Organotypic Slice Cultures as Preclinical Models of Tumor Microenvironment in Primary Pancreatic Cancer and Metastasis.

Realistic preclinical models of primary pancreatic cancer and metastasis are urgently needed to test the therapy response ex vivo and facilitate personalized patient treatment. However, the absence of tumor-specific microenvironment in currently used models, e.g., patient-derived cell lines and xenografts, only allows limited predictive insights. Organotypic slice cultures (OTSCs) comprise intact multicellular tissue, which can be rapidly used for the spatially resolved drug response testing. This protocol describes the generation and cultivation of viable tumor slices of pancreatic cancer and its metastasis. Briefly, tissue is casted in low melt agarose and stored in cold isotonic buffer. Next, tissue slices of 300 µm thickness are generated with a vibratome. After preparation, slices are cultured at an air-liquid interface using cell culture inserts and an appropriate cultivation medium. During cultivation, changes in cell differentiation and viability can be monitored. Additionally, this technique enables the application of treatment to viable human tumor tissue ex vivo and subsequent downstream analyses, such as transcriptome and proteome profiling. OTSCs provide a unique opportunity to test the individual treatment response ex vivo and identify individual transcriptomic and proteomic profiles associated with the respective response of distinct slices of a tumor. OTSCs can be further explored to identify therapeutic strategies to personalize treatment of primary pancreatic cancer and metastasis.

Circadian clock network desynchrony promotes weight gain and alters glucose homeostasis in mice.

OBJECTIVE: A network of endogenous circadian clocks adapts physiology and behavior to recurring changes in environmental demands across the 24-hour day cycle. Circadian disruption promotes weight gain and type 2 diabetes development. In this study, we aim to dissect the roles of different tissue clocks in the regulation of energy metabolism. METHODS: We used mice with genetically ablated clock function in the circadian pacemaker of the suprachiasmatic nucleus (SCN) under different light and feeding conditions to study peripheral clock resetting and the role of the peripheral clock network in the regulation of glucose handling and metabolic homeostasis. RESULTS: In SCN clock-deficient mice, behavioral and non-SCN tissue clock rhythms are sustained under rhythmic lighting conditions but deteriorate quickly in constant darkness. In parallel to the loss of behavioral and molecular rhythms, the animals develop adiposity and impaired glucose utilization in constant darkness. Restoring peripheral clock rhythmicity and synchrony by time-restricted feeding normalizes body weight and glucose metabolism. CONCLUSIONS: These data reveal the importance of an overall synchronized circadian clockwork for the maintenance of metabolic homeostasis.

Heterogeneity of Sweat Gland Stem Cells.

Sweat glands play an important role in skin physiology and are an integral part of the natural skin barrier. In order to maintain functionality throughout life, sweat glands make use of several types of stem cells. This chapter focuses on the classification of different types of stem cells found in the sweat gland and their physiological roles. First, sweat gland formation during skin maturation is addressed in order to give an overview of sweat gland origin and formation in vivo. Then, different kinds of adult sweat gland stem cells are introduced and classified between different potency levels and corresponding physiological roles. Finally, the importance of these cell sources for future developments, including applications in wound healing and cosmetics research, is discussed.

Fabrication of a Co-Culture System with Human Sweat Gland-Derived Cells and Peripheral Nerve Cells.

The interaction of peripheral nerves with different cells of the skin is a relevant aspect of many physiological processes including nociception, temperature control, and wound healing. Here we describe a protocol for the setup of an indirect co-culture system of peripheral nerve cells and sweat gland-derived stem cells, which can be used to quantify neurite outgrowth.

Skin-Derived Stem Cells for Wound Treatment Using Cultured Epidermal Autografts: Clinical Applications and Challenges.

The human skin fulfills important barrier, sensory, and immune functions-all of which contribute significantly to health and organism integrity. Widespread skin damage requires immediate treatment and coverage because massive skin loss fosters the invasion of pathogens, causes critical fluid loss, and may ultimately lead to death. Since the skin is a highly immunocompetent organ, autologous transplants are the only viable approach to permanently close a widespread skin wound. Despite the development of tissue-saving autologous transplantation techniques such as mesh and Meek grafts, treatment options for extensive skin damage remain severely limited. Yet, the skin is also a rich source of stem and progenitor cells. These cells promote wound healing under physiological conditions and are potential sources for tissue engineering approaches aiming to augment transplantable tissue by generating cultured epidermal autografts (CEAs). Here, we review autologous tissue engineering strategies as well as transplantation products based on skin-derived stem cells. We further provide an overview of clinical trial activities in the field and discuss relevant translational and clinical challenges associated with the use of these products.

Sulfur mustard skin lesions: A systematic review on pathomechanisms, treatment options and future research directions.

