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1.  Analysis of machine perfusion benefits in kidney grafts: a preclinical study 
Machine perfusion (MP) has potential benefits for marginal organs such as from deceased from cardiac death donors (DCD). However, there is still no consensus on MP benefits. We aimed to determine machine perfusion benefits on kidney grafts.
We evaluated kidney grafts preserved in ViaspanUW or KPS solutions either by CS or MP, in a DCD pig model (60 min warm ischemia + 24 h hypothermic preservation). Endpoints were: function recovery, quality of function during follow up (3 month), inflammation, fibrosis, animal survival.
ViaspanUW-CS animals did not recover function, while in other groups early follow up showed similar values for kidney function. Alanine peptidase and β-NAG activities in the urine were higher in CS than in MP groups. Oxydative stress was lower in KPS-MP animals. Histology was improved by MP over CS. Survival was 0% in ViaspanUW-CS and 60% in other groups. Chronic inflammation, epithelial-to-mesenchymal transition and fibrosis were lowest in KPS-MP, followed by KPS-CS and ViaspanUW-MP.
With ViaspanUW, effects of MP are obvious as only MP kidney recovered function and allowed survival. With KPS, the benefits of MP over CS are not directly obvious in the early follow up period and only histological analysis, urinary tubular enzymes and red/ox status was discriminating. Chronic follow-up was more conclusive, with a clear superiority of MP over CS, independently of the solution used. KPS was proven superior to ViaspanUW in each preservation method in terms of function and outcome. In our pre-clinical animal model of DCD transplantation, MP offers critical benefits.
PMCID: PMC3038164  PMID: 21266040
Transplantation proceedings  2008;40(2):480-482.
Procurement of donor pancreases for islet isolation and transplantation is not yet widely practiced due to concerns about post-mortem ischemia upon functional islet yields. Perfusion/preservation technology can help to circumvent ischemic injury and is applied in this study to porcine pancreata (Px) prior to islet isolation. Px harvested from adult pigs were assigned to one of three preservation treatment groups:G1) Fresh controls - processed immediately with minimum cold ischemia(<1h) G2) Static Cold Storage-flushed with cold UW-Viaspan and stored at 2–4ºC for 24h, and G3) Hypothermic Machine Perfusion (HMP)-perfused on a pulsatile LifePort® machine with KPS1 solution at 4–7ºC and low pressure(10mmHg) for 24h. Islet isolation was then accomplished using conventional methods and standard accepted product release criteria were used to assess islet yield and function. Islet yield was markedly different between the treatment groups and the increased yield in the HMP group over the static cold storage in UW-Viaspan was statistically significant (p<0.05). Functionally, the islets from each experimental group were equivalent and not significantly different to fresh controls (G1). Dithizone staining for islets showed a consistently more uniform digestion of the Px from G3 compared with G1 and G2, with greater separation of the tissue and less entrapped islets. HMP for 24h is well tolerated leading to moderate edema but no loss of function of the harvested islets. The edema appears to aid in enzymatic digestion producing a greater yield and purity of islets compared with Px subjected to 24h of static cold storage.
PMCID: PMC2413169  PMID: 18374108
3.  The Unfolded Protein Response in Human Corneal Endothelial Cells Following Hypothermic Storage: Implications of a Novel Stress Pathway 
Cryobiology  2011;63(1):46-55.
Human corneal endothelial cells (HCEC) have become increasingly important for a range of eye disease treatment therapies. Accordingly, a more detailed understanding of the processing and preservation associated stresses experienced by corneal cells might contribute to improved therapeutic outcomes. To this end, the unfolded protein response (UPR) pathway was investigated as a potential mediator of corneal cell death in response to hypothermic storage. Once preservation-induced failure had begun in HCECs stored at 4°C, it was noted that necrosis accounted for the majority of cell death but with significant apoptotic involvement, peaking at several hours post-storage (4-8 hours). Western blot analysis demonstrated changes associated with apoptotic activation (caspase 9, caspase 3, and PARP cleavage). Further, the activation of the UPR pathway was observed through increased and sustained levels of ER folding and chaperone proteins (Bip, PDI, and ERO1-Lα) in samples experiencing significant cell death. Modulation of the UPR pathway using the specific inhibitor, salubrinal, resulted in a 2-fold increase in cell survival in samples experiencing profound cold-induced failure. Furthermore, this increased cell survival was associated with increased membrane integrity, cell attachment, and decreased necrotic cell death populations. Conversely, addition of the UPR inducer, tunicamycin, during cold exposure resulted in a significant decrease in HCEC survival during the recovery period. These data implicate for the first time that this novel cell stress pathway may be activated in HCEC as a result of the complex stresses associated with hypothermic exposure. The data suggest that the targeted control of the UPR pathway during both processing and preservation protocols may improve cell survival and function of HCEC thus improving the clinical utility of these cells as well as whole human corneas.
PMCID: PMC3397425  PMID: 21549109
Apoptosis; Cornea; Unfolded Protein Response; Hypothermia; Endoplasmic Reticulum Stress
4.  Oxygen persufflation as adjunct in liver preservation (OPAL): Study protocol for a randomized controlled trial 
Trials  2011;12:234.
Early graft dysfunction due to preservation/reperfusion injury represents a dramatic event after liver transplantation. Enhancement of donor organ criteria, in order to cope with the ever increasing donor shortage, further increases graft susceptibility to ischemic alterations.
