Preserving Tissue Viability Research Articles

A comprehensive preanalytical protocol for fresh solid tumor biospecimens

Nearly seventy percent of diagnostic lab test errors occur due to variability in preanalytical factors. These are the parameters involved with all aspects of tissue processing, starting from the time tissue is collected from the patient in the operating room, until it is received and tested in the laboratory. While there are several protocols for transporting fixed tissue, organs, and liquid biopsies, such protocols are lacking for transport and handling of live solid tumor tissue specimens. There is a critical need to establish preanalytical protocols to reduce variability in biospecimen integrity and improve diagnostics for personalized medicine. Here, we provide a comprehensive protocol for the standard collection, handling, packaging, cold-chain logistics, and receipt of solid tumor tissue biospecimens to preserve tissue viability.

Poloxamer 188 washing of lipoaspirate improves fat graft survival: A comparative study in nude mice

BackgroundAutologous fat transplantation is limited by the uncertainty of grafting retention, impeding its application. Among current strategies of processing lipoaspirates, high-density fat (HDF) due to its enrichment of stem cells and washing before cotton concentration for the simplicity of operation are recommended. Poloxamer 188 (P188) washing has been shown to repair the membranes of damaged cells. This study aimed to investigate the effect of P188-washing on fat graft survival and to identify the best processing technique. MethodsLipoaspirates were prepared by centrifuging to obtain HDF, and by washing with saline or P188 followed by cotton concentration. Tissue integrity, adipocytic activity and viability of SVF in samples from three groups were assessed. Samples were sequenced in vitro by high-throughput RNA-Seq, and differentially expressed genes (DEGs) were validated through qPCR and WB. After transplantation under the dorsum of nude mice for 8 weeks, the grafts were extracted and examined in residual volume, histologic characteristics and vascularization. ResultsHDF and P188 groups showed higher survival rate of SVF, more Ki67-positive cells, intact tissue structure, and less fibrosis. There were no significant differences in the density of SVF and residual volume of grafts. HDF showed significantly improved vascularization during 8 weeks. Through RNA-Seq and bioinformatic analysis, there were noteworthy changes of several related genes after transplantation. ConclusionsP188 treatment can prevent cell from apoptosis and preserve tissue viability, thereby improving graft quality. High-density fat contains large amount of SVF and can be regarded as an excellent grafting material.

Molecular Mechanisms of Ischemic Stroke: A Review Integrating Clinical Imaging and Therapeutic Perspectives.

Ischemic stroke poses a significant global health challenge, necessitating ongoing exploration of its pathophysiology and treatment strategies. This comprehensive review integrates various aspects of ischemic stroke research, emphasizing crucial mechanisms, therapeutic approaches, and the role of clinical imaging in disease management. It discusses the multifaceted role of Netrin-1, highlighting its potential in promoting neurovascular repair and mitigating post-stroke neurological decline. It also examines the impact of blood-brain barrier permeability on stroke outcomes and explores alternative therapeutic targets such as statins and sphingosine-1-phosphate signaling. Neurocardiology investigations underscore the contribution of cardiac factors to post-stroke mortality, emphasizing the importance of understanding the brain-heart axis for targeted interventions. Additionally, the review advocates for early reperfusion and neuroprotective agents to counter-time-dependent excitotoxicity and inflammation, aiming to preserve tissue viability. Advanced imaging techniques, including DWI, PI, and MR angiography, are discussed for their role in evaluating ischemic penumbra evolution and guiding therapeutic decisions. By integrating molecular insights with imaging modalities, this interdisciplinary approach enhances our understanding of ischemic stroke and offers promising avenues for future research and clinical interventions to improve patient outcomes.

