[This corrects the article DOI: 10.1016/j.isci.2023.107660.].

Translating preclinical research findings to clinical applications presents challenges due to the metabolic and anatomical disparities between animal models and humans. Although organ-on-a-chip models replicate the organization of human cells, they lack the complexity of the microenvironment and anatomical fidelity. Developing alternative research models with greater physiological and anatomical relevance to humans holds the potential to enhance translational success and reduce reliance on animal experimentation. In this study, we have showcased the technical details and conditions to maintain the viability of ex vivo perfused large human abdominal fasciocutaneous flaps for up to three weeks. Our efforts involved refining surgical procedures, vascular territory mapping (angiosome analysis), and pedicle exploration; engineering the bioreactor system; and optimizing perfusion media. Angiography via thermal and fluorescent methods was used to confirm the perfusion success. Metabolic activity was closely monitored by tracking glucose consumption and lactate production. Assessments of tissue viability encompassed histological analysis, TUNEL staining, gene expression profiling, measurement of vascular and metabolic reactivity, and in vitro propagation of isolated adipose stem cells and dermal fibroblasts. Furthermore, we harnessed our optimized human skin perfusion model to investigate the dynamics of radiation and chemical-induced injuries. Additionally, we explored the model's utility in studying adipose tissue metabolism and employed human skin tissue to establish melanoma and breast cancer tumor models. To our knowledge, this is the first model capable of preserving the function and viability of a large flap for extended periods, providing a proof-of-concept foundation for diverse research applications.

Abstract Introduction Frostbite is a serious injury that occurs when body tissues are exposed to freezing temperatures, potentially resulting in amputation. Tissue damage arises from a combination of direct freezing and subsequent inflammation, vasoconstriction, and thrombosis. Thrombolytic agents are an important component of acute-phase treatment. Iloprost has recently received FDA approval for managing acute frostbite. However, pooled and comparative evidence on their effectiveness remains limited. This study aimed to systematically evaluate and compare the impact of thrombolytic agents and iloprost on amputation outcomes in patients with severe frostbite. Methods A systematic review and meta-analysis was conducted in accordance with PRISMA guidelines across PubMed, Cochrane Library, EMBASE, and Web of Science. This study was registered on PROSPERO (CRD420250652369). Eligible articles reported on frostbite treatment with either iloprost or thrombolytic agents in 3 or more patients. The primary outcomes were the number of amputated patients and the digital amputation rate. Results Of 7875 studies screened, 12 met the inclusion criteria, including 10 dealing with tPA, 2 with iloprost and 1 reporting about both interventions. They examined 779 patients with severe frostbite (grade 2-4), affecting the upper (50.25%) or lower (49.75%) extremities. Patients were predominantly male (81.64%) with a mean age of 41.63 ± 15.38 years. Thrombolytic therapy significantly reduced the risk of digital amputation in severe frostbite (RR = 0.21; 95% CI: 0.14–0.33; p<.00001; I2 = 0%). Iloprost showed a trend toward reduced digital amputation risk, although heterogeneity was high (RR = 0.09; 95% CI: 0.00–5.04; p=.24; I2 = 88%). Conclusions Amputation is the most feared complication of severe frostbite. Thrombolytic agents emerged as a key acute intervention to prevent tissue loss in eligible patients. Iloprost may contribute to improved amputation outcomes. Additional high-quality comparative studies are needed to determine the most effective strategy for tissue salvage. Applicability of Research to Practice Early thrombolytic therapy should be prioritized in eligible severe frostbite cases to reduce amputation risk. Iloprost may be useful when thrombolysis is contraindicated, though its role is less defined. Standardized protocols and timely intervention are key to improving tissue salvage and patient outcomes. Funding for the study N/A.