Major parts of post-preservation injury, however, occur at the time of warm reperfusion but not during ischemic storage; successful reperfusion of ischemic tissue in turn depends on an adequate redox and intracellular signal homeostasis. The latter has been shown experimentally to be favorably influenced by oxygen persufflation within short time spans. Thus viability of marginally preserved liver grafts could still be augmented by transient hypothermic reconditioning even after normal procurement and static cold storage. The present study is aimed to confirm the conceptual expectations, that hypothermic reconditioning by gaseous oxygen persufflation is a useful method to suppress injurious cellular activation cascades and to improve post-ischemic recovery of marginally preserved liver grafts.
OPAL is a prospective single center randomized proof of concept study, including two parallel groups in a total of 116 liver transplant patients. The effect of an in hospital treatment of the isolated liver graft by 2 hours of oxygen persufflation immediately prior to transplantation will be assesses as compared to standard procedure (cold storage without further intervention). The primary endpoint is the peak transaminase serum level (AST) during the first three days after transplantation as a surrogate readout for parenchymal liver injury. Other outcomes comprise patient and graft survival, time of intensive care requirement, hepatic tissue perfusion 1h after revascularisation, early onset of graft dysfunction based on coagulation parameters, as well as the use of a refined scoring-system for initial graft function based on a multi-parameter (AST, ALT, Quick and bilirubin) score. Furthermore, the effect of OPAL on molecular pathways of autophagy and inflammatory cell activation will be evaluated. Final analysis will be based on all participants as randomized (intention to treat).
Trial Registration
Current Controlled Trials ISRCTN00167887
PMCID: PMC3215181  PMID: 22035223
5.  Development of Pancreas Storage Solutions: Initial Screening of Cytoprotective Supplements for β-Cell Survival and Metabolic Status after Hypothermic Storage 
Biopreservation and Biobanking  2013;11(1):12-18.
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 Belzer's 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.
PMCID: PMC4077006  PMID: 24845250
6.  The sensitivity of human mesenchymal stem cells to vibration and cold storage conditions representative of cold transportation 
In the current study, the mechanical and hypothermic damage induced by vibration and cold storage on human mesenchymal stem cells (hMSCs) stored at 2–8°C was quantified by measuring the total cell number and cell viability after exposure to vibration at 50 Hz (peak acceleration 140 m s−2 and peak displacement 1.4 mm), 25 Hz (peak acceleration 140 m s−2, peak displacement 5.7 mm), 10 Hz (peak acceleration 20 m s−2, peak displacement 5.1 mm) and cold storage for several durations. To quantify the viability of the cells, in addition to the trypan blue exclusion method, the combination of annexin V-FITC and propidium iodide was applied to understand the mode of cell death. Cell granularity and a panel of cell surface markers for stemness, including CD29, CD44, CD105 and CD166, were also evaluated for each condition. It was found that hMSCs were sensitive to vibration at 25 Hz, with moderate effects at 50 Hz and no effects at 10 Hz. Vibration at 25 Hz also increased CD29 and CD44 expression. The study further showed that cold storage alone caused a decrease in cell viability, especially after 48 h, and also increased CD29 and CD44 and attenuated CD105 expressions. Cell death would most likely be the consequence of membrane rupture, owing to necrosis induced by cold storage. The sensitivity of cells to different vibrations within the mechanical system is due to a combined effect of displacement and acceleration, and hMSCs with a longer cold storage duration were more susceptible to vibration damage, indicating a coupling between the effects of vibration and cold storage.
PMCID: PMC3427524  PMID: 22628214
stem cells; mechanical stress; vibration; regenerative medicine; hypothermia; viability
7.  Adenosine 5'triphosphate transport and accumulation during the cold preservation of rat hepatocytes in University of Wisconsin solution 
AIM: We used isolated hepatocytes to investigate how different concentrations of ATP in the University of Wisconsin (UW) solution affected both cellular ATP content and cell viability during the cold storage and the rewarming step. The mechanism involved in ATP transport and accumulation in hypothermia was also determined.
METHODS: The cells were preserved up to 72 h in different conditions: UW solution without ATP (a-group), UW+5 mmol/L ATP (b-group), and UW+10 mmol/L ATP (c-group). The ATP content and the cell viability (LDH release) were determined during the cold storage and the rewarming step. In the groups a and c, the respiratory function of the cells at rewarming was studied. In addition, the cell volume of hepatocytes and the mechanism involved in ATP transport and accumulation were assessed. The extracellular degradation of exogenous nucleotides during transport experiments was investigated by a HPLC technique.
RESULTS: After three days of cold storage a loss of cellular ATP content was observed in hepatocytes preserved either without nucleotides (a-group) or with 5 mmol/L ATP (b-group). In contrast, 10 mmol/L ATP (c-group) was able to maintain a normal ATP cellular content, with only a 6% diminution after 72 h of cold storage. The respiratory function was significantly different in hepatocytes preserved with 10 mmol/L ATP than without ATP. No significant change was detected for the three groups in cellular volume during the cold storage. We also report that the time course accumulation of [3H]-ATP by cold stored hepatocytes is a rapid process that is completed after 180 s with linear dependence on the extracellular ATP concentration (linear fitting results in a slope of 0.5624±0.1179 mmol/L ATP intracell/mmol/L ATP extracell).
CONCLUSION: Our results show that, during hypothermic storage in UW solution, hepatocytes are permeable to ATP by a diffusive mechanism. Also, we found that it is ATP the main extracellular nucleotide available for transport and it is not the breakdown products.
