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.
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.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Background Donation after circulatory death (DCD) grafts are vital for increasing available donor organs. Gradual rewarming during machine perfusion has proven effective in mitigating reperfusion ...injury and enhancing graft quality. Limited data exist on artificial oxygen carriers as an effective solution to meet the increasing metabolic demand with temperature changes. The aim of the present study was to assess the efficacy and safety of utilizing a hemoglobin-based oxygen carrier (HBOC) during the gradual rewarming of DCD rat livers. Methods Liver grafts were procured after 30 min of warm ischemia. The effect of 90 min of oxygenated rewarming perfusion from ice cold temperatures (4 °C) to 37 °C with HBOC after cold storage was evaluated and the results were compared with cold storage alone. Reperfusion at 37 °C was performed to assess the post-preservation recovery. Results Gradual rewarming with HBOC significantly enhanced recovery, demonstrated by markedly lower lactate levels and reduced vascular resistance compared to cold-stored liver grafts. Increased bile production in the HBOC group was noted, indicating improved liver function and bile synthesis capacity. Histological examination showed reduced cellular damage and better tissue preservation in the HBOC-treated livers compared to those subjected to cold storage alone. Conclusion This study suggests the safety of using HBOC during rewarming perfusion of rat livers as no harmful effect was detected. Furthermore, the viability assessment indicated improvement in graft function.
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.
Abstract Introduction Assessment of thermal burns in the acute phase is critical since healing may require surgical intervention depending on the depth of injury. Early depth estimation guides ...debridement and planning care strategy. Current assessment standard has limitations, and biopsies are invasive and painful. There is an urgent need for a rapid, non-invasive, and objective metric of burn depth. Methods We created a model to study intermediate burn depth in Yucatan miniature pigs, which is clinically relevant. Brass blocks of 4cm radius create contact burns by preheating to 63°C for 2nd-degree (intermediate), and 95°C for 3rd-degree (full thickness) burns. Contact duration varied between 15 to 45 seconds for 2-degree burns to create different depths. Burns are evenly distributed and randomized on the dorsal paravertebral region of the pigs. Full-thickness burns were debrided on POD 3. Each wound was assessed using a custom bench-top Resonance Raman Spectroscopy (RRS) device to measure oxygen saturation, hemoglobin content, and autofluorescence spectrum. Measurements with RRS were performed immediately ( < 2 hours), 2 days, and 3 days after injury. Animals received punch biopsies and dressing changes every week for 9 wks and photographic documentation of healing. Post hoc analysis was performed in GraphPad Prism software to observe correlation between RRS readings on POD 0 to 3 and wound healing duration. Results We successfully created intermediate and full-thickness burns, as confirmed by visual observation and histological analysis. We also created different depths of intermediate burns that vary in healing potential (>95% re-epithelialization). Of 9 wounds on 2 pigs, 4 healed in 5 wks, 3 in 7 wks, and 2 did not heal by 9 wks. RRS measurements had 4 technical replicates for each wound on POD 0, 2, and 3, and were correlated with wound type (2nd vs. 3rd) and duration of healing for 2nd-degree burns. We defined Burn Index (BI) as a marker of burn severity- Burn Index B.I. = Oxygen Saturation / (Normalized Laser Induced Fluorescence * Normalized Hemoglobin Content) BI distinguished between 2nd-degree (n=9) and 3rd-degree (n=4) burns on POD 0 with an area under the curve (AUC) for a receiver operating characteristic (ROC) of 79.17%. It could also distinguish wounds taking more than 9 wks to heal (n=2) from those that healed earlier (n=7) by POD 2, with AUC 97.77%, indicating high sensitivity and specificity. By POD 3, BI distinguished healing in less than 5 wks (n=4) from others (n=5) with AUC 79.38%. Conclusions We established a model of intermediate burns with Yucatan miniature pigs. Resonance Raman Spectroscopy (RRS) based Burn index (BI) could distinguish full and intermediate thickness burns by POD 0 and predict healing time by POD 2 and POD 3 with high specificity and sensitivity. Applicability of Research to Practice Rapid, early, non-invasive quantification of burn depth using our portable device can aid decision-making regarding intermediate-depth wound care, such as skin grafting.
Burn wound healing is a complex and long process. Despite extensive experience, plastic surgeons and specialized teams in burn centers still face significant challenges. Among these challenges, the ...extent of the burned soft tissue can evolve in the early phase, creating a delicate balance between conservative treatments and necrosing tissue removal. Thermal burns are the most common type, and burn depth varies depending on multiple parameters, such as temperature and exposure time. Burn depth also varies in time, and the secondary aggravation of the "shadow zone" remains a poorly understood phenomenon. In response to these challenges, several innovative treatments have been studied, and more are in the early development phase. Nanoparticles in modern wound dressings and artificial skin are examples of these modern therapies still under evaluation. Taken together, both burn diagnosis and burn treatments need substantial advancements, and research teams need a reliable and relevant model to test new tools and therapies. Among animal models, swine are the most relevant because of their strong similarities in skin structure with humans. More specifically, Yucatan minipigs show interesting features such as melanin pigmentation and slow growth, allowing for studying high phototypes and long-term healing. This article aims to describe a reliable and reproducible protocol to study multi-depth burn wounds in Yucatan minipigs, enabling long-term follow-up and providing a relevant model for diagnosis and therapeutic studies.
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