Pelvic radiotherapy can lead to loss of bladder compliance, detrusor overactivity, and superficial vascular proliferation. Animal studies have demonstrated a reduction in radiation-induced inflammation and fibrosis following administration of mesenchymal stem cell products. We performed a first-in-human pilot study to assess the safety and feasibility of intravesical adipose stromal vascular fraction injection in an 82-year-old man with a history of brachytherapy for prostate cancer. Following FDA and IRB approval, subcutaneous adipose tissue harvested under local anesthesia was immediately processed using the SVF-2 device (GID Bio). The cell product, consisting of 4.58 × 107 stromal vascular fraction cells resuspended in 20 cc/s of lactated ringers, was injected into the bladder wall using a flexible cystoscope. Urinary symptoms, urologic flow parameters, repeat cystoscopy with bladder biopsy, and MRI were employed during the 16-month follow-up period. There were no postprocedural complications. The patient reported expected symptoms of self-limited dysuria, hematuria, and flank bruising in the 2 weeks following the procedure. Urinary symptoms and flow parameters remained stable for 6 months but progressed slightly at 15 months. A repeat cystoscopy with biopsy showed stabilization of disease without concern for secondary malignancy. The patient's 12-month postprocedural MRI and 15-month urodynamics study were unchanged from prior. This study demonstrates the safety and feasibility of adipose harvest under local anesthesia, followed by point-of-care isolation and administration of an adipose-derived cell product via cystoscopic-guided intravesical injection. This pilot is foundational for further clinical studies to elucidate effective dosing and patient selection in the treatment of bladder fibrosis with cell therapy.

Metformin (Met), a highly hydrophilic drug, exhibits anti-inflammatory and regenerative effects beyond its antidiabetic role. However, its extreme polarity limits passive diffusion through the stratum corneum, necessitating optimized formulations for effective transdermal delivery. This study compared Met permeability through human and porcine ear skin using three lotion formulations containing different permeation enhancers: (1) 6% Met + glycerol (Gly); (2) 6% Met + Gly and propylene glycol (PG); and (3) 10% Met + PG and Transcutol® (PG + T). In human skin, Gly and PG formulations showed minimal permeation, whereas PG + T markedly increased cumulative permeation and flux, confirming the critical role of Transcutol®. Porcine skin displayed consistently higher permeability across all formulations (Gly < PG < PG + T), indicating that it may overestimate human permeability to hydrophilic drugs. Despite limited transdermal flux, Gly- and PG-based lotions produced measurable Met accumulation within the epidermis and dermis, suggesting potential for topical use. Overall, these results emphasize the importance of enhancer selection-particularly Transcutol®-in improving dermal delivery of hydrophilic compounds and caution against relying solely on porcine skin as a human surrogate.

Physiological adaptations to fasting enable humans to survive for prolonged periods without food and involve molecular pathways that may drive life-prolonging effects of dietary restriction in model organisms. Mobilization of fatty acids and glycerol from adipocyte lipid stores by canonical neutral lipases, including the rate limiting adipose triglyceride lipase (Pnpla2/ATGL), is critical to the adaptive fasting response. Here we discovered an alternative mechanism of lipolysis in adipocytes involving a lysosomal program. We functionally tested lysosomal lipolysis with pharmacological and genetic approaches in mice and in murine and human adipocyte and adipose tissue explant culture, establishing dependency on lysosomal acid lipase (LIPA/LAL) and the microphthalmia/transcription factor E (MiT/TFE) family. Our study establishes a model whereby the canonical pathway is critical for rapid lipolytic responses to adrenergic stimuli operative in the acute stage of fasting, while the alternative lysosomal pathway dominates with prolonged fasting. Survival during fasting requires release of adipose tissue lipid stores and is thought to be dependent on canonical lipases, including the rate limiting action of adipose triglyceride lipase. Here the authors show that lysosomes and lysosomal acid lipase play a critical role in adipocyte lipolysis with fasting in mice.

Background Radiation therapy often leads to late radiation-induced skin fibrosis (RISF), causing movement impairment and discomfort. We conducted a comprehensive study to assess the effectiveness of metformin and adipose-derived stem cells (ASCs), whether autologous or allogeneic, individually or in combination therapy, in mitigating RISF. Methods Using a female C57BL/6J mouse model subjected to hind limb irradiation as a representative RISF model, we evaluated metformin, ASCs, or their combination in two contexts: prophylactic (started on day 1 post-irradiation) and therapeutic (initiated on day 14 post-irradiation, coinciding with fibrosis symptoms). We measured limb movement, examined skin histology, and analyzed gene expression to assess treatment efficacy. Results Prophylactic metformin and ASCs, whether autologous or allogeneic, effectively prevented late fibrosis, with metformin showing promising results. However, combination therapy did not provide additional benefits when used prophylactically. Autologous ASCs, alone or with metformin, proved most effective against late-stage RISF. Prophylactic intervention outperformed late therapy for mitigating radiation skin damage. Co-culture studies revealed that ASCs and metformin downregulated inflammation and fibrotic gene expression in both mouse and human fibroblasts. Conclusions Our study suggests metformin's potential as a prophylactic measure to prevent RISF, and the combination of ASCs and metformin holds promise for late-stage RISF treatment. These findings have clinical implications for improving the quality of life for those affected by radiation-induced skin fibrosis.

