Person: Bruinsma, Bote G.
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Bruinsma
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Bote G.
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Bruinsma, Bote G.
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Publication Supercooling as a Viable Non-Freezing Cell Preservation Method of Rat Hepatocytes(Public Library of Science, 2013) Usta, O. Berk; Kim, Yeonhee; Ozer, Sinan; Bruinsma, Bote G.; Lee, Jungwoo; Demir, Esin; Berendsen, Tim A.; Puts, Catheleyne F.; Izamis, Maria-Louisa; Uygun, Korkut; Uygun, Basak; Yarmush, MartinSupercooling preservation holds the potential to drastically extend the preservation time of organs, tissues and engineered tissue products, and fragile cell types that do not lend themselves well to cryopreservation or vitrification. Here, we investigate the effects of supercooling preservation (SCP at -4oC) on primary rat hepatocytes stored in cryovials and compare its success (high viability and good functional characteristics) to that of static cold storage (CS at +4oC) and cryopreservation. We consider two prominent preservation solutions a) Hypothermosol (HTS-FRS) and b) University of Wisconsin solution (UW) and a range of preservation temperatures (-4 to -10 oC). We find that there exists an optimum temperature (-4oC) for SCP of rat hepatocytes which yields the highest viability; at this temperature HTS-FRS significantly outperforms UW solution in terms of viability and functional characteristics (secretions and enzymatic activity in suspension and plate culture). With the HTS-FRS solution we show that the cells can be stored for up to a week with high viability (~56%); moreover we also show that the preservation can be performed in large batches (50 million cells) with equal or better viability and no loss of functionality as compared to smaller batches (1.5 million cells) performed in cryovials.Publication Supercooling preservation and transplantation of the rat liver(Springer Science and Business Media LLC, 2015-02-18) Bruinsma, Bote G.; Berendsen, Tim A; Izamis, Maria-Louisa; Yeh, Heidi; Yarmush, Martin; Uygun, KorkutPublication Successful Supercooled Liver Storage for 4 Days(2014) Berendsen, Tim A.; Bruinsma, Bote G.; Puts, Catheleyne F.; Saeidi, Nima; Usta, O. Berk; Uygun, Basak; Izamis, Maria-Louisa; Toner, Mehmet; Yarmush, Martin; Uygun, KorkutThe realization of long–term human organ preservation will have groundbreaking effects on the current practice of transplantation. Herein we present a novel technique based on sub–zero non–freezing tissue preservation and extracorporeal machine perfusion that allows transplantation of rat livers preserved for up to 4 days, thereby tripling the viable preservation duration.Publication Functional Human Liver Preservation and Recovery by Means of Subnormothermic Machine Perfusion(MyJove Corporation, 2015) Bruinsma, Bote G.; Avruch, James H.; Weeder, Pepijn D.; Sridharan, Gautham V.; Uygun, Basak; Karimian, Negin; Porte, Robert J.; Markmann, James; Yeh, Heidi; Uygun, KorkutThere is currently a severe shortage of liver grafts available for transplantation. Novel organ preservation techniques are needed to expand the pool of donor livers. Machine perfusion of donor liver grafts is an alternative to traditional cold storage of livers and holds much promise as a modality to expand the donor organ pool. We have recently described the potential benefit of subnormothermic machine perfusion of human livers. Machine perfused livers showed improving function and restoration of tissue ATP levels. Additionally, machine perfusion of liver grafts at subnormothermic temperatures allows for objective assessment of the functionality and suitability of a liver for transplantation. In these ways a great many livers that were previously discarded due to their suboptimal quality can be rescued via the restorative effects of machine perfusion and utilized for transplantation. Here we describe this technique of subnormothermic machine perfusion in detail. Human liver grafts allocated for research are perfused via the hepatic artery and portal vein with an acellular oxygenated perfusate at 21 °C.Publication Tissue heterogeneity in structure and conductivity contribute to cell survival during irreversible electroporation ablation by “electric field sinks”(Nature Publishing Group, 2015) Golberg, Alexander; Bruinsma, Bote G.; Uygun, Basak; Yarmush, MartinIrreversible electroporation (IRE) is an emerging, minimally invasive technique for solid tumors ablation, under clinical investigation for cancer therapy. IRE affects only the cell membrane, killing cells while preserving the extracellular matrix structure. Current reports indicate tumors recurrence rate after IRE averaging 31% of the cases, of which 10% are local recurrences. The mechanisms for these recurrences are not known and new explanations for incomplete cell death are needed. Using finite elements method for electric field distribution, we show that presence of vascular structures with blood leads to the redistribution of electric fields leading to the areas with more than 60% reduced electric field strength in proximity to large blood vessels and clustered vessel structures. In an in vivo rat model of liver IRE ablation, we show that cells located in the proximity of larger vessel structures and in proximity of clustered vessel