Lia H. Campbell

Culturing with trehalose produces viable endothelial cells after cryopreservation

Dimethylsulfoxide, the most commonly employed cryoprotectant for cells, has well documented cytotoxic effects in patients. Among the compounds available that may provide protection to cells and tissues during preservation with less cytotoxicity is trehalose. Some animals, such as brine shrimp and tardigrades, accumulate trehalose during periods of extreme environmental stress. In this study, experiments were performed to evaluate the effects of culturing a bovine endothelial cell line (ATCC #CCL-209) in the presence of trehalose prior to preservation by freezing. A number of factors were shown to contribute to cell retention of metabolic activity and proliferative potential including cell culture time with trehalose and the solution conditions during cryopreservation. Using an optimized protocol consisting of 24 h of cell culture with 0.2 M trehalose followed by cryopreservation with 0.2-0.4 M trehalose in sodium bicarbonate buffered Eagles minimum essential medium at pH 7.4 resulted in 87±4% post-preservation cell metabolic activity expressed as relative fluorescence based upon reduction of resazurin to resorufin. This new method provides an alternative preservation strategy to the more classical preservation methods employing dimethylsulfoxide available for cells and tissues.

Interstitial fluid analysis for assessment of organ function.

Abstract:  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 µM (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.

Lessons from nature for preservation of mammalian cells, tissues, and organs

The study of mechanisms by which animals tolerate environmental extremes may provide strategies for preservation of living mammalian materials. Animals employ a variety of compounds to enhance their survival, including production of disaccharides, glycerol, and antifreeze compounds. The cryoprotectant glycerol was discovered before its role in amphibian survival. In the last decade, trehalose has made an impact on freezing and drying methods for mammalian cells. Investigation of disaccharides was stimulated by the variety of organisms that tolerate dehydration stress by accumulation of disaccharides. Several methods have been developed for the loading of trehalose into mammalian cells, including inducing membrane lipid-phase transitions, genetically engineered pores, endocytosis, and prolonged cell culture with trehalose. In contrast, the many antifreeze proteins (AFPs) identified in a variety of organisms have had little impact. The first AFPs to be discovered were found in cold water fish; their AFPs have not found a medical application. Insect AFPs function by similar mechanisms, but they are more active and recombinant AFPs may offer the best opportunity for success in medical applications. For example, in contrast to fish AFPs, transgenic organisms expressing insect AFPs exhibit reduced ice nucleation. However, we must remember that nature’s survival strategies may include production of AFPs, antifreeze glycolipids, ice nucleators, polyols, disaccharides, depletion of ice nucleators, and partial desiccation in synchrony with the onset of winter. We anticipate that it is only by combining several natural low temperature survival strategies that the full potential benefits for mammalian cell survival and medical applications can be achieved.

Serum-free solutions for cryopreservation of cells

With the development of cell-based assays and therapies, the purity of reagents used to grow and maintain cells has become much more important. In particular, the use of fetal calf serum for culturing cells presents a direct path for potential contamination of cell cultures. In recent years, much research has focused on the development of serum-free culturing systems, not only to alleviate difficulties due to availability and cost of fetal calf serum but also to prevent the transmission of potentially fatal diseases to human patients. Additionally, methods need to be developed for long-term storage of cell stocks that also reduce the risk of exposure to harmful diseases. As most methods employ fetal calf serum in their freezing formulations, solutions that avoid the use of fetal calf serum while providing equivalent or better recovery of cells upon thawing would be ideal. In this study, two vascular cell lines have been cryopreserved as adherent cell populations in two widely used cryoprotectants, dimethyl sulfoxide and 1,2-propanediol, and two vehicle solutions, Euro-Collins and Unisol-cryoprotectant vehicle specifically formulated for the maintenance of cell homeostasis at temperatures below 37° C. The addition of serum to these formulations was also evaluated to determine if its presence provided any additional benefit to the cells during cryopreservation. The results demonstrated that using vehicle solutions designed for lower temperatures produced viable cells that retained cell population viability values up to 75% of unfrozen controls. These results also demonstrated that including serum in the formulation provided no additional benefit to the cells and in some cases actually produced lower cell viability after cryopreservation. In conclusion, the development of solutions designed for low-temperature storage of cells provides a viable alternative to more conventional cryopreservation protocols and eliminates the necessity of including serum in these formulations.

Recombinant Dendroides canadensis antifreeze proteins as potential ingredients in cryopreservation solutions.