Adipose tissue regulates metabolic balance, but aging disrupts it, shifting fat from insulin-sensitive subcutaneous to insulin-resistant visceral depots, impacting overall metabolic health. Adipose-derived stem cells (ASCs) are crucial for tissue regeneration, but aging diminishes their stemness and regeneration potential. Our findings reveal that aging is associated with a decrease in subcutaneous adipose tissue mass and an increase in the visceral fat depots mass. Aging is associated with increase in adipose tissue fibrosis but no significant change in adipocyte size was observed with age. Long term caloric restriction failed to prevent fibrotic changes but resulted in significant decrease in adipocytes size. Aged subcutaneous ASCs displayed an increased production of ROS. Using mitochondrial membrane activity as an indicator of stem cell quiescence and senescence, we observed a significant decrease in quiescence ASCs with age exclusively in subcutaneous adipose depot. In addition, aged subcutaneous adipose tissue accumulated more senescent ASCs having defective autophagy activity. However, long-term caloric restriction leads to a reduction in mitochondrial activity in ASCs. Furthermore, caloric restriction prevents the accumulation of senescent cells and helps retain autophagy activity in aging ASCs. These results suggest that caloric restriction and caloric restriction mimetics hold promise as a potential strategy to rejuvenate the stemness of aged ASCs. Further investigations, including in vivo evaluations using controlled interventions in animals and human studies, will be necessary to validate these findings and establish the clinical potential of this well-established approach for enhancing the stemness of aged stem cells.

PURPOSE: Autologous fat grafting is a widely used technique for soft tissue augmentation in facial and breast contouring. However, a significant drawback is that, on average, nearly half of the grafted volume is reabsorbed over time, necessitating the need for repetitive procedures. Here we have designed a cryopreservation device and optimized a protocol for on-site storage of the excess lipoasirate. METHODS: This study presents an innovative device designed for on-site cryopreservation of lipoaspirate featuring a cylindrical vessel with female Luer ports at both ends compatible with fat harvest and processing equipment. It operates as a closed system for fat washing, reducing contamination risks. We conducted evaluations involving various cryoprotectant combinations, freezing temperatures, and durations. To assess cryopreservation outcomes, we measured cell viability by trypan blue and Calcein-Am staining at different time points post-cryopreservation. RESULTS: In vitro viability analyses indicated that a cryopreservation agent consisting of 10% DMSO and 2% human serum albumin, stored at -80°C, provided optimal cryopreservation results following a three month period compared to fresh fat. For in-vivo graft retention analysis, Nu/Nu athymic mice were utilized, and human fat cryopreserved for seven days, 21 days, three months, or 11 months was engrafted. After three months, assessments of fat graft weight, volume retention, and histology revealed no significant differences between the comparison groups and freshly harvested fat. CONCLUSION: This study highlights the clinical application of our device to minimize the need for repeated harvest and to effectively preserve lipoasirate for up to one year.

Burn injuries are a significant global health concern, leading to high morbidity and mortality. Deep burn injuries often result in delayed healing and scar formation, necessitating effective treatment options. Regenerative medicine, particularly cell therapy using adipose-derived stem cells (ASCs), has emerged as a promising approach to improving burn wound healing and reducing scarring. Both in vitro and preclinical studies have demonstrated the efficacy of ASCs and the stromal vascular fraction (SVF) in addressing burn wounds. The application of ASCs for burn healing has been studied in various forms, including autologous or allogeneic cells delivered in suspension or within scaffolds in animal burn models. Additionally, ASC-derived non-cellular components, such as conditioned media or exosomes have shown promise. Injection of ASCs and SVF at burn sites have been demonstrated to enhance wound healing by reducing inflammation and promoting angiogenesis, epithelialization, and granulation tissue formation through their paracrine secretome. This review discusses the applications of adipose tissue derivatives in burn injury treatment, encompassing ASC transplantation, as well as the utilization of non-cellular components utilization for therapeutic benefits. The application of ASCs in burn healing in the future will require addressing donor variability, safety, and efficacy for successful clinical application.