PMCID: PMC4305717  PMID: 15800986
UW; ATP; Hepatocytes; Cold preservation; Hypothermia; Oxygen
8.  Storage Temperatures for Cold-Chain Delivery in Cell Therapy: A Study of Alginate-Encapsulated Liver Cell Spheroids Stored at −80°C or −170°C for Up to 1 Year 
A bioartificial liver comprising alginate-encapsulated liver cell spheroids (ELS) could bridge the gap to transplant or spontaneous recovery in acute liver failure, but will be required for emergency use, necessitating cryopreservation. A cryopreservation protocol has been developed, but beyond this, the feasibility of cold-chain storage is considered here. Cryopreservation will be increasingly required for timely delivery of tissue and bioengineered products, and significant, but often, over-looked factors that impact on cost and ease of clinical application are the storage temperature and useful preservation time. Storage in the vapor phase of liquid nitrogen (∼−170°C) is the gold standard, but for safety and economic purposes, storing ELS in electric freezers at −80°C may be preferable.
ELS were cryopreserved using an optimized protocol and stored at either −80°C or at −170°C for up to 1 year. ELS were removed from storage after 1, 2, 3, 6, 9, or 12 months, and recovery was assessed 24 h postwarming. Cell recovery was assessed using viability (fluorescent staining with image analysis), cell number (nuclei count), and functional (hepatospecific protein enzyme-linked immunosorbent assay) assays.
Viability, the viable cell number, and function of ELS stored at −170°C were maintained at similar values throughout the year. In contrast, ELS stored at −80°C exhibited decreased viability, viable cell numbers, and function by as early as 1 month. Progressive deterioration was subsequently observed. After 12 months of storage at −80°C, viable cell recovery of ELS was ∼15% that of ELS stored at −170°C.
While convenience and cost might support the use of −80°C for storage of multicellular bioengineered products such as ELS, results indicate rapid deterioration in functional recoveries after only a few weeks. This study demonstrates that storage temperature is an important consideration in regenerative medicine and caution should be applied by limiting storage at −80°C to only a few weeks.
PMCID: PMC3557435  PMID: 22834979
9.  Aldose Reductase Inhibition Prevents Allergic Airway Remodeling through PI3K/AKT/GSK3β Pathway in Mice 
PLoS ONE  2013;8(2):e57442.
Long-term and unresolved airway inflammation and airway remodeling, characteristic features of chronic asthma, if not treated could lead to permanent structural changes in the airways. Aldose reductase (AR), an aldo-sugar and lipid aldehyde metabolizing enzyme, mediates allergen-induced airway inflammation in mice, but its role in the airway remodeling is not known. In the present study, we have examined the role of AR on airway remodeling using ovalbumin (OVA)-induced chronic asthma mouse model and cultured human primary airway epithelial cells (SAECs) and mouse lung fibroblasts (mLFs).
Airway remodeling in chronic asthma model was established in mice sensitized and challenged twice a week with OVA for 6 weeks. AR inhibitor, fidarestat, was administered orally in drinking water after first challenge. Inflammatory cells infiltration in the lungs and goblet cell metaplasia, airway thickening, collagen deposition and airway hyper-responsiveness (AHR) in response to increasing doses of methacholine were assessed. The TGFβ1-induced epithelial-mesenchymal transition (EMT) in SAECs and changes in mLFs were examined to investigate AR-mediated molecular mechanism(s) of airway remodeling.
In the OVA-exposed mice for 6 wks inflammatory cells infiltration, levels of inflammatory cytokines and chemokines, goblet cell metaplasia, collagen deposition and AHR were significantly decreased by treatment with AR inhibitor, fidarestat. Further, inhibition of AR prevented TGFβ1-induced altered expression of E-cadherin, Vimentin, Occludin, and MMP-2 in SAECs, and alpha-smooth muscle actin and fibronectin in mLFs. Further, in SAECs, AR inhibition prevented TGFβ1- induced activation of PI3K/AKT/GSK3β pathway but not the phosphorylation of Smad2/3.
Our results demonstrate that allergen-induced airway remodeling is mediated by AR and its inhibition blocks the progression of remodeling via inhibiting TGFβ1-induced Smad-independent and PI3K/AKT/GSK3β-dependent pathway.
PMCID: PMC3584054  PMID: 23460857
10.  Hepatocyte viability and ATP content decrease linearly over time during conventional cold storage of rat liver grafts 
Transplantation proceedings  2011;43(5):1484-1488.
The gold standard in organ preservation is static cold storage (SCS) using University of Wisconsin solution (UW). While it’s known that there is a finite limit to SCS preservation, and that there is a correlation between the ATP levels and organ function post preservation, a quantitative relationship has not been established, which is important in understanding the fundamental limitations to preservation, minimizing cold ischemic injury and hence maximizing utilization of the donor organ pool.
To determine the time limits of cellular viability and metabolic function during SCS, and to characterize the relationship between cellular viability and energetic state, using clinically relevant techniques in organ preservation.
Rat livers were procured and stored using conventional storage in UW solution at 4°C. Viability was assessed by determining the amount of viable hepatocytes and intracellular ATP content after 0, 24, 48, 72 and 120 hours of storage.
Numbers of viable hepatocytes that were isolated from these livers decreased steadily during SCS. After 5 days, viable hepatocytes decreased from 25.95×106 to 0.87×106 cells/gram tissue. Intracellular ATP content decreased from 9.63 to 0.93 μmoles/gram tissue. Statistical analysis (ANOVA) established a linear relation for both parameters as a function of time (P<0.05).
The linear correlation between hepatocyte viability, ATP content, and storage time suggests a shared physiological foundation. These findings confirm ATP as direct predictor for organ quality in the context of liver preservation, which will aid quantitative assessment of donor organs for various applications.