Sulfur mustard (SM) is a chemical warfare, which has been used for one hundred years. However, its exact pathomechanisms are still incompletely understood and there is no specific therapy available so far. In this systematic review, studies published between January 2000 and July 2017 involving pathomechanisms and experimental treatments of SM-induced skin lesions were analyzed to summarize current knowledge on SM pathology, to provide an overview on novel treatment options, and to identify promising targets for future research to more effectively counter SM effects. We suggest that future studies should focus on (I) systemic effects of SM intoxication due to its distribution throughout the body, (II) removal of SM depots that continuously release active compound contributing to chronic skin damage, and (III) therapeutic options that counteract the pleiotropic effects of SM.

Collection of Wound Exudate From Human Digit Tip Amputations Does Not Impair Regenerative Healing: A Randomized Trial.

The regrowth of amputated digit tips represents a unique regenerative healing in mammals with subcutaneous volume regrowth, restoration of dactylogram, and suppression of scar formation. Although factor analysis in amphibians and even in mice is easy to obtain, safety of harvesting biomaterial from human digit tip amputations for analysis has not yet been described.The aim of this study was to evaluate if recovering wound exudate does hamper clinical outcome or influence microbiologic or inflammation status.A predefined cohort of 18 patients with fresh digit tip amputations was randomly assigned to receive standard therapy (debridement, occlusive dressing) with (n = 9) or without (n = 9) collection of the whole wound exudate in every dressing change. Primary endpoint (lengthening) and secondary endpoints (regeneration of dactylogram, nail bed and bone healing, time to complete wound closure, scar formation, 2-point discrimination, microbiologic analysis, inflammatory factors interleukin (IL)-1α, tumor necrosis factor-α, IL-4, and IL-6) were determined by an independent, blinded observer.Patients' characteristics showed no significant differences between the groups. All patients completed the study to the end of 3 months follow-up. Exudate collection did not influence primary and secondary endpoints. Furthermore, positive microbiologic findings as well as pus- and necrosis-like appearance neither impaired tissue restoration nor influenced inflammatory factor release.Here, the authors developed an easy and safe protocol for harvesting wound exudate from human digit tip amputations. For the first time, it was shown that harvesting does not impair regenerative healing. Using this method, further studies can be conducted to analyze regeneration associated factors in the human digit tip.DRKS.de Identifier: DRKS00006882 (UTN: U1111-1166-5723).

Electrocautery Devices With Feedback Mode and Teflon-Coated Blades Create Less Surgical Smoke for a Quality Improvement in the Operating Theater.

Monopolar electrocautery is a fast and elegant cutting option. However, as it creates surgical smoke containing polycyclic aromatic hydrocarbons (PAHs), it may be hazardous to the health of the surgical team. Although new technologies, such as feedback mode (FM) and Teflon-coated blades (TBs), reduce tissue damage, their impact on surgical smoke creation has not yet been elucidated. Therefore, we analyzed the plume at its source.The aim of this study was to evaluate if electrocautery FM and TBs create less surgical smoke.Porcine tissue containing skin was cut in a standardized manner using sharp-edged Teflon-coated blades (SETBs), normal-shaped TBs, or stainless steel blades (SSBs). Experiments were performed using FM and pure-cut mode. Surgical smoke was sucked through filters or adsorption tubes. Subsequently, filters were scanned and analyzed using a spectrophotometer. A high-performance liquid chromatography (HPLC-UV) was performed to detect benzo[a]pyrene (BaP) and phenanthrene as 2 of the most critical PAHs. Temperature changes at the cutting site were measured by an infrared thermometer.In FM, more surgical smoke was created using SSB compared with TBs (P < 0.001). Furthermore, differences between FM and pure-cut mode were found for SSB and TB (P < 0.001), but not for SETB (P = 0.911). Photometric analysis revealed differences in the peak heights of the PAH spectrum. In HLPC-UV, the amount of BaP and phenanthrene detected was lower for TB compared with SSB. Tissue temperature variations increased when SSB was used in FM and pure-cut mode. Furthermore, different modes revealed higher temperature variations with the use of SETB (P = 0.004) and TB (P = 0.005) during cutting, but not SSB (P = 0.789).We found that the use of both TBs and FM was associated with reduced amounts of surgical smoke created during cutting. Thus, the surgical team may benefit from the adoption of such new technologies, which could contribute to the primary prevention of smoke-related diseases.

Co-culture systems of human sweat gland derived stem cells and peripheral nerve cells: an in vitro approach for peripheral nerve regeneration.