An Evaluation of the Efficacy and Safety of TAMIXAM®, Based on Hyaluronic Acid and Tamarind Seed Extract, for Esophageal Mucosal Protection from Acid Insult

TAMIXAM® is a novel technology that combines hyaluronic acid and tamarind seed extract in its formulation. It is designed to protect the esophageal mucosa by creating a barrier through its filmogenic properties. The aim of this study is to evaluate the safety and efficacy of this technology through mucoadhesion tests, a cell viability assay, TEER measurements, and morphological analysis on reconstructed esophageal mucosa exposed to 10% hydrochloric acid before and after treatment. The mucoadhesion test highlighted the synergistic bioadhesive effect of the technology’s components. Cell viability assays revealed the substantial mucoprotective and barrier effects of the technology, preserving tissue viability when applied before exposure to acid insult. A morphological analysis illustrated TAMIXAM®’s efficacy in countering acid-induced damage, reducing erosion, necrosis, and tissue degeneration compared to the positive control, both pre- and post-acid insult. An evaluation of epithelial integrity through TEER measurements indicated a minimal reduction in tissues treated with the invention before acid exposure, demonstrating its ability to maintain epithelial integrity in the presence of an acid insult. However, this effect was less pronounced in tissues treated with the technology after the acid insult, implying a potential partial recovery of epithelial integrity. Furthermore, comprehensive in vitro and in vivo studies supported the safety profile of the invention. In conclusion, TAMIXAM® emerged as a compelling solution, providing enhanced mechanical action to maintain epithelial balance and shield the esophageal mucosa from acid-induced damage.

Do Basic Neurologic Assessments Tell Us More Than We Realize?

Neurologic assessments using a monofilament and a tuning fork are routinely performed to screen for peripheral neuropathy and to identify foot ulceration and amputation risks. We investigated whether assessments commonly used to monitor sensation in the feet may illuminate a more holistic perspective of a person's overall health status. Recruitment of 50 participants for foot health screening was facilitated via a promotional event for Foot Health Week. Participants were aged 52 to 92 years (31 women and 19 men). Monofilament and tuning fork assessments were used to determine each participant's neurologic status. Participants also completed a modified Foot Health Status Questionnaire. Data were analyzed to identify correlations between neurologic assessment results and questionnaire responses. For participants self-reporting an "excellent" health rating, a significant relationship was identified with adequate vibration sensation (P < .01). Significant correlations were also identified between a greater number of sites detected using a 10-g monofilament assessment and a person's experience of having a lot of energy (P = .03), limited interference with social activities (P = .03), and greater confidence completing a variety of functional tasks. Significant correlations were observed between basic neurologic assessments and a participant's perception of their overall health. Although these findings reflect a correlational rather than a causational relationship, they may provide a stimulus for clinicians to reflect on the holistic value of peripheral neurologic assessment. Although the immediate focus for a practitioner is minimizing risk and preserving tissue viability, neurologic test results may be useful to stimulate further discussion about a patient's health outcomes by exploring issues beyond the presenting condition.

Enhancing oxidative phosphorylation over glycolysis for energy production in cultured mesenchymal stem cells.

ObjectiveStrokes represent as one of the leading causes of death and disability in the USA, however, there is no optimal treatment to reduce the occurrence or improve prognosis. Preconditioning of tissues triggers ischemic tolerance, a physiological state that may involve a metabolic switch (i.e. from glycolysis to oxidative phosphorylation or OxPhos) to preserve tissue viability under an ischemic insult. Here, we hypothesized that metabolic switching of energy source from glucose to galactose in cultured mesenchymal stem cells (MSCs) stands as an effective OxPhos-enhancing strategy.MethodsMSCs were grown under ambient condition (normal MSCs) or metabolic switching paradigm (switched MSCs) and then assayed for oxygen consumption rates (OCR) and extracellular acidification rate (ECAR) using the Seahorse technology to assess mitochondrial respiration.ResultsNormal MSCs showed a lower OCR/ECAR ratio than switched MSCs at baseline (P < 0.0001), signifying that there were greater levels of OxPhos compared to glycolysis in switched MSCs. By modulating the mitochondrial metabolism with oligomycin (time points 4–6), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (7–9), and rotenone and antimycin (time points 10–12), switched MSCs greater reliance on OxPhos was further elucidated (time points 5–12; P < 0.0001; time point 4; P < 0.001).ConclusionThe metabolic switch from glycolytic to oxidative metabolism amplifies the OxPhos potential of MSCs, which may allow these cells to afford more robust therapeutic effects against neurological disorders that benefit from ischemic tolerance.

