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Organ transplantation is a life-saving procedure affecting over 100,000 people on the transplant waitlist. Ischemia reperfusion injury (IRI) is a major challenge in the field as it can cause post-transplantation complications and limit the use of organs from extended criteria donors. Machine perfusion technology has the potential to mitigate IRI; however, it currently fails to achieve its full potential due to a lack of highly sensitive and specific assays to assess organ quality during perfusion. We developed a real-time and non-invasive method of assessing organs during perfusion based on mitochondrial function and injury using resonance Raman spectroscopy. It uses a 441 nm laser and a high-resolution spectrometer to quantify the oxidation state of mitochondrial cytochromes during perfusion. This index of mitochondrial oxidation, or 3RMR, was used to understand differences in mitochondrial recovery of cold ischemic rodent livers during machine perfusion at normothermic temperatures with an acellular versus cellular perfusate. Measurement of the mitochondrial oxidation revealed that there was no difference in 3RMR of fresh livers as a function of normothermic perfusion when comparing acellular versus cellular-based perfusates. However, following 24 h of static cold storage, 3RMR returned to baseline faster with a cellular-based perfusate, yet 3RMR progressively increased during perfusion, indicating injury may develop over time. Thus, this study emphasizes the need for further refinement of a reoxygenation strategy during normothermic machine perfusion that considers cold ischemia durations, gradual recovery/rewarming, and risk of hemolysis.

Models using 3D cell culture techniques are increasingly accepted as the most biofidelic in vitro representations of tissues for research. These models are generated using biomatrices and bulk populations of cells derived from tissues or cell lines. We present an alternate method to culture individually selected cells in relative isolation from the rest of the population under physiologically relevant matrix conditions. Matrix gel islands are spotted on a cell culture dish to act as support for receiving and culturing individual single cells; a glass capillary-based microfluidic setup is used to extract each desired single cell from a population and seed it on top of an island. Using examples of breast and colorectal cancers, we show that individual cells evolve into tumors or aspects of tumors displaying different characteristics of the initial cancer type and aggressiveness. By implementing a morphometry assay with luminal A breast cancer, we demonstrate the potential of the proposed approach to study phenotypic heterogeneity. Results reveal that intertumor heterogeneity increases with time in culture and that varying degrees of intratumor heterogeneity may originate from individually seeded cells. Moreover, we observe that a positive relationship exists between fast growing tumors and the size and heterogeneity of their nuclei.

We present a two-tiered microchip system to capture and retrieve rare cells from blood samples with high purity. The first module of the system is a high throughput microfluidic interface that is used to immunomagnetically isolate targeted rare cells from whole blood, and discard > 99.999% of the unwanted leukocytes. The second module is a microwell array that furthers the purification by magnetically guiding each cell into a separate well concurrently, and allows individual retrieval of each cell. We demonstrate the design of the system as well as its characterization by experiments using model cell lines that represent circulating fetal trophoblasts. Our results show that single cells can be retrieved with efficiencies and purities as high as 100% within 145 mins.

Background Accurate diagnosis of burn depth in the early post-burn phase is critical for guiding treatment decisions and optimal healing outcomes in patients. Current clinical assessment methods are often subjective, delayed, and prone to low diagnostic accuracy. A rapid and objective method of assessment to distinguish burn depths could significantly improve the standard of care. Methods We developed a non-invasive Resonance Raman Spectroscopic (RRS) protocol to assess burn depth using a compact and portable device. It provides Hemoglobin Index (HI), a metric of hemoglobin concentration in the wound bed. Since blood supply to the wound can depend on the severity of vascular damage, HI can be used to categorize burns by depth. We tested this approach in a clinically relevant Yucatan mini-pig model of multi-depth burns, where we created a total of 24 wounds of different depths over three animals to perform this analysis. We also performed visual and histological analysis of the wound damage in the acute phase. Additionally, we performed a histological analysis of wound healing up to post-burn day 64 and observed changes in Raman-associated Fluorescence Index between different wound categories. Results We successfully created superficial, superficial partial-thickness, deep partial-thickness, and full-thickness burns in Yucatan mini-pigs, as confirmed with the visual and histological analysis. With HI-based analysis, we found a high accuracy of diagnosis on post-burn day 3 in a binary classifier model for superficial partial-thickness and deep partial-thickness burns (AUC: 0.85, 95% CI: 0.56-1, n  = 4–5), with nearly perfect classification in the broader categories (AUC: 1, 95% CI: 1–1, n  = 4–9). Simultaneously, we observed histological features of healing that were consistent with the burn depth categories, along with trends in Resonance Raman-associated Fluorescence Index that might suggest a role in longitudinal wound monitoring with further development. Conclusions Non-invasive measurements with our device showed a high accuracy of burn depth classification using the Hemoglobin Index by post-burn 3. These results indicate a high potential for clinical translation of our approach for burn depth diagnosis in patients.