PMCID: PMC3121302  PMID: 21693222
11.  Etanercept Enhances Preservation of Osteochondral Allograft Viability 
Osteochondral allografts are an increasingly popular treatment for the repair of articular cartilage lesions. Current tissue bank protocols require bacteriological testing that takes from 21 to 28 days to process. During this time, tumor necrosis factor-alpha TNF-α (a pro-apoptotic cytokine) is upregulated resulting in loss of chondrocyte viability. To date, etanercept (a cytokine inhibitor) has not been studied in the current storage paradigm with the intention of preserving cell viability.
To assess whether or not the addition of Etanercept can improve the chondrocytic viability of osteochondral allograft during storage.
Controlled, randomized and blinded in vitro laboratory study.
Osteochondral allografts were harvested from eight Boer goat femurs and placed into storage media and stored at 4°C for 28 days. The experimental group was supplemented with 10 µg/mL of Etanercept. After storage, cell viability was assessed by live/dead staining and confocal microscopy. Specimens were also analyzed histologically and underwent histomorphological analysis. TNF-α expression was measured with semi-quantitative PCR.
At 28 days, the percent viability of the superficial zone in etanercept-treated allografts was maintained at significantly higher levels than those measured in the untreated group (69.3 ± 9.4 compared to 47.8 ± 19.1, p=0.01). No difference was found histologically between the etanercept and the untreated group (i.e. safranin-O staining for GAG expression). Histomorphologic assessment showed no difference in indentation stiffness or roughness between groups. TNF-α expression was significantly decreased in the etanercept group compared to the untreated group.
Etanercept was able to maintain cell viability of osteochondral allografts significantly better than the current storage paradigm after 28 days storage.
Maintaining the viability of the superficial zone will benefit outcomes by facilitating joint articulation via improved lubrication. Additionally, maintaining the cellular viability for increased periods of time may allow a greater window of time in which a suitable recipient may be found.
PMCID: PMC3506262  PMID: 21389186
Osteochondral; Allograft; Cartilage; Etanercept
12.  Cold Storage of Rat Hepatocyte Suspensions for One Week in a Customized Cold Storage Solution – Preservation of Cell Attachment and Metabolism 
PLoS ONE  2012;7(7):e40444.
Background & Aims
Primary hepatocytes are of great importance for basic research as well as cell transplantation. However, their stability, especially in suspension, is very low. This feature severely compromises storage and shipment. Based on previous studies with adherent cells, we here assessed cold storage injury in rat hepatocyte suspensions and aimed to find a cold storage solution that preserves viability, attachment ability and functionality of these cells.
Rat hepatocyte suspensions were stored in cell culture medium, organ preservation solutions and modified TiProtec solutions at 4°C for one week. Viability and cell volume were determined by flow cytometry. Thereafter, cells were seeded and density and metabolic capacity (reductive metabolism, forskolin-induced glucose release, urea production) of adherent cells were assessed.
Cold storage injury in hepatocyte suspensions became evident as cell death occurring during cold storage or rewarming or as loss of attachment ability. Cell death during cold storage was not dependent on cell swelling and was almost completely inhibited in the presence of glycine and L-alanine. Cell attachment could be greatly improved by use of chloride-poor solutions and addition of iron chelators. Using a chloride-poor, potassium-rich storage solution containing glycine, alanine and iron chelators, cultures with 75% of the density of control cultures and with practically normal cell metabolism could be obtained after one week of cold storage.
In the solution presented here, cold storage injury of hepatocyte suspensions, differing from that of adherent hepatocytes, was effectively inhibited. The components which acted on the different injurious processes were identified.
PMCID: PMC3392233  PMID: 22792326
Transplantation proceedings  2010;42(7):2771-2776.
We previously demonstrated that adding pyruvate to Perfadex® increased graft metabolism during 24 hours storage and improved reperfusion lung function. This increased metabolism was associated with progressively lower storage solution pH over the preservation interval. We hypothesized that more effective pH regulation would result in further improvements in lung survival after hypothermic storage. Rat lungs were stored for 24 hours in Perfadex, Perfadex with N-2-hydroxyethylpiperazine-propanesulfonic acid buffer (HEPES), pyruvate-modified Perfadex, and pyruvate-modified Perfadex with HEPES. pH change in the storage solution was measured. Structural lung injury was evaluated on hematoxylin-eosin stained tissue sections. Cell death was quantified by measuring necrotic cells by trypan blue exclusion and apoptotic cells by TUNEL assay. Lungs stored in Perfadex® demonstrated the greatest degree of cell death. Lungs from the Pyruvate group exhibited decreased cell death despite greater acidosis. The addition of HEPES reduced cell death and preservation solution acidosis in both Perfadex and pyruvate-modified Perfadex (p<.05). Almost all cell death resulted from necrosis. Adding pyruvate to the preservation solution increases acid formation during storage but reduces cell death. HEPES ameliorates this acidosis and reduces allograft cell destruction. Increasing the preservation solution buffering capacity may be a simple strategy to improve lung preservation for transplantation.
PMCID: PMC2954414  PMID: 20832585
The Journal of General Physiology  1948;31(5):385-410.
