Assessment of stored red blood cells through lab-on-a-chip technologies for precision transfusion medicine.

Transfusion of red blood cells (RBCs) is one of the most valuable and widespread treatments in modern medicine. Lifesaving RBC transfusions are facilitated by the cold storage of RBC units in blood banks worldwide. Currently, RBC storage and subsequent transfusion practices are performed using simplistic workflows. More specifically, most blood banks follow the "first-in-first-out" principle to avoid wastage, whereas most healthcare providers prefer the "last-in-first-out" approach simply favoring chronologically younger RBCs. Neither approach addresses recent advances through -omics showing that stored RBC quality is highly variable depending on donor-, time-, and processing-specific factors. Thus, it is time to rethink our workflows in transfusion medicine taking advantage of novel technologies to perform RBC quality assessment. We imagine a future where lab-on-a-chip technologies utilize novel predictive markers of RBC quality identified by -omics and machine learning to usher in a new era of safer and precise transfusion medicine.

Zebrafish as a high throughput model for organ preservation and transplantation research.

Despite decades of effort, the preservation of complex organs for transplantation remains a significant barrier that exacerbates the organ shortage crisis. Progress in organ preservation research is significantly hindered by suboptimal research tools that force investigators to sacrifice translatability over throughput. For instance, simple model systems, such as single cell monolayers or co-cultures, lack native tissue structure and functional assessment, while mammalian whole organs are complex systems with confounding variables not compatible with high-throughput experimentation. In response, diverse fields and industries have bridged this experimental gap through the development of rich and robust resources for the use of zebrafish as a model organism. Through this study, we aim to demonstrate the value zebrafish pose for the fields of solid organ preservation and transplantation, especially with respect to experimental transplantation efforts. A wide array of methods were customized and validated for preservation-specific experimentation utilizing zebrafish, including the development of assays at multiple developmental stages (larvae and adult), methods for loading and unloading preservation agents, and the development of viability scores to quantify functional outcomes. Using this platform, the largest and most comprehensive screen of cryoprotectant agents (CPAs) was performed to determine their toxicity and efficiency at preserving complex organ systems using a high subzero approach called partial freezing (i.e., storage in the frozen state at -10°C). As a result, adult zebrafish cardiac function was successfully preserved after 5 days of partial freezing storage. In combination, the methods and techniques developed have the potential to drive and accelerate research in the fields of solid organ preservation and transplantation.

Cryogenic enrichment of Plasmodium falciparum gametocytes from spiked whole blood.

Each individual cell type typically requires a unique set of conditions for optimal cryopreservation outcome, which relates to its specific response to cryoprotective agent (CPA) toxicity, osmotic behavior and sensitivity to ice crystallization. Cryopreservation of heterogenous cell populations is therefore exceedingly difficult as it requires separate and often conflicting conditions for each cell type. Conversely, these contrasting conditions could be utilized to favor cryogenic preference of a single cell population within a heterogenous sample, leading to its enrichment by elimination of remaining cells. To establish proof-of-concept for this overall approach, a protocol was developed for the cryogenic enrichment of Plasmodium falciparum gametocytes from whole blood. To accomplish this goal, we evaluated the effects of CPAs and cooling conditions during cryopreservation of whole blood samples spiked with P. falciparum gametocytes. We identified that cooling to -80 °C at a rate of -1 °C/min in the presence of 11 % glycerol selectively favors recovery of gametocytes. This protocol eliminates 95.3 ± 1.7 % of total blood cells and recovers 43.2 ± 6.5 % of parasites, leading to a 19-fold enrichment as assessed by microscopic examination of blood smears. This protocol is tunable, where gametocyte enrichment 900-fold may be feasible, however there is an apparent tradeoff in overall parasite recovery. Although translation of this protocol for point-of-care testing for malaria presents many challenges, the overall approach of cryogenic purification may prove useful for alternative diagnostic applications.