WE7.4 A Systematic Review of Splenic Viability Following Arterial Embolisation in Trauma

Abstract The spleen is the most commonly injured organ in blunt abdominal trauma. Patients with these injuries are increasingly being managed nonoperatively, with use of Splenic Artery Embolisation (SAE). Many of these patients receive lifelong prophylactic antibiotics as though they were asplenic. The aim is to seek robust evidence of splenic vascular integrity and immunocompetence following SAE, and consider whether lifelong antibiotic prophylaxis is necessary for trauma patients managed in this way. 2 separate literature reviews were carried out using the Medline database. One to assess literature pertaining to splenic tissue viability following embolisation, the other to find literature reviewing splenic immune function after embolisation. In the vascular viability arm, 21 articles were reviewed. 12 studies observed no splenic infarcts at all. 18 of the studies noted &amp;lt;10% splenic infarcts in their populations. Distal embolisation resulted in more infarcted areas of splenic tissue than proximal. 9 studies performed follow-up imaging and confirmed splenic tissue viability post-procedure. For the review of immunocompetence, the literature search resulted in 13 full text articles. There was no consistency of the immunologic marker used to test splenic immunocompetence post-SAE, but 11 of 13 studies confirmed preserved immunocompetence by their respective markers. The data do suggest preserved tissue viability and immunocompetence. Agreement is yet to be reached as to which immune marker definitively assesses immunocompetence, and the length of follow-up needed to investigate it. More research is required to confirm the suggestion from these data that lifelong antibiotic prophylaxis is unnecessary for trauma patients treated with SAE.

P-445 Recovery of metabolic activity and VEGF secretion after thawing of cryopreserved, slow frozen ovarian tissue

Abstract Study question Is slow-frozen ovarian cortical tissue limited in its metabolical activity after thawing, especially with respect to VEGF secretion? Summary answer Slow-frozen ovarian cortical tissue showed decreased metabolic activity and VEGF directly after thawing, but recovered in the course of 48h in in vitro culture. What is known already Cryopreservation of ovarian cortical tissue has become an important method in fertility preservation. Freezing and thawing processes had been optimized to preserve tissue viability. However, the major obstacle of retransplantation is the rapid angiogenesis to accomplish sufficient blood supply. As freezing and thawing entails constraints in cellular metabolic activity, we aimed to assess the metabolic activity and the secretion of the angiogenic factor VEGF of frozen-thawed ovarian cortical tissue over time. Study design, size, duration Ex vivo gained ovarian tissue was cultured over 48h. Thereby, non-frozen fresh tissue was compared to slow-frozen-thawed ovarian tissue. Participants/materials, setting, methods Provided a signed consent, biopsies of ovarian tissue from patients undergoing fertility preservation were cultured without freezing (n = 13) or after slow-freezing and thawing (n = 12). VEGF secretion during 48h was measured by ELISA. Applying CellTiterBlue assay, metabolic activity was assessed in biopsies without freezing or after thawing from the same patients (n = 4-7) after 0, 24 and 48h. Similarly, VEGF secretion was measured after 0, 24 and 48h as well (n = 7) to capture temporarily changes. Main results and the role of chance Although VEGF secretion varies among patients and biopsies, there was a significant lower VEGF content in supernatant from frozen-thawed tissue compared to fresh ovarian cortical tissue after 48h of culture (p &amp;lt; 0.01). Directly after thawing and 24h thereafter, metabolic activity was significantly reduced (p &amp;lt; 0.05) compared to fresh ovarian cortex. In the course of 48h, the metabolic activity recovered (p &amp;lt; 0.01). Similarly, VEGF secretion increased dramatically during 24h after thawing (p &amp;lt; 0.05). Limitations, reasons for caution This in vitro study might not resemble the metabolic and secretory recovery after retransplantation. Wider implications of the findings We showed that ovarian tissue recovers metabolically over 48h. This recovery included an increase in VEGF secretion. As latter induces angiogenesis a culture step after thawing might support VEGF secretion and angiogenesis. However, the recovery period could also be useful as a temporal bridge until sufficient supplies are available. Trial registration number not applicable

Autoamputation of the appendix and survival of the amputated part: a rare case report and literature review