INTRODUCTION: Radiation-induced skin fibrosis is one of the main adverse effects of radiation therapy for cancer treatment. Radiation fibrosis syndrome is caused by the overactivation of TGF-B that promotes fibroblast that induces collagenases and breaks down type III collagen and replaces it with type I collagen. The inhibition of P53 and the accumulation of ROS are the main mechanisms of radiation-induced damage. To fully understand these mechanisms, we tested the use of our novel human skin perfusion model to recreate the damage caused by radiation and as a platform to test possible therapeutic and/or prophylactic treatments to prevent fibrosis. METHODS: We use our perfusion model, which consists of a human tissue sample recovered from abdominoplasty. We dissect the superficial inferior epigastric artery and cannulated it and perfuse it with special culture media. On the first day after cannulation, we exposed the skin to a single targeted irradiation dose of 20gy and 40gy and took punch biopsies on days 3, 6, 12, and 16 for histological and gene expression analyses. The histological samples were stained with H&E, and Masson´s Trichrome stain to determine the morphology changes and extracellular matrix deposition respectively. TUNEL and DAPI immunofluorescent staining was performed to analyze for apoptotic changes in the epidermis/dermis. Expression of inflammatory, fibrotic and apoptotic genes was analyzed by real-time quantitative PCR. RESULTS: The morphological changes in the skin were significant. The radiation-exposed skin started peeling compared to the control group. The H&E staining showed an increase of inflammation in the dermis along with epidermis/dermis separation as well as papillary dermis containing fibrin deposition, accumulation of inflammatory cells, reactive changes in the endothelial cells, and abundant necrotic keratinocytes. Masson’s Trichrome staining revealed a increased deposition of extracellular matrix between the papillary and reticular dermis in the irradiated skin. The TUNEL and DAPI stain shows an increase in apoptotic cells in the radiation group that correlates with the damage induced by radiation. Gene expression analyses revealed upregulation of inflammatory and anti-apoptotic genes expression. CONCLUSIONS: Our perfusion model was stable for 19 days and was able to recreate the radiation-induced deposition of collagen shown in the H&E and Masson´s trichrome as well as the radiation-induced damage in the TUNEL and DAPI staining. Our system is reliable and can be used as a platform for developing therapeutics to mitigate the effects of radiation on the skin.

PURPOSE: Autologous fat transfer is an effective treatment for soft tissue reconstruction. The main challenge is that on average, 56% of the graft volume takes; this necessitates repeat procedures. Therefore, preserving harvested tissue on-site for future injections is a clinical need. This study investigates different cryopreservation methods and applies the best results for a clinically usable device. METHODS: Different cryoprotectant combinations, freezing temperatures, and conditions were tested, and the outcome of the cryopreservation was assessed by measuring cell viability using trypan blue and Calcin-Am staining two days post-freezing.In vitro validation of optimized conditions was tested for up to 3 months. For in-vivo testing, Nu/Nu athymic mice were used, and human fat cryopreserved for seven days, 21 days, three months, or 11 months was compared to fresh fat for graft weight and volume retention, histology, vacuole formation, and inflammation at nine weeks post grafting. At +4 degrees celsius for three months, stored combination compared to fresh. RESULTS: A combination of 10% DMSO and 2% human serum albumin at -80 degrees celsius provided optimum cryopreservation. We observed no significant differences in the cell viability of cryopreserved fat for up to 3 months compared to the fresh fat. Cryopreserved fat grafts showed weight, volume retention, histological morphology, vacuole formation, and inflammation comparable to fresh fat grafts. The cryopreservation solution was stable during storage. CONCLUSION: The result of this study will enable the development of devices with clinically compatible appendages and a defined protocol for clinical use for long cryopreservation of fat tissue at -80 degrees celsius within a closed system.