BACKGROUND/AIMS: The treatment of peripheral nerve lesions still represents a clinical challenge. Several approaches such as novel biomaterials for nerve guides, addition of growth factors or cellular supplements moved in the focus of research. Especially the application of autologous stem cells is highly promising for future applications. Human sweat gland derived stem cells (hSGSCs) represent an easy accessible source of autologous adult stem cells and did already show a beneficial effect in dermal wound healing. METHODS: In this study, the effect of hSGSCs on neurite outgrowth of primary adult or prenatal Dorsal root ganglia (DRG) neurons was analysed in an indirect co-culture model. Additionally, direct co-cultures with hSGSCs as a feeder layer were performed. RESULTS: Adult and prenatal DRG neurons showed increased neurite outgrowth after 24 h co-culture with hSGSCs. The outgrowth increased significantly by the factors 5.6 and 2.6 respectively. Direct co-cultures revealed neurite alignment along the hSGSCs orientation. CONCLUSION: The paracrine influence of hSGSCs on neurite outgrowth, but also their ability to operate as a feeder layer with guidance properties shows great potential for future applications in peripheral nerve regeneration.

Electrophysiological characterization of spontaneously contracting cell aggregates obtained from rainbow trout larvae with multielectrode arrays.

BACKGROUND/AIMS: Safety pharmacology requires novel model systems for the detection of cardiac side effects. Ranging from cell-based systems to model organisms, no model available to date reflects the complexity of the human heart and evokes the great need for improved and more affordable systems. Many drugs interact with hERG potassium channels and consequently cause life threatening ventricular arrhythmias, further highlighting the importance of suitable model systems. METHODS: Spontaneously Contracting Cell aggregates (SCC) as a 3D in vitro heart-syncytium obtained from rainbow trout larvae represent a novel model system for cardiac safety pharmacology. SCCs can be harvested cost-effectively and kept in culture for several weeks while retaining their functionality and displaying contraction rates similar to the human heart. RESULTS: Extracellular field potential recordings with multielectrode arrays revealed significant prolongation of field potential duration upon administration of common hERG potassium channel blockers. Infusion of 1 µM Dofetilide and 10 µM Terfenadine prolonged field potentials 10 fold and 2 fold, respectively. In addition, SCCs enabled analysis of autonomous contraction frequencies. CONCLUSION: Thus, SCCs represent a novel and low-cost cardiac model system of the human heart for application in safety pharmacology.

Multipotent nestin-positive stem cells reside in the stroma of human eccrine and apocrine sweat glands and can be propagated robustly in vitro.

Human skin harbours multiple different stem cell populations. In contrast to the relatively well-characterized niches of epidermal and hair follicle stem cells, the localization and niches of stem cells in other human skin compartments are as yet insufficiently investigated. Previously, we had shown in a pilot study that human sweat gland stroma contains Nestin-positive stem cells. Isolated sweat gland stroma-derived stem cells (SGSCs) proliferated in vitro and expressed Nestin in 80% of the cells. In this study, we were able to determine the precise localization of Nestin-positive cells in both eccrine and apocrine sweat glands of human axillary skin. We established a reproducible isolation procedure and characterized the spontaneous, long-lasting multipotent differentiation capacity of SGSCs. Thereby, a pronounced ectodermal differentiation was observed. Moreover, the secretion of prominent cytokines demonstrated the immunological potential of SGSCs. The comparison to human adult epidermal stem cells (EpiSCs) and bone marrow stem cells (BMSCs) revealed differences in protein expression and differentiation capacity. Furthermore, we found a coexpression of the stem cell markers Nestin and Iα6 within SGSCs and human sweat gland stroma. In conclusion the initial results of the pilot study were confirmed, indicating that human sweat glands are a new source of unique stem cells with multilineage differentiation potential, high proliferation capacity and remarkable self renewal. With regard to the easy accessibility of skin tissue biopsies, an autologous application of SGSCs in clinical therapies appears promising.

Organotypic slice culture from human adult ventricular myocardium.

AIMS: Cardiovascular research requires complex and functionally intact experimental models. Due to major differences in the cellular and subcellular composition of the myocardium between species, the use of human heart tissue is highly desirable. To enhance the experimental use of the human myocardium, we established methods for the preparation of vital tissue slices from the adult ventricular myocardium as well as conditions for their long-term preservation in organotypic culture. METHODS AND RESULTS: Human ventricular heart samples were derived from surgical specimens excised during a therapeutic Morrow myectomy and cut into 300 µm thick slices. Slices were either characterized in acute experiments or cultured at a liquid-air interface. Viability and functionality were proven by viability staining, enzyme activity tests, intracellular potential recordings, and force measurements. Precision-cut slices showed high viability throughout 28 days in culture and displayed typical cardiomyocyte action potential characteristics, which enabled pharmacological safety testing on the rapid component of the delayed rectifier potassium current (I(Kr)) and ATP-dependent potassium channels throughout the whole culture period. Constant expression of major ion channels was confirmed by quantitative PCR. Acute slices developed excitation-dependent contractions with a clear preload dependency and a ß-adrenergic response. Contractility and myosin light chain expression decreased during the first days in culture but reached a steady state with reactivity upon ß-adrenergic stimulation being preserved. CONCLUSION: Organotypic heart slices represent a multicellular model of the human myocardium and a novel platform for studies ranging from the investigation of molecular interactions to tissue engineering.