Skin pigmentation can pose challenges for physicians to diagnose pathologies. In Vascularized Composite Allotransplantation (VCA), this increases the difficulty of diagnosing rejection by clinical observation, which could be improved by noninvasive monitoring, thereby completely avoiding or aiding in guiding location for invasive diagnostics. In this study, pigmented and non-pigmented allogeneic and non-pigmented syngeneic control transplant recipients underwent daily thermal assessment using infrared (IR) gun and forward-looking IR (FLIR) imaging of VCAs using a rodent partial hindlimb transplant model. Daily clinical assessment was performed, and biopsies were taken on postoperative day (POD) 1, 3, and 7. Clinical and histological assessments indicated signs of rejection on POD 3. In contrast, thermal assessment using the IR gun detected significant differences as early as POD 1, notably a decrease in temperature, when comp ared to syngeneic control transplants. This demonstrates the capability of thermal assessments to identify early signs of rejection before clinical symptoms become apparent. The findings suggest that thermal assessments can serve as a non-contact, objective adjunct tool for early detection of graft rejection, with consideration of skin pigmentation. This approach may reduce the need for invasive biopsies, thereby improving patient comfort and reducing potential complications associated with current diagnostic methods.

Ischemia reperfusion injury (IRI) is a critical problem in liver transplantation that can lead to life-threatening complications and substantially limit the utilization of livers for transplantation. However, because there are no early diagnostics available, fulminant injury may only become evident post-transplant. Mitochondria play a central role in IRI and are an ideal diagnostic target. During ischemia, changes in the mitochondrial redox state form the first link in the chain of events that lead to IRI. In this study we used resonance Raman spectroscopy to provide a rapid, non-invasive, and label-free diagnostic for quantification of the hepatic mitochondrial redox status. We show this diagnostic can be used to significantly distinguish transplantable versus non-transplantable ischemically injured rat livers during oxygenated machine perfusion and demonstrate spatial differences in the response of mitochondrial redox to ischemia reperfusion. This novel diagnostic may be used in the future to predict the viability of human livers for transplantation and as a tool to better understand the mechanisms of hepatic IRI.

Models using 3D cell culture techniques are increasingly accepted as the most biofidelic in vitrorepresentations of tissues for research. These models are generated using biomatrices and bulk populations of cells derived from tissues or cell lines. This thesis study focuses on an alternate method to culture individually selected cells in relative isolation from the rest of the population under physiologically relevant matrix conditions. Matrix gel islands are spotted on a cell culture dish to act as support for receiving and culturing individual single cells; a glass capillary-based microfluidic setup is used to extract each desired single cell from a population and seed it on top of an island. Using examples of breast and colorectal cancers, we show that individual cells evolve into tumors or aspects of tumors displaying different characteristics of the initial cancer type and aggressiveness. By implementing a morphometry assay with luminal A breast cancer, we demonstrate the potential of the proposed approach to studying phenotypic heterogeneity. Results reveal that intertumor heterogeneity increases with time in culture and that varying degrees of intratumor heterogeneity may originate from individually seeded cells. Moreover, we observe a positive correlation between fast-growing tumors and the size and heterogeneity of their nuclei.