BackgroundAutoamputation of the appendix, i.e., complete separation of a part of the appendix without any surgical intervention, has been rarely documented in the literature in recent years. Herein, we report a case where the amputated part of the appendix was viable after autoamputation and reviewed the related literature.Case presentationA 39-year-old female patient was admitted to our hospital complaining of abdominal pain and subsequently underwent an emergency laparoscopic appendectomy (LA). Intraoperatively, we found an abnormally short appendix protruding from the cecum and a strip-like tissue attached to the mesoappendix, considered a duplex appendix, was resected. Finally, in conjunction with the histopathology findings and the past medical history, the patient was diagnosed with “Pseudo-duplication of the Appendix”.ConclusionsAutoamputation of the appendix resulting in preserved tissue viability and absence of necrosis at both ends, can be termed as “Pseudo-duplication of the Appendix”. This condition is very rare in clinical practice and has not been reported in China, to the best of our knowledge. It has been established that the autoamputated appendix can produce chronic inflammation, intestinal fistulae and even cancer, affecting the patient’s quality of life. Accordingly, a clear diagnosis and timely management are essential. In this report, we established a novel classification for “Pseudo-duplication of the Appendix”, hoping that our report will help surgeons better understand this anatomical anomaly of the appendix, to help during the differential diagnosis process and avoid confusion.

Abstract P6-06-03: Mechanobiology of cancer cells and tumour microenvironment jointly regulates cancer cell invasion and onset of metastasis

Abstract Mechanical properties of cancer cells and their microenvironment play a critical role in cancer progression and metastatic dissemination. We have established an in vitro assay using native human basement membrane (BM) to study interactions with normal epithelial cells in terms of BM composition, architecture and stiffness. We showed that these are required to act jointly in order to achieve apico-basal polarity, tissue barrier formation and specific stiffness to maintain healthy epithelial tissues. In contrast, in cancer the onset of metastasis occurs when cancer cells invade and breach the BM. For example, it is well known that cancer cells can perforate BM using proteolysis. However, recently we have demonstrated that in presence of carcinoma-associated fibroblasts (CAFs) isolated from cancer patients, moderately invasive cancer cells were able to invade into surrounding stromal matrix in a metalloproteinase-independent manner. In fact cell invasion was associated with decrease in stiffness and correlated to changes in cell and BM morphology. Using live imaging and atomic force microscopy (AFM), we could visualise CAFs actively pulling, stretching and softening the BM, forming gaps through which cancer cells can migrate. By exerting contractile forces, CAFs altered the organisation and physical properties of native BM, making it permissive for cancer cell invasion. Also recently we showed that from the epithelial side, structural centrosome aberrations known to accompany breast cancer progression can create local stiffness heterogeneity that triggers the escape of soft cells undergoing mitosis. In particular, we demonstrated that structural centrosome aberrations can trigger cell dissemination through a non-cell-autonomous mechanism, raising the prospect that centrosome aberrations might facilitate dissemination of soft metastatic cells. In summary, physical interactions between cancer cells and the extracellular matrix (ECM) components contribute to softening of specific cancer cells, physical activation of stromal cells such as CAFs to facilitate cancer invasion and metastases. By extending these findings to a routine clinical setting we measured nanomechanical profiles of biopises from breast cancer patients in a physiological tissue protective solution thus preserving tissue viability and allowing for further pathohistological analysis of the same specimens. Conclusively, we demonstrated how detecting highly deformable cancer cells in patient tissues can serve as a unique marker of cancer aggressiveness thus potentially enabling treatment optimization and development of novel treatment strategies. Citation Format: Marija Plodinec, Philipp Oertle, Alexandre Glentis, Danijela Vignjevic, Olivier Ganier, Eric Nigg, Tobias Appenzeller, Ahmed Jizawi, Flora Scott, Cristian Raez, Marko Loparic, Zlatko Marusic, Ellen C. Obermann, Rosemarie Burian, Roderick Y.H. Lim. Mechanobiology of cancer cells and tumour microenvironment jointly regulates cancer cell invasion and onset of metastasis [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-06-03.