Expanding cryopreservation methods to include a wider range of cell types, such as those sensitive to freezing, is needed for maintaining the viability of cell-based regenerative medicine products. Conventional cryopreservation protocols, which include use of cryoprotectants such as dimethylsulfoxide (Me2SO), have not prevented ice-induced damage to cell and tissue matrices during freezing. A family of antifreeze proteins (AFPs) produced in the larvae of the beetle, Dendroides canadensis allow this insect to survive subzero temperatures as low as -26°C. This study is an assessment of the effect of the four hemolymph D. canadensis AFPs (DAFPs) on the supercooling (nucleating) temperature, ice structure patterns and viability of the A10 cell line derived from the thoracic aorta of embryonic rat. Cryoprotectant solution cocktails containing combinations of DAFPs in concentrations ranging from 0 to 3mg/mL in Unisol base mixed with 1M Me2SO were first evaluated by cryomicroscopy. Combining multiple DAFPs demonstrated significant supercooling point depressing activity (∼9°C) when compared to single DAFPs and/or conventional 1M Me2SO control solutions. Concentrations of DAFPs as low as 1 μg/mL were sufficient to trigger this effect. In addition, significantly improved A10 smooth muscle cell viability was observed in cryopreservation experiments with low DAFP-6 and DAFP-2 concentrations in combination with Me2SO. No significant improvement in viability was observed with either DAFP-1 or DAFP-4. Low and effective DAFP concentrations are advantageous because they minimize concerns regarding cell cytotoxicity and manufacturing cost. These findings support the potential of incorporating DAFPs in solutions used to cryopreserve cells and tissues.

MitoQ Blunts Mitochondrial and Renal Damage during Cold Preservation of Porcine Kidneys

Cold preservation has greatly facilitated the use of cadaveric kidneys for transplantation but damage occurs during the preservation episode. It is well established that oxidant production increases during cold renal preservation and mitochondria are a key target for injury. Our laboratory has demonstrated that cold storage of renal cells and rat kidneys leads to increased mitochondrial superoxide levels and mitochondrial electron transport chain damage, and that addition of Mitoquinone (MitoQ) to the preservation solutions blunted this injury. In order to better translate animal studies, the inclusion of large animal models is necessary to develop safe preclinical protocols. Therefore, we tested the hypothesis that addition of MitoQ to cold storage solution preserves mitochondrial function by decreasing oxidative stress, leading to less renal tubular damage during cold preservation of porcine kidneys employing a standard criteria donor model. Results showed that cold storage significantly induced oxidative stress (nitrotyrosine), renal tubular damage, and cell death. Using High Resolution Respirometry and fresh porcine kidney biopsies to assess mitochondrial function we showed that MitoQ significantly improved complex II/III respiration of the electron transport chain following 24 hours of cold storage. In addition, MitoQ blunted oxidative stress, renal tubular damage, and cell death after 48 hours. These results suggested that MitoQ decreased oxidative stress, tubular damage and cell death by improving mitochondrial function during cold storage. Therefore this compound should be considered as an integral part of organ preservation solution prior to transplantation.

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.

Development of pancreas storage solutions: Initial screening of cytoprotective supplements for β-cell survival and metabolic status after hypothermic storage.

Insulin-dependent diabetes mellitus is one of the leading causes of death world-wide. Donor-derived pancreas and Islet of Langerhans transplantation are potential cures; however, postmortem ischemia impacts islet quality. The murine βt3 cell line was employed as a model to study cell viability and proliferation after hypothermic storage by comparing Belzers Machine Perfusion Solution with Unisol™ Solution. The objective was to determine which of these solutions provided the best base line support for βt3 cells and to screen potential cytoprotective additives to the solutions. Initial βt3 cell viability was similar in the two storage solutions; however, better proliferation was observed after storage in Unisol Solution. The caspase inhibitor, Q-VD-OPH, and α-tocopherol improved viability in both storage solutions, suggesting that apoptotic pathways may be responsible for cell death during hypothermic storage of βt3 cells. Analysis of apoptosis markers, caspase activity, and DNA laddering showed a reduction in apoptosis when these additives were included. The effects of Q-VD-OPH and α-tocopherol were also synergistic when employed together during either hypothermic exposure, post-hypothermic physiologic incubation, or combinations of hypothermic exposure and physiologic incubation. These results suggest that both supplements should be included in pancreas hypothermic storage solutions and in islet culture media during post-isolation culture prior to transplantation.

Best practices in cell culture: an overview

This overview describes a series of articles to provide an unmet need for information on best practices in animal cell culture. The target audience primarily consists of entry-level scientists with minimal experience in cell culture. It also include scientists, journalists, and educators with some experience in cell culture, but in need of a refresher in best practices. The articles will be published in this journal over a six-month period and will emphasize best practices in: (1) media selection; (2) use and evaluation of animal serum as a component of cell culture medium; (3) receipt of new cells into the laboratory; (4) naming cell lines; (5) authenticating cell line identity; (6) detecting and mitigating risk of cell culture contamination; (7) cryopreservation and thawing of cells; and (8) storing and shipping viable cells.

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.