissues and organs at subzero temperatures is the single most critical factor that severely restricts the extent to which tissues can survive procedures involving freezing and thawing. In recent years, this major problem has been effectively circumvented in some tissues by using ice-free cryopreservation techniques based upon vitrification. Nevertheless, despite these promising advances there remain several recognized hurdles to be overcome before deep-subzero cryopreservation, either by classic freezing and thawing or by vitrification, can provide the much-needed means for biobanking complex tissues and organs for extended periods of weeks, months, or even years. In many cases, the approaches outlined here, including new underexplored paradigms of high-subzero preservation, are novel and inspired by mechanisms of freeze tolerance, or freeze avoidance, in nature. Others apply new bioengineering techniques such as nanotechnology, isochoric pressure preservation, and non-Newtonian fluids to circumvent currently intractable problems in cryopreservation.

Plasticity and Aggregation of Juvenile Porcine Islets in Modified Culture: Preliminary Observations.

Diabetes is a major health problem worldwide, and there is substantial interest in developing xenogeneic islet transplantation as a potential treatment. The potential to relieve the demand on an inadequate supply of human pancreata is dependent upon the efficiency of techniques for isolating and culturing islets from the source pancreata. Porcine islets are favored for xenotransplantation, but mature pigs (>2 years) present logistic and economic challenges, and young pigs (3-6 months) have not yet proven to be an adequate source. In this study, islets were isolated from 20 juvenile porcine pancreata (~3 months; 25 kg Yorkshire pigs) immediately following procurement or after 24 h of hypothermic machine perfusion (HMP) preservation. The resulting islet preparations were characterized using a battery of tests during culture in silicone rubber membrane flasks. Islet biology assessment included oxygen consumption, insulin secretion, histopathology, and in vivo function. Islet yields were highest from HMP-preserved pancreata (2,242 ± 449 IEQ/g). All preparations comprised a high proportion (>90%) of small islets (<100 µm), and purity was on average 63 ± 6%. Morphologically, islets appeared as clusters on day 0, loosely disaggregated structures at day 1, and transitioned to aggregated structures comprising both exocrine and endocrine cells by day 6. Histopathology confirmed both insulin and glucagon staining in cultures and grafts excised after transplantation in mice. Nuclear staining (Ki-67) confirmed mitotic activity consistent with the observed plasticity of these structures. Metabolic integrity was demonstrated by oxygen consumption rates = 175 ± 16 nmol/min/mg DNA, and physiological function was intact by glucose stimulation after 6-8 days in culture. In vivo function was confirmed with blood glucose control achieved in nearly 50% (8/17) of transplants. Preparation and culture of juvenile porcine islets as a source for islet transplantation require specialized conditions. These immature islets undergo plasticity in culture and form fully functional multicellular structures. Further development of this method for culturing immature porcine islets is expected to generate small pancreatic tissue-derived organoids termed "pancreatites," as a therapeutic product from juvenile pigs for xenotransplantation and diabetes research.

Nonenzymatic cryogenic isolation of therapeutic cells: novel approach for enzyme-free isolation of pancreatic islets using in situ cryopreservation of islets and concurrent selective freeze destruction of acinar tissue.

Cell-based therapies, which all involve processes for procurement and reimplantation 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 treatment of type 1 diabetes by transplantation. To avoid the inherent pitfalls of these enzymatic methods, we have conceptualized an alternative approach based on the hypothesis that cryobiological techniques can be used for differential freeze destruction of the pancreas (Px) to release islets that are selectively cryopreserved in situ. Pancreata were procured from juvenile pigs using approved procedures. The concept of cryoisolation is based on differential processing of the pancreas in five stages: 1) infiltrating islets in situ preferentially with a cryoprotectant (CPA) cocktail via antegrade perfusion of the major arteries; 2) retrograde ductal infusion of water to distend the acinar; 3) freezing the entire Px solid to < -160°C for storage in liquid nitrogen; 4) mechanically crushing and pulverizing the frozen Px into small fragments; 5) thawing the frozen fragments, filtering, and washing to remove the CPA. Finally, the filtered effluent (cryoisolate) was stained with dithizone for identification of intact islets and with Syto 13/PI for fluorescence viability testing and glucose-stimulated insulin release assessment. As predicted, the cryoisolate contained small fragments of residual tissue comprising an amorphous mass of acinar tissue with largely intact and viable (>90%) embedded islets. Islets were typically larger (range 50-500 µm diameter) than their counterparts isolated from juvenile pigs using conventional enzyme digestion techniques. Functionally, the islets from replicate cryoisolates responded to a glucose challenge with a mean stimulation index = 3.3 ± 0.7. An enzyme-free method of islet isolation relying on in situ cryopreservation of islets with simultaneous freeze destruction of acinar tissue is feasible and proposed as a new and novel method that avoids the problems associated with conventional collagenase digestion methods.

Current state of hypothermic machine perfusion preservation of organs: The clinical perspective.