Fertilized rabbit ova at the 2-blastomere stage kept in rabbit serum were stored at low temperatures for various lengths of time. They were then cultured at 38°C. for about 24 hours to determine their viability. A number of the viable ova were finally transplanted into recipient does. It was found that rapid cooling of ova to 5° or to 0°C. was more harmful to the subsequent viability of ova than slow cooling. Rapid cooling was not more lethal to the ova than slow cooling, but did prevent their future normal cleavage. There was no difference between those ova cooled rapidly or slowly to 10°C. It was concluded that temperature shock has an adverse effect on ova, especially at the lower temperatures, though temperature shock can be remedied by acclimatization (slow cooling). Thus, the physiological significance of temperature shock would seem to be broadened. The optimal temperature for the storage of ova was investigated. It was found that 10°C. was the best temperature; at this temperature viable ova were obtained after storage for 144 to 168 hours. At 0°, 5°, or 15°C. the ova were viable for 96 to 120 hours, while at 22–24°C., only for 24 to 48 hours. The percentage of dead ova was low at a favorable temperature, increasing only at the end of the storage period. At an unfavorable temperature, however, the rate of death increased steadily from beginning to end of storage. The percentage of abnormally cleaved ova (arrested cleavage and fragmentation) remained at a low level at first at a favorable temperature, but then increased just before or during death of the ova. A critical time for the viability, the abnormal cleavage, and the death of ova was characteristic of each temperature. About 24 to 28 per cent of the viable ova remaining after being stored at 0–15°C. for 2 to 4 days and cultured at 38°C. for 24 hours were capable of development into normal young. The compatibility of serum and ova, the absence of a correlation between the viability of the ova and the source of the fertilizing spermatozoa, and the fertilization of superovulated ova (i.e., the percentage of fertile does in follicular phase and in luteal phase, the percentage of unfertilized ova and of fertilized ova at different stages, the percentage of does that had produced a normal number of ova or had produced a large number of ova, etc.), are reported. The possibility of a more efficient utilization of the germ cells of valuable animals by means of the present techniques, and the possibility of a new approach to the experimental investigation of mammalian genetics and development, have been mentioned.
PMCID: PMC2147119  PMID: 18917023
Transplantation proceedings  2008;40(5):1306-1309.
Extending transplant criteria to include livers obtained from donor after cardiac death (DCD) could increase the liver donor pool, but conventional simple cold storage of these ischemic organs can lead to poor graft function after transplantation. Experimental normothermic machine perfusion has previously proven to be useful for the recovery and preservation of DCD livers, but it is more complicated than conventional cold storage, and is therefore perhaps not practical during the entire preservation period. In clinical situations, the combined use of simple cold storage and normothermic perfusion preservation of DCD livers might be more realistic, but even a brief period of cold storage prior to normothermic preservation has been suggested to have a negative impact on graft viability. In this study we show that rat livers subjected to 45 minutes of ex-vivo warm ischemia followed by 2 hours of simple cold storage can be reclaimed by 4 hours of normothermic machine perfusion. These livers could be orthotopically transplanted into syngeneic recipients with 100% survival after 4 weeks (N=10), similar to the survival of animals that received fresh livers that were stored on ice in University of Wisconsin (UW) solution for 6 hours (N=6). On the other hand, rats that received ischemic livers preserved on ice in UW solution for 6 hours (N = 6) all died within 12 hours after transplantation. These results suggest that normothermic perfusion can be used to reclaim DCD livers subjected to an additional period of cold ischemia during hypothermic storage.
PMCID: PMC2583139  PMID: 18589093
16.  Autophagy and Apoptosis Have a Role in the Survival or Death of Stallion Spermatozoa during Conservation in Refrigeration 
PLoS ONE  2012;7(1):e30688.
Apoptosis has been recognized as a cause of sperm death during cryopreservation and a cause of infertility in humans, however there is no data on its role in sperm death during conservation in refrigeration; autophagy has not been described to date in mature sperm. We investigated the role of apoptosis and autophagy during cooled storage of stallion spermatozoa. Samples from seven stallions were split; half of the ejaculate was processed by single layer centrifugation, while the other half was extended unprocessed, and stored at 5°C for five days. During the time of storage, sperm motility (CASA, daily) and membrane integrity (flow cytometry, daily) were evaluated. Apoptosis was evaluated on days 1, 3 and 5 (active caspase 3, increase in membrane permeability, phosphatidylserine translocation and mitochondrial membrane potential) using flow cytometry. Furthermore, LC3B processing was investigated by western blotting at the beginning and at the end of the period of storage. The decrease in sperm quality over the period of storage was to a large extent due to apoptosis; single layer centrifugation selected non-apoptotic spermatozoa, but there were no differences in sperm motility between selected and unselected sperm. A high percentage of spermatozoa showed active caspase 3 upon ejaculation, and during the period of storage there was an increase of apoptotic spermatozoa but no changes in the percentage of live sperm, revealed by the SYBR-14/PI assay, were observed. LC3B was differentially processed in sperm after single layer centrifugation compared with native sperm. In processed sperm more LC3B-II was present than in non-processed samples; furthermore, in non-processed sperm there was an increase in LC3B-II after five days of cooled storage. These results indicate that apoptosis plays a major role in the sperm death during storage in refrigeration and that autophagy plays a role in the survival of spermatozoa representing a new pro-survival mechanism in spermatozoa not previously described.
PMCID: PMC3266901  PMID: 22292020
17.  Optimization of Storage Temperature for Cultured ARPE-19 Cells 
Journal of Ophthalmology  2013;2013:216359.
Purpose. The establishment of future retinal pigment epithelium (RPE) replacement therapy is partly dependent on the availability of tissue-engineered RPE cells, which may be enhanced by the development of suitable storage methods for RPE. This study investigates the effect of different storage temperatures on the viability, morphology, and phenotype of cultured RPE. Methods. ARPE-19 cells were cultured under standard conditions and stored in HEPES-buffered MEM at nine temperatures (4°C, 8°C, 12°C, 16°C, 20°C, 24°C, 28°C, 32°C, and 37°C) for seven days. Viability and phenotype were assessed by a microplate fluorometer and epifluorescence microscopy, while morphology was analyzed by scanning electron microscopy. Results. The percentage of viable cells preserved after storage was highest in the 16°C group (48.7% ± 9.8%; P < 0.01 compared to 4°C, 8°C, and 24°C–37°C; P < 0.05 compared to 12°C). Ultrastructure was best preserved at 12°C, 16°C, and 20°C. Expression of actin, ZO-1, PCNA, caspase-3, and RPE65 was maintained after storage at 16°C compared to control cells that were not stored. Conclusion. Out of nine temperatures tested between 4°C and 37°C, storage at 12°C, 16°C, and 20°C was optimal for maintenance of RPE cell viability, morphology, and phenotype. The preservation of RPE cells is critically dependent on storage temperature.
