Zhenzhen Chen

Vitreous Preservation of Rabbit Articular Cartilage

The growing need for improved methods of viable tissue cryopreservation has stimulated debate regarding the relative merits of traditional freezing methods versus ice-free vitrification methods. Articular cartilage has proved refractory to satisfactory cryopreservation using conventional freezing methods. Therefore, full-thickness rabbit femoral head articular cartilage was used to compare a freezing method of cryopreservation and an ice-free, vitrification method of cryopreservation with fresh controls in vitro. Chondrocyte viability was determined in vitro using vital fluorescent staining with calcein-acetoxymethyl ester and an oxidation-reduction assay with alamarBlue™ (Accumed International, Westlake, Ohio). Cryosubstitution with 1% osmium tetroxide in 100% methanol was used to detect ice during cryopreserved storage. Cryosubstitution studies of frozen and vitrified articular cartilage pieces revealed negligible ice in the vitrified specimens and extensive ice formation in frozen specimens with the ex...

Cryopreservation of carotid artery segments via vitrification subject to marginal thermal conditions: Correlation of freezing visualization with functional recovery

Cryopreservation is a well-established technique for long-term storage of viable cells and tissues. However, in recent years, application of established cryobiological principles to the preservation of multicellular tissues and organs has demanded considerable attention to ways of circumventing the deleterious effects of ice and thermal stresses in bulky tissues. As part of a multidisciplinary research program designed to study the interactions of thermo-physical events with tissue preservation, we report here on the implementation of a slow cooling (3 degrees C/min) and slow warming (62 degrees C/min) regimen towards scale-up of vitreous preservation of large tissue samples. Specifically, the correlation of thermo-physical events during vitrification of carotid artery segments with function recovery is reported using marginal thermal conditions for achieving vitrification in bulky samples. Moreover, the outcome is compared with a similar study reported previously using a 3-fold higher rate of rewarming (186+/-13 degrees C/min). Tissue vitrification using an 8.4M cryoprotectant cocktail solution (VS55) was achieved in 1 ml samples by imposing a low (2.6+/-0.1 degrees C/min) cooling rate, between -40 degrees C and -100 degrees C, and a low rewarming rate (62+/-4 degrees C/min) between -100 degrees C and -40 degrees C. Following cryoprotectant removal, the artery segments were cut into 3-4mm rings for function testing on a contractility apparatus by measuring isometric responses to four agonist and antagonists (norepinephrine, phenylepinephrine, calcium ionophore and sodium nitroprusside). In addition, non-specific metabolic function of the vessel rings was determined using the REDOX indicator alamarBlue. Contractile function, normalized to untreated control samples, in response to the agonists norepinephrine and phenylepinephrine was significantly better in the slowly rewarmed group of carotid segments (74+/-9% and 62+/-11%, respectively) than for the more rapidly warmed group 31+/-7% and 45+/-15%, respectively). However, EC(50) sensitivities were not significantly different between the groups. Thermo-physical events such as ice formation and fractures were monitored throughout the cooling and warming phases using cryomacroscopy with the aid of a purpose-built borescope device. This technique allowed a direct observation of the visual impact of ice formation on specific zones along the blood vessel segment where, in most cases, no ice formation or fractures were observed in the vicinity of the artery segments. However, in specific instances when some ice crystallization was observed to impact the artery segment, the subsequent testing of function revealed a total loss of contractility. The successful vitrification of blood vessel segments using marginal conditions of slow cooling and rewarming, provide essential information for the development of scale-up protocols that is necessary when clinically relevant size samples need to be cryopreserved in an essentially ice-free state. This information can further be integrated into computer simulations of heat transfer and thermo-mechanical stress, where the slowest cooling rate anywhere in the simulated domain must exceed the critical values identified in the current study.

Vitrification of heart valve tissues.

Application of the original vitrification protocol used for pieces of heart valves to intact heart valves has evolved over time. Ice-free cryopreservation by Protocol 1 using VS55 is limited to small samples where relatively rapid cooling and warming rates are possible. VS55 cryopreservation typically provides extracellular matrix preservation with approximately 80 % cell viability and tissue function compared with fresh untreated tissues. In contrast, ice-free cryopreservation using VS83, Protocols 2 and 3, has several advantages over conventional cryopreservation methods and VS55 preservation, including long-term preservation capability at -80 °C; better matrix preservation than freezing with retention of material properties; very low cell viability, reducing the risks of an immune reaction in vivo; reduced risks of microbial contamination associated with use of liquid nitrogen; improved in vivo functions; no significant recipient allogeneic immune response; simplified manufacturing process; increased operator safety because liquid nitrogen is not used; and reduced manufacturing costs.