Surgical Swine Model of Chronic Cardiac Ischemia Treated by Off-Pump Coronary Artery Bypass Graft Surgery

Chronic cardiac ischemia that impairs cardiac function, but does not result in infarct, is termed hibernating myocardium (HM). A large clinical subset of coronary artery disease (CAD) patients have HM, which in addition to causing impaired function, puts them at higher risk for arrhythmia and future cardiac events. The standard treatment for this condition is revascularization, but this has been shown to be an imperfect therapy. The majority of pre-clinical cardiac research focuses on infarct models of cardiac ischemia, leaving this subset of chronic ischemia patients largely underserved. To address this gap in research, we have developed a well-characterized and highly reproducible model of hibernating myocardium in swine, as swine are ideal translational models for human heart disease. In addition to creating this unique disease model, we have optimized a clinically relevant treatment model of coronary artery bypass surgery in swine. This allows us to accurately study the effects of bypass surgery on heart disease, as well as investigate additional or alternate therapies. This model surgically induces single vessel stenosis by implanting a constrictor on the left anterior descending (LAD) artery in a young pig. As the pig grows, the constrictor creates a gradual stenosis, resulting in chronic ischemia with impaired regional function, but preserving tissue viability. Following the establishment of the hibernating myocardium phenotype, we perform off-pump coronary artery bypass graft surgery to revascularize the ischemic region, mimicking the gold-standard treatment for patients in the clinic.

Surgical Swine Model of Chronic Cardiac Ischemia Treated by Off-Pump Coronary Artery Bypass Graft Surgery.

Chronic cardiac ischemia that impairs cardiac function, but does not result in infarct, is termed hibernating myocardium (HM). A large clinical subset of coronary artery disease (CAD) patients have HM, which in addition to causing impaired function, puts them at higher risk for arrhythmia and future cardiac events. The standard treatment for this condition is revascularization, but this has been shown to be an imperfect therapy. The majority of pre-clinical cardiac research focuses on infarct models of cardiac ischemia, leaving this subset of chronic ischemia patients largely underserved. To address this gap in research, we have developed a well-characterized and highly reproducible model of hibernating myocardium in swine, as swine are ideal translational models for human heart disease. In addition to creating this unique disease model, we have optimized a clinically relevant treatment model of coronary artery bypass surgery in swine. This allows us to accurately study the effects of bypass surgery on heart disease, as well as investigate additional or alternate therapies. This model surgically induces single vessel stenosis by implanting a constrictor on the left anterior descending (LAD) artery in a young pig. As the pig grows, the constrictor creates a gradual stenosis, resulting in chronic ischemia with impaired regional function, but preserving tissue viability. Following the establishment of the hibernating myocardium phenotype, we perform off-pump coronary artery bypass graft surgery to revascularize the ischemic region, mimicking the gold-standard treatment for patients in the clinic.

Monitoring Dopamine ex Vivo during Electrical Stimulation Using Liquid-Microjunction Surface Sampling.

Liquid-microjunction surface sampling (LMJ-SS) is an ambient ionization technique based on the continuous flow of solvent using an in situ microextraction device in which solvent moves through the probe, drawing in the analytes in preparation for ionization using an electrospray ionization source. However, unlike traditional mass spectrometry (MS) techniques, it operates under ambient pressure and requires no sample preparation, thereby making it ideal for rapid sampling of thicker tissue sections for electrophysiological and other neuroscientific research studies. Studies interrogating neural synapses, or a specific neural circuit, typically employ thick, ex vivo tissue sections maintained under near-physiological conditions to preserve tissue viability and maintain the neural networks. Deep brain stimulation (DBS) is a surgical procedure used to treat the neurological symptoms that are associated with certain neurodegenerative and neuropsychiatric diseases. Parkinson's disease (PD) is a neurological disorder which is commonly treated with DBS therapy. PD is characterized by the degeneration of dopaminergic neurons in the substantia nigra pars compacta portion of the brain. Here, we demonstrate that the LMJ-SS methodology can provide a platform for ex vivo analysis of the brain during electrical stimulation, such as DBS. We employ LMJ-SS in the ex vivo analysis of mouse brain tissue for monitoring dopamine during electrical stimulation of the striatum region. The mouse brain tissue was sectioned fresh post sacrifice and maintained in artificial cerebrospinal fluid to create near-physiological conditions before direct sampling using LMJ-SS. A selection of metabolites, including time-sensitive metabolites involved in energy regulation in the brain, were identified using standards, and the mass spectral database mzCloud was used to assess the feasibility of the methodology. Thereafter, the intensity of m/z 154 corresponding to protonated dopamine was monitored before and after electrical stimulation of the striatum region, showing an increase in signal directly following a stimulation event. Dopamine is the key neurotransmitter implicated in PD, and although electrochemical detectors have shown such increases in dopamine post-DBS, this is the first study to do so using MS methodologies.