PMCID: PMC3819763  PMID: 24251032
18.  Protective effect of Lifor solution in experimental renal ischemia-reperfusion injury 
The Journal of surgical research  2010;164(2):e291-e297.
Improved kidney preservation methods are needed to reduce ischemia-reperfusion (IR) injury in kidney allografts. Lifor is an artificial preservation solution comprised of nutrients, growth factors, and a non-protein oxygen and nutrient carrier. The current study compared the effectiveness of Lifor to University of Wisconsin solution (UW) in protecting rat kidneys from warm IR and cold storage injury.
Materials and Methods
In a warm IR model, rat kidneys were perfused in situ with either saline, UW, or Lifor for 45 minutes. Renal function and histology were assessed 24 hours later. In a cold IR model, kidney slices were cold stored in saline, UW, or Lifor at 4°C. Kidney injury was assessed by the release of lactate dehydrogenase (LDH) and immunoblot analysis for cleaved caspase-3.
Lifor perfusion significantly mitigated renal dysfunction and tubular injury at 24 hours compared to saline or UW. Lifor and UW prevented LDH release in hypoxic kidney slices in vitro, however activation of caspase-3 following hypoxia-reoxygenation was attenuated only with Lifor. Cold storage with Lifor or UW significantly decreased LDH release from kidney slices or normal rat kidney cells in comparison to storage in saline or culture media. After 24 hours of cold storage there was a significant decrease in cleaved caspase-3 in Lifor stored slices compared that seen following cold storage in saline or UW solution.
Lifor solution mitigates both warm and cold renal IR and appears to provide greater protection from apoptosis as compared to UW solution.
PMCID: PMC2991539  PMID: 20888590
renal ischemia-reperfusion; cold preservation; UW solution; Lifor; apoptosis
19.  Material Properties of Fresh Cold-stored Allografts for Osteochondral Defects at 1 Year 
Little is known about the long-term properties of fresh cold-stored osteochondral allograft tissue. We hypothesized fresh cold-stored tissue would yield superior material properties in an in vivo ovine model compared to those using freeze-thawed acellular grafts. In addition, we speculated that a long storage time would yield less successful grafts. We created 10-mm defects in medial femoral condyles of 20 sheep. Defects were reconstructed with allograft plugs stored at 4°C for 1, 14, and 42 days; control specimens were freeze-thawed or defect-only. At 52 weeks, animals were euthanized and retrieved grafts were analyzed for cell viability, gross morphology, histologic grade, and biomechanical and biochemical analysis. Explanted cold-stored tissue had superior histologic scores over freeze-thawed and defect-only grafts. Specimens stored for 1 and 42 days had higher equilibrium moduli and proteoglycan content than freeze-thawed specimens. We observed no difference among any of the cold-stored specimens for chondrocyte viability, histology, equilibrium aggregate modulus, proteoglycan content, or hypotonic swelling. Reconstructing cartilage defects with cold-stored allograft resulted in superior histologic and biomechanical properties compared with acellular freeze-thawed specimens; however, storage time did not appear to be a critical factor in the success of the transplanted allograft.
PMCID: PMC2584258  PMID: 18528743
20.  In vitro and in vivo development of mice morulae after storage in non-frozen conditions 
Interchange of genetically modified (GM) mice between laboratories using embryos provides several advantages. Not only is transport stress avoided, but also the health status of the recipient colony is not compromised. Embryos do not need to be shipped in frozen stage, which requires expensive packaging in addition to a certain degree of expertise in order to freeze and thaw them correctly. The aim of this study was to examine different storage conditions and their effect on embryo viability in order to establish the feasibility of practical, non-frozen conditions for embryo shipment.
Mouse morulae developed in vivo (collected from donors 2.5d post coitum) or in vitro (zygotes cultured until morulae stage) were stored, combining two different media (KSOMeq or KSOM-H) and temperatures (4 degrees C, 15 degrees C and 37 degrees C) throughout 24 or 48 hours. After storage in vitro viability was assessed determining percentage of development to blastocyst and total cell number. In vivo viability was determined based on the number of implantations and living fetuses after embryo transfer of stored embryos. The storage effect at the molecular level was assessed by studying a gene pool involved in early development by quantitative RT-PCR.
In vivo-produced morulae stored for 24 hours did not show differences in development up to the blastocyst stage, regardless of the storage type. Even though a decrease in the total cell number in vivo was observed, embryo development after embryo transfer was not affected. All 24 hour storage conditions tested provided a similar number of implantations and fetuses at day 14 of pregnancy. Morulae obtained from in vitro embryo culture collected at the 1-cell stage showed a decreased ability to develop to blastocyst after 24 hours of storage at 15degrees C both in KSOMeq and KSOM-H. Concomitantly, a significant decrease of embryo implantation rates after transfer to recipients was also found. In order to further characterize the effect of non-frozen storage combining a molecular approach with the ordinary in vitro culture evaluation, embryos collected at the morula stage were submitted to the same storage conditions described throughout 48 hours. In vitro culture of those embryos showed a significant decrease in their developmental rate to blastocyst in both KSOMeq and KSOM-H at 15degrees C, which also affected the total number of cells. Gene transcription studies confirmed significant alterations in retrotransposons (Erv4 and Iap) after 48 h of storage at 15degrees C.