Polyethylene glycol and caspase inhibitor emricasan alleviate cold injury in primary rat hepatocytes.

Current methods of storing explanted donor livers at 4 °C in University of Wisconsin (UW) solution result in loss of graft function and ultimately lead to less-than-ideal outcomes post transplantation. Our lab has previously shown that supplementing UW solution with 35-kilodalton polyethylene glycol (PEG) has membrane stabilizing effects for cold stored primary rat hepatocytes in suspension. Expanding on past studies, we here investigate if PEG has the same beneficial effects in an adherent primary rat hepatocyte cold storage model. In addition, we investigated the extent of cold-induced apoptosis through treating cold-stored hepatocytes with pan caspase inhibitor emricasan. In parallel to storage at the current cold storage standard of 4 °C, we investigated the effects of lowering the storage temperature to -4 °C, at which the storage solution remains ice-free due to the supercooling phenomenon. We show the addition of 5 % PEG to the storage medium significantly reduced the release of lactate dehydrogenase (LDH) in plated rat hepatocytes and a combinatorial treatment with emricasan maintains hepatocyte viability and morphology following recovery from cold storage. These results show that cold-stored hepatocytes undergo multiple mechanisms of cold-induced injury and that PEG and emricasan treatment in combination with supercooling may improve cell and organ preservation.

Exceeding the Limits of Static Cold Storage in Limb Transplantation Using Subnormothermic Machine Perfusion.

BACKGROUND: For 50 years, static cold storage (SCS) has been the gold standard for solid organ preservation in transplantation. Although logistically convenient, this preservation method presents important constraints in terms of duration and cold ischemia-induced lesions. We aimed to develop a machine perfusion (MP) protocol for recovery of vascularized composite allografts (VCA) after static cold preservation and determine its effects in a rat limb transplantation model. METHODS: Partial hindlimbs were procured from Lewis rats and subjected to SCS in Histidine-Tryptophan-Ketoglutarate solution for 0, 12, 18, 24, and 48 hours. They were then either transplanted (Txp), subjected to subnormothermic machine perfusion (SNMP) for 3 hours with a modified Steen solution, or to SNMP + Txp. Perfusion parameters were assessed for blood gas and electrolytes measurement, and flow rate and arterial pressures were monitored continuously. Histology was assessed at the end of perfusion. For select SCS durations, graft survival and clinical outcomes after transplantation were compared between groups at 21 days. RESULTS: Transplantation of limbs preserved for 0, 12, 18, and 24-hour SCS resulted in similar survival rates at postoperative day 21. Grafts cold-stored for 48 hours presented delayed graft failure (p = 0.0032). SNMP of limbs after 12-hour SCS recovered the vascular resistance, potassium, and lactate levels to values similar to limbs that were not subjected to SCS. However, 18-hour SCS grafts developed significant edema during SNMP recovery. Transplantation of grafts that had undergone a mixed preservation method (12-hour SCS + SNMP + Txp) resulted in better clinical outcomes based on skin clinical scores at day 21 post-transplantation when compared to the SCS + Txp group (p = 0.01613). CONCLUSION: To date, VCA MP is still limited to animal models and no protocols are yet developed for graft recovery. Our study suggests that ex vivo SNMP could help increase the preservation duration and limit cold ischemia-induced injury in VCA transplantation.

Optimization of Ex Vivo Machine Perfusion and Transplantation of Vascularized Composite Allografts.