Abstract 457: Rad-deletion Provides Dual Therapeutic Benefits for the Heart

Rationale: Myocardial infarction (MI) compromises the overall mechanical function of the heart, reducing cardiac output and triggering decompensatory remodeling. Rad GTPase is an L-type calcium channel complex (LTCC) constituent that governs trigger calcium in the myocardium. Rad deletion in mice results in increased Ca 2+ handling and a sustained non-pathological improvement in left ventricular function compared to wildtype. Hypothesis: Rad-ablation attenuates post-ischemic loss of function, resulting in reduced remodeling and improved long-term contractility. Methods and Results: We subjected Rad-deficient (Rad -/- ) mice to ligation of the left anterior descending (LAD) coronary artery, and monitored cardiac structure and function using echocardiography and single cell Ca 2+ measurements. We found that Rad deletion reduces both mortality and contractile dysfunction after MI, as well as ventricular dilation over five weeks. This improvement is also accompanied by preserved Ca 2+ handling in isolated myocytes. Histological and MRI examination of ex vivo global ischemia and in vivo 24 hour post-MI myocardium revealed that initial infarct size and area at risk are comparable between knockout and wildtype. Rad loss reduced scar spread independent of preserving tissue viability. mRNA microarray findings implicated differential inflammatory pathway activation with Rad-deletion. Rad -/- showed reduced neutrophil extravasation and reduced inflammatory cytokines in the left ventricle 24 hours after MI. Conclusion: Rad deletion results in reduced cardiac remodeling, diminished myocardial inflammation, and improved contractile function before and after MI. These results suggest that Rad-deletion is a novel therapeutic direction that may serve as a combined positive inotrope and regulator of acute MI inflammatory response.

The macrophage stimulating anti-cancer agent, RRx-001, protects against ischemia-reperfusion injury

ABSTRACTBackground: RRx-001, a clinical macrophage-stimulating anti-cancer agent that also produces nitric oxide (NO) was studied in a model of ischemia-reperfusion injury.Methods: The production of NO is dependent on the oxygen tension because nitric oxide synthases convert l-arginine to NO and l-citrulline in the presence of O2. Since the P450 enzymes, which metabolize nitrate esters such as nitroglycerin are dependent on oxygen, the generation of ‘exogenous’ NO is also sensitive to alterations in tissue PO2. I/R injury was studied in a hamster chamber window, with compression of the periphery of the window for 1 h to induce ischemia. Animals received RRx-001 (5 mg/kg) 24 h before ischemia and sodium nitrite (10 nmols/kg) was supplemented 10 min after the start of reperfusion. Vessel diameter, blood flow, adherent leukocytes, and functional capillary density were assessed by intravital microscopy at 0.5, 2, and 24 h following the release of the ischemia.Results: The results demonstrated that, compared to control, RRx-001 preconditioning increased blood flow and functional capillary density, and preserved tissue viability in the absence of side effects over a sustained time period.Conclusion: Thus, RRx-001 may serve as a long-lived protective agent during postsurgical restoration of flow and other ischemia-reperfusion associated conditions, increasing blood flow and functional capillary density as well as preserving tissue viability in the absence of side effects.