Our results show that both KSOMeq and KSOM-H can be equally used, and that several temperature conditions allow good survival rates in vitro and in vivo. Some of these storage conditions can substitute freezing in order to maintain embryo viability for 24–48 hours, providing a reliable and less demanding technical alternative for embryo interchanges.
PMCID: PMC3517773  PMID: 22913368
Embryo; Mouse; Storage
21.  Release of mitochondrial cytochrome C in both apoptosis and necrosis induced by beta-lapachone in human carcinoma cells. 
Molecular Medicine  1999;5(4):232-239.
BACKGROUND: There are two fundamental forms of cell death: apoptosis and necrosis. Molecular studies of cell death thus far favor a model in which apoptosis and necrosis share very few molecular regulators. It appears that apoptotic processes triggered by a variety of stimuli converge on the activation of a member of the caspase family, such as caspase 3, which leads to the execution of apoptosis. It has been suggested that blocking of caspase activation in an apoptotic process may divert cell death to a necrotic demise, suggesting that apoptosis and necrosis may share some upstream events. Activation of caspase is preceded by the release of mitochondrial cytochrome C. MATERIALS AND METHODS: We first studied cell death induced by beta-lapachone by MTT and colony-formation assay. To determine whether the cell death induced by beta-lapachone occurs through necrosis or apoptosis, we used the PI staining procedure to determine the sub-G1 fraction and the Annexin-V staining for externalization of phophatidylserine. We next compared the release of mitochondrial cytochrome C in apoptosis and necrosis. Mitochondrial cytochrome C was determined by Western blot analysis. To investigate changes in mitochondria that resulted in cytochrome C release, the mitochondrial membrane potential (delta psi) was analyzed by the accumulation of rhodamine 123, a membrane-permeant cationic fluorescent dye. The activation of caspase in apoptosis and necrosis were measured by using a profluorescent substrate for caspase-like proteases, PhiPhiLuxG6D2. RESULTS: beta-lapachone induced cell death in a spectrum of human carcinoma cells, including nonproliferating cells. It induced apoptosis in human ovary, colon, and lung cancer cells, and necrotic cell death in four human breast cancer cell lines. Mitochondrial cytochrome C release was found in both apoptosis and necrosis. This cytochrome C release occurred shortly after beta-lapachone treatment when cells were fully viable by trypan blue exclusion and MTT assay, suggesting that cytochrome C release is an early event in beta-lapachone induced apoptosis as well as necrosis. The mitochondrial cytochrome C release induced by beta-lapachone is associated with a decrease in mitochondrial transmembrane potential (delta psi). There was activation of caspase 3 in apoptotic cell death, but not in necrotic cell death. This lack of activation of CPP 32 in human breast cancer cells is consistent with the necrotic cell death induced by beta-lapachone as determined by absence of sub-G1 fraction, externalization of phosphatidylserine. CONCLUSIONS: beta-lapachone induces either apoptotic or necrotic cell death in a variety of human carcinoma cells including ovary, colon, lung, prostate, and breast, suggesting a wide spectrum of anti-cancer activity in vitro. Both apoptotic and necrotic cell death induced by beta-lapachone are preceded by a rapid release of cytochrome C, followed by the activation of caspase 3 in apoptotic cell death but not in necrotic cell death. Our results suggest that beta-lapachone is a potential anti-cancer drug acting on the mitochondrial cytochrome C-caspase pathway, and that cytochrome C is involved in the early phase of necrosis.
PMCID: PMC2230323  PMID: 10448645
22.  Characterization of Apoptosis Induced by Emodin and Related Regulatory Mechanisms in Human Neuroblastoma Cells 
Emodin (1,3,8-trihydroxy-6-methylanthraquinone), a major constituent of rhubarb, has a wide range of therapeutic applications. Recent studies have shown that emodin can induce or prevent cell apoptosis, although the precise molecular mechanisms underlying these effects are unknown. Experiments from the current study revealed that emodin (10–20 μM) induces apoptotic processes in the human neuroblastoma cell line, IMR-32, but exerts no injury effects at treatment doses below 10 μM. Treatment with emodin at concentrations of 10–20 μM led to a direct increase in the reactive oxygen species (ROS) content in IMR-32 cells, along with significant elevation of cytoplasmic free calcium and nitric oxide (NO) levels, loss of mitochondrial membrane potential (MMP), activation of caspases-9 and -3, and cell death. Pretreatment with nitric oxide (NO) scavengers suppressed the apoptotic biochemical changes induced by 20 μM emodin, and attenuated emodin-induced p53 and p21 expression involved in apoptotic signaling. Our results collectively indicate that emodin at concentrations of 10–20 μM triggers apoptosis of IMR-32 cells via a mechanism involving both ROS and NO. Based on the collective results, we propose a model for an emodin-triggered apoptotic signaling cascade that sequentially involves ROS, Ca2+, NO, p53, caspase-9 and caspase-3.
PMCID: PMC3821607  PMID: 24113589
emodin; apoptosis; oxidative stress; calcium; nitric oxide
23.  Neutrophil preservation: the effect of short-term storage on in vivo kinetics. 
Journal of Clinical Investigation  1977;59(3):475-480.