BACKGROUND: Machine perfusion is gaining interest as an efficient method of tissue preservation of Vascularized Composite Allografts (VCA). The aim of this study was to develop a protocol for ex vivo subnormothermic oxygenated machine perfusion (SNMP) on rodent hindlimbs and to validate our protocol in a heterotopic hindlimb transplant model. METHODS: In this optimization study we compared three different solutions during 6 h of SNMP (n = 4 per group). Ten control limbs were stored in a preservation solution on Static Cold Storage [SCS]). During SNMP we monitored arterial flowrate, lactate levels, and edema. After SNMP, muscle biopsies were taken for histology examination, and energy charge analysis. We validated the best perfusion protocol in a heterotopic limb transplantation model with 30-d follow up (n = 13). As controls, we transplanted untreated limbs (n = 5) and hindlimbs preserved with either 6 or 24 h of SCS (n = 4 and n = 5). RESULTS: During SNMP, arterial outflow increased, and lactate clearance decreased in all groups. Total edema was significantly lower in the HBOC-201 group compared to the BSA group (P = 0.005), 4.9 (4.3-6.1) versus 48.8 (39.1-53.2) percentage, but not to the BSA + PEG group (P = 0.19). Energy charge levels of SCS controls decreased 4-fold compared to limbs perfused with acellular oxygen carrier HBOC-201, 0.10 (0.07-0.17) versus 0.46 (0.42-0.49) respectively (P = 0.002). CONCLUSIONS: Six hours ex vivo SNMP of rodent hindlimbs using an acellular oxygen carrier HBOC-201 results in superior tissue preservation compared to conventional SCS.

Epigenetic regulation by DNA methyltransferases during torpor in the thirteen-lined ground squirrel Ictidomys tridecemlineatus.

The thirteen-lined ground squirrel, Ictidomys tridecemlineatus, is a mammal capable of lowering its Tb to almost 0 °C while undergoing deep torpor bouts over the winter. To decrease its metabolic rate to such a drastic extent, the squirrel must undergo multiple physiological, biological, and molecular alterations including downregulation of almost all nonessential processes. Epigenetic regulation allows for a dynamic range of transient phenotypes, allowing the squirrel to downregulate energy-expensive and nonessential pathways during torpor. DNA methylation is a prominent form of epigenetic regulation; therefore, the DNA methyltransferase (DNMT) family of enzymes were studied by measuring expression and activity levels of the five major proteins during torpor bouts. Additionally, specific cytosine marks on genomic DNA were quantified to further elucidate DNA methylation during hibernation. A tissue-specific response was observed that highlighted variant degrees of DNA methylation and DNMT expression/activity, demonstrating that DNA methylation is a highly complex form of epigenetic regulation and likely one of many regulatory mechanisms that enables metabolic rate depression in response to torpor.

Modulating Nrf2 transcription factor activity: Revealing the regulatory mechanisms of antioxidant defenses during hibernation in 13-lined ground squirrels.

Mammalian hibernators undergo major behavioural, physiological and biochemical changes to survive hypothermia, ischaemia-reperfusion and finite fuel reserves during days or weeks of continuous torpor. During hibernation, the 13-lined ground squirrel (Ictidomys tridecemlineatus) undergoes a global suppression of energetically expensive processes such as transcription and translation, while selectively upregulating certain genes/proteins to mitigate torpor-related damage. Antioxidant defenses are critical for preventing damage caused by reactive oxygen species (ROS) during torpor and arousal, and Nrf2 is a critical regulator of these antioxidant genes. This study analysed the relative protein expression levels of Nrf2, KEAP1, small Mafs (MafF, MafK and MafG) and catalase and the regulation of Nrf2 transcription factors by post-translational modifications (PTMs) and protein-protein interactions with a negative regulator (KEAP1) during hibernation. It was found that a significant increase in MafK during late torpor predicated an increase in relative Nrf2 and catalase levels seen in arousal. Additionally, Nrf2-KEAP1 protein-protein interactions and Nrf2 PTMs, including serine phosphorylation and lysine acetylation, were responsive to cycles of torpor-arousal with peak responses occurring during arousal. These peaks seen during arousal correspond to a surge in oxygen consumption, which causes increased ROS production. Thus, these regulatory mechanisms could be important during hibernation because they provide mechanisms for mitigating the deleterious effects of oxidative stress by modifying Nrf2 expression and function in an energetically inexpensive manner.