Abstract 448: The Loss of Rad GTPase Protects Against Infarction-induced Myocardial Remodeling, Scar Formation, and Decompensatory Dilation

Rationale: Myocardial infarction (MI) is a leading cause of death in the U.S. A non-contractile infarct compromises the overall mechanical function of the heart, reducing cardiac output and triggering decompensatory ventricular dilation. Rad GTPase, a member of the small GTPase RGK (Rem, Gem, Kir) family, is a calcium channel blocker that is endogenously expressed in the myocardium. We have previously shown that Rad deletion in mice results in increased Ca 2+ handling and a sustained non-pathological improvement in left ventricular function compared to wildtype. Hypothesis: Rad-ablation attenuates post-ischemic loss of function, resulting in reduced remodeling and improved long-term contractility. Methods and Results: We subjected Rad-deficient mice to ligation of the left anterior descending (LAD) coronary artery, and monitored cardiac function using echocardiography. We found that Rad deletion reduces both mortality and contractile dysfunction after MI, as well as ventricular dilation over five weeks. This improvement is also accompanied by preserved calcium handling in isolated myocytes. Histological and MRI examination of both ex vivo global ischemia and in vivo 24 hour LAD ligated myocardium revealed that initial infarct size is comparable between knockout and wildtype. We found that Rad loss reduced scar development and elongation independent of preserving tissue viability. Investigation of inflammatory pathways to account for this revealed increased expression of the anti-inflammatory protein thrombospondin accompanied by a reduction in neutrophil infiltration into the myocardium after MI. Conclusion: Rad deletion results in reduced cardiac remodeling, diminished myocardial inflammation, and improved contractile function after MI.

Abstract P4-04-19: Primary breast cancer culture in a perfusion-based bioreactor suitable for in vitro testing of immune blockade therapy

Abstract Introduction: Interaction between cancer cells and immune system critically affects development, progression and treatment of human malignancies. Two-dimensional (2D) in vitro culture systems and in vivo animal models are the primary tools used to test cancer cell response to drugs but they are not suited for the development of immune-mediated therapies. Here we present an innovative method to culture breast cancer tissue in porous 3D scaffolds by using a perfusion-based bioreactor system that allows the maintenance and expansion of tumor microenvironment. Experimental procedures: Freshly excised breast cancer specimens were fragmented and cultured in a 3D "sandwich-like format" between two layers of porous collagen scaffold under perfusion flow (U-CUP). DMEM/F12, supplemented with 10% autologous human serum, was used as a culture medium. We assessed the ability of tumor and non-malignant cells to survive and expand into the scaffold in perfusion culture, as well as their capacity to recapitulate features of the original breast cancer tissue. The maintenance of immune-infiltrating cells allowed testing of immune blockade therapy in vitro using anti-PD-L1 and anti-CTLA4 antibodies alone or in combination. Results: The U-CUP culture system preserved tissue viability better compared to a static culture and promoted the expansion of breast cancer cells from surgical specimens together with accompanying stromal and immune cells into the porous scaffold. Tumor tissues were viable after 21 days and mostly recapitulating the initial histology with formation of glands. Administration of anti-PDL1 antibody, alone or in combination with anti-CTLA4, to the culture medium was associated with increased expression of markers of immune-activation (i.e. IFNg) and decreased expression of immunosuppressive cytokine IL10. Conclusions: Our results show that culture of breast cancer tissue in a 3D perfusion-based bioreactor might represent a promising system for the pre-clinical evaluation of immune-mediated therapies. Preserving malignant, interstitial and immunocompetent cells comprised in surgically excised breast cancer samples might allow a direct evaluation of the effects of various treatments on the complex tumor microenvironment. This engineered in vitro model could be extended as a platform allowing the testing of innovative approaches for the treatment of human malignancies, possibly in the direction of personalized medicine. Citation Format: Muraro MG, Muenst S, Mele V, Spagnoli GC, Oertli D, Weber WP, Soysal SD. Primary breast cancer culture in a perfusion-based bioreactor suitable for in vitro testing of immune blockade therapy. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-04-19.