Impact of Hypothermia upon Chondrocyte Viability and Cartilage Matrix Permeability after 1 Month of Refrigerated Storage

Background: The purpose of this research was to assess the extracellular matrix and chondrocytes of articular cartilage during refrigerated storage and to determine whether changes could be detected in the time frame that cartilage is stored for clinical use. Methods: Porcine cartilage was stored as either bisected femoral heads with bone attached or plugs without the underlying bone in culture medium with fetal bovine serum for 1 month at 4 °C. Metabolic activity was tested using a resazurin reduction method on intact tissue and viable cell recovery after enzymatic tissue digestion at each time point. Cartilage plug permeability was evaluated by measuring electrical conductivity. Results: Storage in culture medium provided good cartilage viability and metabolic function for 7 days; however, significant changes were observed in femoral heads (p < 0.05). All mean chondrocyte assessment values were <30% of fresh controls at 28 days. Cartilage plugs tended to perform better after 7 days of storage than the femoral heads and retained significantly higher metabolic activity (mean = 94.5% vs. 70.5%; p < 0.05). Cartilage plugs demonstrated consistent changes in electrical conductivity after 28 days of storage (p < 0.05). Conclusion: Refrigerated storage of cartilage results in both loss of chondrocyte viability and matrix permeability.

Tissue engineering of a collagen-based vascular media: Demonstration of functionality.

The property of vasoactivity is important for both resistance vessels and larger arteries. Evaluation of smooth muscle cell phenotype is often done in place of functional testing in engineered tissues, assuming a direct correlation between cell phenotype and tissue contractile force. In this study we look at a large panel of vasoactive agents to determine the functionality of our collagen-based tissue. The engineered vascular media elicited a measurable change in force in response to seven of the nine agents used. As part of this characterization, TGF-β1 and TNF-α were used to promote a more contractile and synthetic cell phenotype respectively. Both smooth muscle α-actin and vasoconstriction were evaluated in ring sections. Due to large differences in cell-compaction and cell distribution in the tissues, no correlation was found between α-actin expression and contractile strength. This highlights the need for functional testing of engineered tissue and the importance of cell-matrix interactions in vasoactivity.

Ex vivo evaluation of porcine livers post-hypothermic machine perfusion at 4-6ºC and 12-14ºC

Background: The goal of our research is the development of a clinical hypothermic machine perfusion method and device for liver preservation during storage and transport prior to transplantation. The purpose of this study was comparison of two hypothermic temperature ranges. Methods: Heart beating donor pig livers with 2h of static cold storage on ice were employed. Thirteen experimental livers were perfused, at either 4-6oC or 12-14oC with oxygenation, employing a prototype device. They were compared with six control 2h static cold stored livers during normothermic ex vivo blood perfusion. Physiological measurements and biochemical perfusate analyses were performed to assess the impact of storage conditions on liver functions. Statistics were assessed by one way analysis of variance, p<0.05 was regarded as significant. Results: During hypothermic perfusion perfusate solutes (Na + , K + and Ca ++ ) varied little with time. A single 12-14oC perfused liver exhibited a Ca ++ spike at 22 hours. Temperature dependent metabolic activity was observed in both groups. Lactate levels were ≤4.5mmol/L for 22h. During normothermic ex vivo testing most controls and perfusion-treated experimental livers produced bile within an hour. 10h perfusion liver lactate dehydrogenase, hyaluronic acid uptake, and total bile production were not significantly different from controls. There were trends for albumin, glucose and lactate. Significantly different Factor V, indocyanine green clearance, and blood urinary nitrogen concentrations were observed in both 10h perfusion groups. Bilirubin kinetics was perturbed in all perfusion groups, however the peak concentrations in the 10h perfusion groups were not significantly different, while the 22h 12-14oC perfusion group was significantly less than controls. A significant difference in alanine aminotransferase values between the 2 perfusion groups, was observed, however this may be due to washout during 12-14°C perfusion. Lactate dehydrogenase was doubled in both perfusion groups at 22h and individual 22h livers exhibited other assay values outside control and 10h perfusion ranges. Conclusions: Significant differences were observed between controls and both perfused groups during post hypothermic storage ex vivo assessment. These results suggest that both temperature ranges tested, with further optimization, may be suitable for liver support for up to 10h of oxygenated hypothermic perfusion. Since there were no clear advantages to 12-14°C perfusion we will continue development of a 4-6°C perfusion device for storage and transport of livers for transplantation.