The efficacy of HBOC-201 in ex situ gradual rewarming kidney perfusion in a rat model.

Gradual rewarming from hypothermic to normothermic is a novel perfusion modality with superior outcome to sudden rewarming to normothermic. However, the identification of an oxygen carrier that could function at a temperature range from 4 to 7°C or whether it is necessary to use oxygen carrier during kidney rewarming, remains unresolved. This study was designed to test the use of a hemoglobin-based oxygen carrier (HBOC) during gradual kidney rewarming as an alternative to simple dissolved oxygen. In this study, 10 rat kidneys were randomly divided into the control and the HBOC group. In the control group, no oxygen carrier was used during rewarming perfusion and the perfusion solution was oxygenated only by applying diffused carbogen flow. The protocol mimicked a donor after circulatory death (DCD) kidney transplantation, where after 30 minutes warm ischemia and 120 minutes cold storage in University of Wisconsin solution, the DCD kidneys underwent gradual rewarming from 10 to 37°C during 90 minutes with or without HBOC. This was followed by 30 minutes of warm ischemia in room temperature to mimic the anastomosis time and 120 minutes of reperfusion at 37°C to mimic the early post-transplant state of the graft. The HBOC group demonstrated superior kidney function which was highlighted by higher ultrafiltrate production, better glomerular filtration rate and improved sodium reabsorption. There was no significant difference between the 2 groups regarding the hemodynamics, tissue injury, and adenosine triphosphate levels. In conclusion, this study suggests better renal function recovery in DCD kidneys after rewarming with HBOC compared to rewarming without an oxygen carrier.

Molecular control of protein synthesis, glucose metabolism, and apoptosis in the brain of hibernating thirteen-lined ground squirrels.

Thirteen-lined ground squirrels (Ictidomys tridecemlineatus) are excellent models for studying acute brain ischemia because they show high resistance to reductions in blood flow and oxygen delivery without evidence of neurological damage. In this study, we analyzed the insulin signaling pathway and regulation of mitochondrial substrate oxidation in three regions of ground squirrel brain (forebrain, cerebellum, and brainstem), comparing summer, late torpor, and interbout arousal conditions. We found select decreases in phospho-Akt in the cerebellum during torpor compared with summer animals, as well as select increases in the forebrain during interbout arousal, suggesting that Akt may influence either metabolism or cytoprotective pathways. The phosphoprotein abundance of glycogen synthase kinase 3 beta (GSK3ß) showed the most consistent trend across all three brain regions, with peak increases observed during deep torpor, suggesting a crucial role for this protein during hibernation. Furthermore, all three regions of the brain showed increased phospho-protein abundance of pyruvate dehydrogenase at serine 232 during both deep torpor and interbout arousal, and serine 300 during interbout arousal only, whereas other phosphorylation sites showed a region-specific expression pattern. Information collected from these studies sheds light on the molecular controls governing insulin signaling and fuel utilization in the brain of hibernating ground squirrels.

Bulk Droplet Vitrification: An Approach to Improve Large-Scale Hepatocyte Cryopreservation Outcome.