In Vivo Therapeutic Potential of Mesenchymal Stromal Cells Depends on the Source and the Isolation Procedure

SummaryOver the last several years, mesenchymal stromal cells (MSCs) have been isolated from different tissues following a variety of different procedures. Here, we comparatively assess the ex vivo and in vivo properties of MSCs isolated from either adipose tissue or bone marrow by different purification protocols. After MSC transplantation into a mouse model of hindlimb ischemia, clinical and histological analysis revealed that bone marrow MSCs purified on adhesive substrates exerted the best therapeutic activity, preserving tissue viability and promoting formation of new arterioles without directly transdifferentiating into vascular cells. In keeping with these observations, these cells abundantly expressed cytokines involved in vessel maturation and cell retention. These findings indicate that the choice of MSC source and purification protocol is critical in determining the therapeutic potential of these cells and warrant the standardization of an optimal MSC isolation procedure in order to select the best conditions to move forward to more effective clinical experimentation.

Impact of hemoglobin nitrite to nitric oxide reductase on blood transfusion for resuscitation from hemorrhagic shock.

Background:Transfusion of blood remains the gold standard for fluid resuscitation from hemorrhagic shock. Hemoglobin (Hb) within the red blood cell transports oxygen and modulates nitric oxide (NO) through NO scavenging and nitrite reductase.Aims:This study was designed to examine the effects of incorporating a novel NO modulator, RRx-001, on systemic and microvascular hemodynamic response after blood transfusion for resuscitation from hemorrhagic shock in a hamster window chamber model. In addition, to RRx-001 the role of low dose of nitrite (1 × 10−9 moles per animal) supplementation after resuscitation was studied.Materials and Methods:Severe hemorrhage was induced by arterial controlled bleeding of 50% of the blood volume (BV) and the hypovolemic state was maintained for 1 h. The animals received volume resuscitation by an infusion of 25% of BV using fresh blood alone or with added nitrite, or fresh blood treated with RRx-001 (140 mg/kg) or RRx-001 (140 mg/kg) with added nitrite. Systemic and microvascular hemodynamics were followed at baseline and at different time points during the entire study. Tissue apoptosis and necrosis were measured 8 h after resuscitation to correlate hemodynamic changes with tissue viability.Results:Compared to resuscitation with blood alone, blood treated with RRx-001 decreased vascular resistance, increased blood flow and functional capillary density immediately after resuscitation and preserved tissue viability. Furthermore, in RRx-001 treated animals, both mean arterial pressure (MAP) and met Hb were maintained within normal levels after resuscitation (MAP >90 mmHg and metHb <2%). The addition of nitrite to RRx-001 did not significantly improve the effects of RRx-001, as it increased methemoglobinemia and lower MAP.Conclusion:RRx-001 alone enhanced perfusion and reduced tissue damage as compared to blood; it may serve as an adjunct therapy to the current gold standard treatment for resuscitation from hemorrhagic shock.

A method to investigate radial glia cell behavior using two-photon time-lapse microscopy in an ex vivo model of spinal cord development.

The mammalian central nervous system (CNS) develops from multipotent progenitor cells, which proliferate and differentiate into the various cell types of the brain and spinal cord. Despite the wealth of knowledge from progenitor cell culture studies, there is a significant lack of understanding regarding dynamic progenitor cell behavior over the course of development. This is in part due to shortcomings in the techniques available to study these processes in living tissues as they are occurring. In order to investigate cell behavior under physiologically relevant conditions we established an ex vivo model of the developing rat spinal cord. This method allows us to directly observe specific populations of cells ex vivo in real time and over extended developmental periods as they undergo proliferation, migration, and differentiation in the CNS. Previous investigations of progenitor cell behavior have been limited in either spatial or temporal resolution (or both) due to the necessity of preserving tissue viability and avoiding phototoxic effects of fluorescent imaging. The method described here overcomes these obstacles. Using two-photon and confocal microscopy and transfected organotypic spinal cord slice cultures we have undertaken detailed imaging of a unique population of neural progenitors, radial glial cells. This method uniquely enables analysis of large populations as well as individual cells; ultimately resulting in a 4D dataset of progenitor cell behavior for up to 7 days during embryonic development. This approach can be adapted to study a variety of cell populations at different stages of development using appropriate promoter driven fluorescent protein expression. The ability to control the tissue micro-environment makes this ex vivo method a powerful tool to elucidate the underlying molecular mechanisms regulating cell behavior during embryonic development.