Background: Polytrauma patients present unique surgical and medical challenges, with increased risk of complications due to physiological instability and associated injuries. Aim: To determine the incidence, type, and risk factors for complications in polytrauma patients undergoing orthopedic surgery. Methods: This prospective observational study was conducted at the Department of Orthopaedics, Pt. B.D. Sharma PGIMS, Rohtak, from May 2024 to April 2025. A total of 120 polytrauma patients (Injury Severity Score >16) who underwent orthopedic surgical intervention were included. Data on demographics, injury characteristics, operative details, and postoperative complications were collected. Complications were categorized as early (<2 weeks) and late (>2 weeks). Statistical analysis was done using SPSS v26. Results: The mean age was 36.4 ± 12.7 years, with a male-to-female ratio of 3.2:1. Road traffic accidents accounted for 78.3% of injuries. Early complications included surgical site infection (15.8%), deep vein thrombosis (6.7%), acute respiratory distress syndrome (5.8%), implant failure (4.2%), and fat embolism syndrome (2.5%). Late complications included non-union (8.3%), malunion (6.7%), joint stiffness (10.8%), and chronic osteomyelitis (3.3%). Overall complication rate was 42.5%, with mortality of 5.0%. Higher Injury Severity Score (>25), surgery within 24 h in unstable patients, and ICU stay >7 days were significantly associated with increased complications (p < 0.05). Conclusion: Polytrauma patients undergoing orthopedic surgery have a high complication rate. Careful patient optimization, adherence to damage control orthopaedics principles, and vigilant perioperative monitoring may reduce adverse outcomes.

Mitochondrial dysfunction is a critical factor in several diseases, but current in situ assessment methods are severely limited. Non-invasive monitoring of mitochondrial redox state using resonance Raman Spectroscopy (RRS) offers a promising solution. This study aims to demonstrate RRS utility with liver models of warm ischemia-reperfusion injury in organ transplantation. Lewis rat (female) and Yorkshire pig (both sexes) livers were evaluated during reperfusion by subnormothermic machine perfusion, with 3-6 replicates per study group, and statistical comparisons using unpaired two-tailed Student's t-tests with Welch's correction for potentially unequal variance. RRS provides in situ quantification of the overall mitochondrial redox state, and herein further refined to resolve the redox state of individual complex III and IV. Here we show that RRS can differentiate non-viable rat livers (3 h warm ischemia, WI) from viable 1 h WI and fresh controls as early as 30 mins into reperfusion. RRS also identifies dysfunction at complex III characterized by hyperoxidation during reperfusion. This guides us to test methylene blue, which acts as an alternate electron donor to bypass complex III, as treatment rescuing mitochondria from WI-induced reperfusion injury. When tested on pig marginal livers with extended WI (30-45 mins), our RRS-guided treatment enables recovery of hemodynamics and oxygen/lactate values that approached controls without WI. RRS assessment and guided treatment with methylene blue provide two lines of evidence indicating that mitochondrial hyperoxidation, specifically at complex III, is a critical mechanism underlying warm ischemia-reperfusion injury. This study demonstrates the potential of RRS for transplantation and broader applications.

Ischemia reperfusion injury (IRI) is a critical problem in liver transplantation that can lead to life-threatening complications and substantially limit the utilization of livers for transplantation. However, because there are no early diagnostics available, fulminant injury may only become evident post-transplant. Mitochondria play a central role in IRI and are an ideal diagnostic target. During ischemia, changes in the mitochondrial redox state form the first link in the chain of events that lead to IRI. In this study we used resonance Raman spectroscopy to provide a rapid, non-invasive, and label-free diagnostic for quantification of the hepatic mitochondrial redox status. We show this diagnostic can be used to significantly distinguish transplantable versus non-transplantable ischemically injured rat livers during oxygenated machine perfusion and demonstrate spatial differences in the response of mitochondrial redox to ischemia reperfusion. This novel diagnostic may be used in the future to predict the viability of human livers for transplantation and as a tool to better understand the mechanisms of hepatic IRI.