Loss of hepatocyte viability and metabolic function after cryopreservation is still a major issue. Although vitrification is a promising alternative, it has generally been proven to be unsuitable for vitrification of large cell volumes which is required for clinical applications. Here, we propose a novel bulk droplet (3-5 mm diameter) vitrification method which allows high throughput volumes (4 mL/min), while using a low preincubated CPA concentration (15% v/v) to minimize toxicity and loss of cell viability and function. We used rapid (1.25 s) osmotic dehydration to concentrate a low preincubated intracellular CPA concentration ahead of vitrification, without the need of fully equilibrating toxic CPA concentrations. We compared direct postpreservation viability, long-term viability, and metabolic function of bulk droplet vitrified, cryopreserved, and fresh hepatocytes. Simulations and cooling rate measurements confirmed an adequate concentration of the intracellular CPA concentration (up to 8.53 M) after dehydration in combination with high cooling rates (960-1320 °C/min) for successful vitrification. In comparison to cryopreserved hepatocytes, bulk droplet vitrified hepatocytes had a significantly higher viability, directly after preservation and after 1 day in culture. Moreover, bulk droplet vitrified hepatocytes had evidently better morphology and showed significantly higher metabolic activity than cryopreserved hepatocytes in long-term collagen sandwich cultures. In conclusion, we developed a novel bulk droplet vitrification method of which we validated the theoretical background and demonstrated the feasibility to use this method to vitrify large cell volumes. Moreover, we showed that this method results in improved hepatocyte viability and metabolic function as compared to cryopreservation.

Leveraging the zebrafish to model organ transplantation.

PURPOSE OF REVIEW: The availability of organs for transplant fails to meet the demand and this shortage is growing worse every year. As the cost of not getting a suitable donor organ can mean death for patients, new tools and approaches that allows us to make advances in transplantation faster and provide a different vantage point are required. To address this need, we introduce the concept of using the zebrafish (Danio rerio) as a new model system in organ transplantation. The zebrafish community offers decades of research experience in disease modeling and a rich toolbox of approaches for interrogating complex pathological states. We provide examples of how already existing zebrafish assays/tools from cancer, regenerative medicine, immunology, and others, could be leveraged to fuel new discoveries in pursuit of solving the organ shortage. RECENT FINDINGS: Important innovations have enabled several types of transplants to be successfully performed in zebrafish, including stem cells, tumors, parenchymal cells, and even a partial heart transplant. These innovations have been performed against a backdrop of an expansive and impressive list of tools designed to uncover the biology of complex systems that include a wide array of fluorescent transgenic fish that label specific cell types and mutant lines that are transparent, immune-deficient. Allogeneic transplants can also be accomplished using immune suppressed and syngeneic fish. Each of these innovations within the zebrafish community would provide several helpful tools that could be applied to transplant research. SUMMARY: We highlight some examples of existing tools and assays developed in the zebrafish community that could be leveraged to overcome barriers in organ transplantation, including ischemia-reperfusion, short preservation durations, regeneration of marginal grafts, and acute and chronic rejection.

Advances in machine perfusion, organ preservation, and cryobiology: potential impact on vascularized composite allotransplantation.

PURPOSE OF REVIEW: In this review, we discuss novel strategies that allow for extended preservation of vascularized composite allografts and their potential future clinical implications for the field of vascularized composite allotransplantation (VCA). RECENT FINDINGS: The current gold standard in tissue preservation - static cold preservation on ice - is insufficient to preserve VCA grafts for more than a few hours. Advancements in the field of VCA regarding matching and allocation, desensitization, and potential tolerance induction are all within reasonable reach to achieve; these are, however, constrained by limited preservation time of VCA grafts. Although machine perfusion holds many advantages over static cold preservation, it currently does not elongate the preservation time. More extreme preservation techniques, such as cryopreservation approaches, are, however, specifically difficult to apply to composite tissues as the susceptibility to ischemia and cryoprotectant agents varies greatly by tissue type. SUMMARY: In the current scope of extended preservation protocols, high subzero approaches of VCA grafts will be particularly critical enabling technologies for the implementation of tolerance protocols clinically. Ultimately, advances in both preservation techniques and tolerance induction have the potential to transform the field of VCA and eventually lead to broad applications in reconstructive transplantation.

Enhanced Isolation and Release of Circulating Tumor Cells Using Nanoparticle Binding and Ligand Exchange in a Microfluidic Chip.