structures appear less affected by IRE ablation than cells in the tissue parenchyma or in the proximity of small, more isolated vessels. These findings suggest a role for “electric field sinks” in local tumors recurrences after IRE and emphasize the importance of the precise mapping of the targeted organ structure and conductivity for planning of electroporation procedures.Publication Warm Ischemic Injury Is Reflected in the Release of Injury Markers during Cold Preservation of the Human Liver(Public Library of Science, 2015) Bruinsma, Bote G.; Wu, Wilson; Ozer, Sinan; Farmer, Adam; Markmann, James; Yeh, Heidi; Uygun, KorkutBackground: Liver transplantation plays a pivotal role in the treatment of patients with end-stage liver disease. Despite excellent outcomes, the field is strained by a severe shortage of viable liver grafts. To meet high demands, attempts are made to increase the use of suboptimal livers by both pretransplant recovery and assessment of donor livers. Here we aim to assess hepatic injury in the measurement of routine markers in the post-ischemic flush effluent of discarded human liver with a wide warm ischemic range. Methods: Six human livers discarded for transplantation with variable warm and cold ischemia times were flushed at the end of preservation. The liver grafts were flushed with NaCl or Lactated Ringer’s, 2 L through the portal vein and 1 L through the hepatic artery. The vena caval effluent was sampled and analyzed for biochemical markers of injury; lactate dehydrogenase (LDH), alanine transaminase (ALT), and alkaline phosphatase (ALP). Liver tissue biopsies were analyzed for ATP content and histologically (H&E) examined. Results: The duration of warm ischemia in the six livers correlated significantly to the concentration of LDH, ALT, and ALP in the effluent from the portal vein flush. No correlation was found with cold ischemia time. Tissue ATP content at the end of preservation correlated very strongly with the concentration of ALP in the arterial effluent (P<0.0007, R2 = 0.96). Conclusion: Biochemical injury markers released during the cold preservation period were reflective of the duration of warm ischemic injury sustained prior to release of the markers, as well as the hepatic energy status. As such, assessment of the flush effluent at the end of cold preservation may be a useful tool in evaluating suboptimal livers prior to transplantation, particularly in situations with undeterminable ischemic durations.Publication Metabolic profiling during ex vivo machine perfusion of the human liver(Nature Publishing Group, 2016) Bruinsma, Bote G.; Sridharan, Gautham V.; Weeder, Pepijn D.; Avruch, James H.; Saeidi, Nima; Özer, Sinan; Geerts, Sharon; Porte, Robert J.; Heger, Michal; van Gulik, Thomas M.; Martins, Paulo N.; Markmann, James; Yeh, Heidi; Uygun, KorkutAs donor organ shortages persist, functional machine perfusion is under investigation to improve preservation of the donor liver. The transplantation of donation after circulatory death (DCD) livers is limited by poor outcomes, but its application may be expanded by ex vivo repair and assessment of the organ before transplantation. Here we employed subnormothermic (21 °C) machine perfusion of discarded human livers combined with metabolomics to gain insight into metabolic recovery during machine perfusion. Improvements in energetic cofactors and redox shifts were observed, as well as reversal of ischemia-induced alterations in selected pathways, including lactate metabolism and increased TCA cycle intermediates. We next evaluated whether DCD livers with steatotic and severe ischemic injury could be discriminated from ‘transplantable’ DCD livers. Metabolomic profiling was able to cluster livers with similar metabolic patterns based on the degree of injury. Moreover, perfusion parameters combined with differences in metabolic factors suggest variable mechanisms that result in poor energy recovery in injured livers. We conclude that machine perfusion combined with metabolomics has significant potential as a clinical instrument for the assessment of preserved livers.Publication Surgical models of Roux-en-Y gastric bypass surgery and sleeve gastrectomy in rats and mice(Springer Science and Business Media LLC, 2015-02-26) Bruinsma, Bote G.; Uygun, Korkut; Yarmush, Martin; Saeidi, NimaBariatric surgery is the only definitive solution to the present obesity pandemic. These operations typically involve reconfiguration of gastrointestinal tract anatomy and impose profound metabolic and physiological benefits, such as substantially reducing body weight and ameliorating type II diabetes. Therefore, animal models of these surgeries offer unique and exciting opportunities to delineate the underlying mechanisms that contribute to the resolution of obesity and diabetes. Here we describe a standardized procedure for mouse and rat models of Roux-en-Y gastric bypass (80-90 minutes operative time) and sleeve gastrectomy (30-45 minutes operative time), which, to a high degree resemble operations in human. We also provide detailed protocols for both pre- and post-operative techniques that ensure a high success rate in the operations. These protocols provide the opportunity to mechanistically investigate the systemic effects of the surgical interventions, such as regulation of body weight, glucose homeostasis, and gut microbiome.