A rabbit model was developed to study the in vivo viability of neutrophils stored in vitro for up to 72 h. Acid-citrate-dextrose anticoagulated whole blood was obtained from rabbits previously injected with tritiated thymidine ([3H]thymidine), stored under varying conditions, and then injected into recipient rabbits. Neutrophil viability and function were assessed by measuring the ability of the tagged neutrophils to circulate and to migrate into subcutaneous polyvinyl sponges. Unstored neutrophils disappeared exponentially from the circulation with a t1/2 of 3.2 h and gave a zero time recovery of 30.5%. Storage of cells at either room temperature or 4 degrees C for 24 h or longer resulted in temporary sequestration of cells from active circulation. With cells stored for up to 72 h at 4 degrees C, recovery returned to normal values after 1-2 h. Room temperature stored cells, in contrast, showed evidence of irreversible damage at 48-h storage with low recovery for the entire time span studied. With unstored blood, 8.1+/-0.9% of the injected neutrophil label was present in the subcutaneous sponges. The accumulated label progressively decreased as cell storage time increased reaching at 72 h 5.1+/-0.6 and 2.6+/-0.3% for 4 degrees C and room temperature-stored cells, respectively. The results of this study indicate that 4 degrees C storage of rabbit neutrophils is superior to storage at room temperature. The data suggest that it may be feasible to store neutrophils at least a few days without loss of in vivo functions.
PMCID: PMC333384  PMID: 838860
24.  Aldose reductase regulates acrolein-induced cytotoxicity in human small airway epithelial cells 
Free radical biology & medicine  2013;65:10.1016/j.freeradbiomed.2013.06.008.
Aldose reductase (AR), a glucose metabolizing enzyme, reduces lipid aldehydes and their glutathione conjugates with more than 1000-fold efficiency (Km aldehydes 5-30μM) than glucose. Acrolein, a major endogenous lipid peroxidation product as well as component of environmental pollutant and cigarette smoke, is known to be involved in various pathologies including atherosclerosis, airway inflammation, COPD, and age-related disorders but the mechanism of acrolein-induced cytotoxicity is not clearly understood. We have investigated the role of AR in acrolein-induced cytotoxicity in primary human small airway epithelial cells SAECs. Exposure of SAECs to varying concentrations of acrolein caused cell-death in a concentration- and time-dependent manner. AR inhibition by fidarestat prevented the low (5 to 10 μM) but not high (>10 μM) concentrations of acrolein-induced SAECs cell death. AR inhibition protected SAECs from low dose (5 μM) acrolein-induced cellular reactive oxygen species (ROS). Inhibition of acrolein-induced apoptosis by fidarestat was confirmed by decreased condensation of nuclear chromatin, DNA fragmentation, comet tail-moment, and annexin-V fluorescence. Further, fidarestat inhibited acrolein-induced translocation of pro-apoptotic proteins Bax and Bad from cytosol to the mitochondria, and that of Bcl2 and BclXL from mitochondria to cytosol. Acrolein-induced cytochrome c release from mitochondria was also prevented by AR inhibition. The mitogen-activated protein kinases (MAPK) such as extracellular signal-regulated kinases 1 and 2 (ERK1/2), stress-activated protein kinases/c-jun NH2-terminal kinases (SAPK/JNK) and p38MAPK, and c-jun were transiently activated in airway epithelial cells by acrolein in a concentration and time-dependent fashion, which were significantly prevented by AR inhibition. These results suggest that AR inhibitors could prevent acrolein-induced cytotoxicity in the lung epithelial cells.
PMCID: PMC3830659  PMID: 23770200
Aldose reductase; acrolein; apoptosis; cytotoxicity; COPD
Experimental eye research  2013;111:95-104.
There are currently no validated minimally invasive objective metrics for the classification and evaluation of ocular surface diseases and/or for evaluating treatment efficacy. We thus sought to establish a standardized methodology for determining the relative amount of the inflammatory biomarker HLA-DR on the ocular surface and to evaluate the precision, reliability and repeatability of its use for large multicenter clinical trials and translational research studies of ocular surface disease. Multiple studies were conducted to establish a Standard Operating Procedure (SOP) for utilizing HLA-DR expression as a minimally invasive, objective, ocular surface inflammatory biomarker. The established SOPs provide specific guidelines for HLA-DR collection and analysis, in order to incorporate it reliably into multicenter clinical trials and/or translational research. Duplicate cell samples from impression cytology (IC) samples of both normal and dry eye individuals were collected and split to assess repeatability (between the splits and between the duplicate samples). To determine storage capability, one duplicate was stained immediately and the other after 30 days cold storage. To demonstrate the feasibility of the use of the SOP for a multicenter clinical trial, clinicians out-of-state were trained to collect IC samples, and the samples shipped to our Biomarker Laboratory, logged, processed and analyzed. Demonstration of the ability to incorporate of IC into a randomized double masked clinical trial of dry eye disease (DED) was performed. In all cases, processing and analyses were performed by a masked independent observer. The validity/viability of the SOPs was established by demonstrating that: 1) sufficient numbers of cells can be collected via IC; 2) the precision/repeatability of the relative biomarker expression quantified in samples; 3) personnel at distant sites can be taught to collect, store and ship samples successfully; 4) samples can be stored for up to 30 days (refrigeration) before processing without affecting results; 5) IC can be incorporated into a double blind randomized clinical trial (RCT) of DED; and 6) the Biomarker Laboratory can track a large number of masked samples reliably. In conclusion, our standard operating procedure for impression cytology analysis of HLA-DR expression appears to be repeatable and reproducible for use in multicenter clinical trials, providing a minimally invasive objective biomarker of inflammation of the ocular surface.
PMCID: PMC4086244  PMID: 23567204
Impression Cytology; HLA-DR; Dry Eye; Precision; Reliability; Repeatability; Storage; Feasibility

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