The detection of rare circulating tumor cells (CTCs) in the blood of cancer patients has the potential to be a powerful and noninvasive method for examining metastasis, evaluating prognosis, assessing tumor sensitivity to drugs, and monitoring therapeutic outcomes. In this study, we have developed an efficient strategy to isolate CTCs from the blood of breast cancer patients using a microfluidic immune-affinity approach. Additionally, to gain further access to these rare cells for downstream characterization, our strategy allows for easy detachment of the captured CTCs from the substrate without compromising cell viability or the ability to employ next generation RNA sequencing for the identification of specific breast cancer genes. To achieve this, a chemical ligand-exchange reaction was engineered to release cells attached to a gold nanoparticle coating bound to the surface of a herringbone microfluidic chip (NP-HBCTC-Chip). Compared to the use of the unmodified HBCTC-Chip, our approach provides several advantages, including enhanced capture efficiency and recovery of isolated CTCs.

Regulation of the insulin-Akt signaling pathway and glycolysis during dehydration stress in the African clawed frog Xenopus laevis.

Estivation is an adaptive stress response utilized by some amphibians during periods of drought in the summer season. In this study, we examine the regulation of the insulin signaling cascade and glycolysis pathway in the African clawed frog Xenopus laevis during the dehydration stress induced state of estivation. We show that in the brain and heart of X. laevis, dehydration reduces the phosphorylation of the insulin growth factor-1 receptor (IGF-1R), and this is followed by similar reductions in the phosphorylation of the Akt and mechanistic target of rapamycin (mTOR) kinase. Interestingly, phosphorylation levels of IGF-1R and mTOR were not affected in the kidney, and phosphorylation levels of P70S6K and the ribosomal S6 protein were elevated during dehydration stress. Animals under estivation are also susceptible to periods of hypoxia, suggesting that glycolysis may also be affected. We observed that protein levels of many glycolytic enzymes remained unchanged during dehydration; however, the hypoxia response factor-1 alpha (HIF-1α) protein was elevated by greater than twofold in the heart during dehydration. Overall, we provide evidence that shows that the insulin signaling pathway in X. laevis is regulated in a tissue-specific manner during dehydration stress and suggests an important role for this signaling cascade in mediating the estivation response.

MAP kinase signaling and Elk1 transcriptional activity in hibernating thirteen-lined ground squirrels.

BACKGROUND: The thirteen-lined ground squirrel (I. tridecemlineatus) becomes hypometabolic during hibernation; characterized by reductions in energy expensive processes like transcription during periods of low temperature and starvation. We were interested in elucidating the mechanisms of transcriptional control by studying the MAPK pathway and downstream transcription factors in skeletal muscle. We were also interested in how environmental factors, such as body temperature, that fluctuate during hibernation, can affect transcriptional regulation. METHODS: Protein abundance of MAPKs and effectors were quantified using immunoblotting and Luminex® multiplex assays. DNA-protein interaction (DPI)-ELISA was used to quantitatively assess the binding of transcription factors to DNA promoter sequences under environmental conditions which are reflective of those during torpor. RESULTS: JNK2/3 showed increases in protein abundance during early arousal. The transcription factor, Elk1, showed increases in phosphorylation of S383 (which is indicative of increased activity) during arousal that accompany decreases in total protein levels. Further analysis on the relative binding of this transcription factor to its consensus sequence during euthermia and torpor showed that its binding is significantly different when environmental conditions such as temperature, [urea], and [Ca2+] were changed. CONCLUSION: We show evidence of Elk1 regulation during hibernation, and its activity could be influenced by environmental factors such as temperature, [urea] and [Ca2+], as well as post-translational modifications via JNK2/3. GENERAL SIGNIFICANCE: I. tridecemlineatus has natural mechanisms of transcriptional regulation during hibernation through phosphorylation and changes to the cellular environment. We identify regulation of JNK and Elk1 in the skeletal muscle of hibernating I. tridecemlineatus.

The role of global histone post-translational modifications during mammalian hibernation.