This review focuses on the application of hypothermic perfusion technology as a topic of current interest with the potential to have a salutary impact on the mounting clinical challenges to improve the quantity and quality of donor organs and the outcome of transplantation. The ex vivo perfusion of donor organs on a machine prior to transplant, as opposed to static cold storage on ice, is not a new idea but is being re-visited because of the prospects of making available more and better organs for transplantation. The rationale for pursuing perfusion technology will be discussed in relation to emerging data on clinical outcomes and economic benefits for kidney transplantation. Reference will also be made to on-going research using other organs with special emphasis on the pancreas for both segmental pancreas and isolated islet transplantation. Anticipated and emerging benefits of hypothermic machine perfusion of organs are: (i) maintaining the patency of the vascular bed, (ii) providing nutrients and low demand oxygen to support reduced energy demands, (iii) removal of metabolic by-products and toxins, (iv) provision of access for administration of cytoprotective agents and/or immunomodulatory drugs, (v) increase of available assays for organ viability assessment and tissue matching, (vi) facilitation of a change from emergency to elective scheduled surgery with reduced costs and improved outcomes, (vii) improved clinical outcomes as demonstrated by reduced PNF and DGF parameters, (viii) improved stabilization or rescue of ECD kidneys or organs from NHBD that increase the size of the donor pool, (ix) significant economic benefit for the transplant centers and reduced health care costs, and (x) provision of a technology for ex vivo use of non-transplanted human organs for pharmaceutical development research.

Modulating biochemical perturbations during 72-hour machine perfusion of kidneys: role of preservation solution.

This study documents renal biochemistry during hypothermic machine perfusion of kidneys. It is intended to demonstrate that a comprehensive evaluation of organ viability during ex-vivo preservation is needed to increase the number of organs available for transplantation and to reduce the current renal discard rate. Porcine kidneys were hypothermically machine perfused for 72 h with either Unisol-UHK or Belzer-Machine Perfusion Solution, (Belzer-MPS). Renal perfusate samples were periodically collected and biochemically analyzed. Significant differences were measured in the renal metabolic activity between the two experimental groups while similar values for traditional parameters such as renal flow rate and vascular resistance values were recorded. The effluent of UHK perfused kidneys showed strong metabolites and NH(4)(+) dynamics (P<0.05 vs. baseline), while the Belzer-MPS kidneys metabolic activity led to little or no change of the effluent biochemistry relative to baseline.

The role of preservation solution on acid-base regulation during machine perfusion of kidneys.

To meet the current clinical organ demand, efficient preservation methods and solutions are needed to increase the number of viable kidneys for transplantation. In the present study, the influence of perfusion solution buffering strength on renal pH dynamics and regulation mechanisms during kidney ex vivo preservation was determined. Porcine kidneys were hypothermically machine perfused for 72 h with either Unisol-UHK or Belzer-Machine Perfusion solution, Belzer-MP solution. Renal perfusate samples were periodically collected and biochemically analyzed. The UHK solution, a Hepes-based solution (35 mM), provided a more efficient control of renal pH that, in turn, resulted in minor changes in the perfusate pH relative to baseline, in response to tissue CO2 and HCO3- production. In the perfusate of Belzer-MP kidney group a wider range of pH values were recorded and a pronounced pH reduction was seen in response to significant rises in pCO2 and HCO3- concentrations. The Belzer-MP solution, containing phosphate (25 mM) as its main buffer, and only 10 mM Hepes, had a greater buffering requirement to attenuate larger pH changes.

Interstitial fluid analysis for assessment of organ function.

Evaluation methods are required for non-heart-beating donor (NHBD) kidneys to ensure the success of transplantation. In this study, the microdialysis technique was employed for the ex-vivo assessment of hypothermically preserved NHBD kidney function. Microdialysis probes were placed in the renal cortex of 2 h warm ischaemic porcine kidneys to monitor interstitial pyruvate dynamics during hypothermic machine perfusion with perfusate containing 29.4 mM fructose-1,6-diphosphate (FDP). The presence of exogenous FDP in the perfusate induced no changes in the renal flow rate and vascular resistance, renal artery effluent biochemistry, or pyruvate concentration relative to untreated control kidneys. Significant increases in pyruvate production (P < 0.05), however, were observed after 12 h of perfusion in the interstitial fluid of FDP-treated kidneys relative to control kidneys. After 24 h of perfusion, interstitial fluid concentrations of pyruvate were 149.1 +/- 58.4 vs. 55.6 +/- 17.9 micro M (P < 0.05) in the FDP and control group, respectively. The microdialysis probe collected the interstitial fluid directly from the cellular sites of metabolic and synthetic activity, where perfusate dilution was minimal. Consequently, the biochemical changes induced by the organ metabolic activity were detected only at the interstitial level, in the microdialysates. Interstitial fluid pyruvate may be a good indicator of kidney function. The addition of FDP to the perfusion solution during ischaemic kidney preservation resulted in enhanced pyruvate production in the extracellular space, indirectly reflecting an increase in anaerobic ATP production. The pyruvate will be transformed during organ reperfusion into acetyl Co-A enzyme allowing an immediate start of aerobic metabolism. This in turn can increase the amount of ATP available to the cells and may help prevent reperfusion injury upon transplantation.

Acid-base buffering in organ preservation solutions as a function of temperature: new parameters for comparing buffer capacity and efficiency.

Control of acidity and preventing intracellular acidosis are recognized as critical properties of an effective organ preservation solution. Buffer capacity and efficiency are therefore important for comparing the relative merits of preservation fluids for optimum hypothermic storage, but these parameters are not available for the variety of organ preservation solutions of interest in transplantation today. Moreover, buffer capacity is dependent upon both concentration and pH such that buffer capacity is not easily predicted for a complex solution containing multiple buffer species. Using standard electrometric methods to measure acid dissociation constants, this study was undertaken to determine the maximum and relative buffer capacities of a variety of new and commonly used hypothermic preservation solutions as a function of temperature. The reference data provided by these measurements show that comparative buffer capacity and efficiency vary widely between the commonly used solutions. Moreover, the fluids containing zwitterionic sulfonic acid buffers such as Hepes possess superior buffering for alpha-stat pH regulation in the region of physiological importance.