Mammalian hibernators must cope with hypothermia, ischemia-reperfusion, and finite fuel reserves during days or weeks of continuous torpor. One means of lowering ATP demands during hibernation involves substantial transcriptional controls. The present research analyzed epigenetic regulatory factors as a means of achieving transcriptional control over cycles of torpor-arousal. This study analyzes differential regulation of select histone modifications (e.g. phosphorylation, acetylation, methylation), and identifies post-translational modifications on purified histones using mass spectrometry from thirteen-lined ground squirrels (Ictidomys tridecemlineatus). Post-translational modifications on histone proteins were responsive to torpor-arousal, suggesting a potential mechanism to dynamically alter chromatin structure. Furthermore, proteomic sequencing data of ground squirrel histones identified lysine 19 and 24 acetylation on histone H3, while acetylation sites identified on H2B were lysine 6, 47, 110, and 117. The present study provides a new glimpse into the epigenetic mechanisms which may play a role in transcriptional regulation during mammalian hibernation.

Controlled ice nucleation using freeze-dried Pseudomonas syringae encapsulated in alginate beads.

The control of ice nucleation is of fundamental significance in many process technologies related to food and pharmaceutical science and cryobiology. Mechanical perturbation, electromagnetic fields and ice-nucleating agents (INAs) have been known to induce ice nucleation in a controlled manner. But these ice-nucleating methods may suffer from cumbersome manual operations, safety concerns of external fields, and biocompatibility and recovery issues of INA particles, especially when used in living systems. Given the automatic ice-seeding nature of INAs, a promising solution to overcome some of the above limitations is to engineer a biocomposite that accommodates the INA particles but minimizes their interactions with biologics, as well as enabling the recovery of used particles. In this study, freeze-dried Pseudomonas syringae, a model ice-nucleating agent, was encapsulated into microliter-sized alginate beads. We evaluated the performance of the bacterial hydrogel beads to initiate ice nucleation in water and aqueous glycerol solution by investigating factors including the size and number of the beads and the local concentration of INA particles. In the aqueous sample of a fixed volume, the total mass of the INA particles (m) was found to be the governing parameter that is solely responsible for determining the ice nucleation performance of the bacterial hydrogel beads. The freezing temperature has a strong positive linear correlation with log10m. The findings in this study provide an effective, predictable approach to control ice nucleation, which can improve the outcome and standardization of many ice-assisted process technologies.

Regulation of pyruvate dehydrogenase (PDH) in the hibernating ground squirrel, (Ictidomys tridecemlineatus).

Pyruvate dehydrogenase (PDH) is a vital regulatory enzyme that catalyzes the conversion of pyruvate into acetyl-CoA and connects anaerobic glycolysis to aerobic TCA cycle. Post-translational inhibition of PDH activity via three serine phosphorylation sites (pS232, pS293, and pS300) regulate the metabolic flux through the TCA cycle, decrease glucose utilization, and facilitate lipid metabolism during times of nutrient deprivation. As metabolic readjustment is necessary to survive hibernation, the purpose of this study was to explore the post-translational regulation of pyruvate dehydrogenase and the expression levels of four mitochondrial serine/threonine kinases (PDHKs), during torpor-arousal cycles in liver, heart, and skeletal muscle of 13-lined ground squirrels. A combination of Luminex multiplex technology and western immunoblotting were used to measure the protein expression levels of total PDH, three phosphorylation sites, S232, 293, 300, and the expression levels of the corresponding PDH kinases (PDHK1-4) during euthermic control, entrance, late torpor, and interbout arousal. Liver and heart showed strong inhibitory PDH regulation, indicating a possible decrease in glucose utilization and a possible preference for ß-oxidation of fatty acids during periods of low temperature and starvation. On the contrary, skeletal muscle showed limited PDH regulation via phosphorylation, possibly due to alternate controls. Phosphorylation of PDH may play an important role in regulating aerobic and anaerobic metabolic responses during hibernation in the 13-lined ground squirrel.