Insulin-dependent glucose consumption dynamics in 3D primary human liver cultures measured by a sensitive and specific glucose sensor with nanoliter input volume.

The liver plays a central role in glucose homeostasis and hepatic insulin resistance constitutes a key feature of type 2 diabetes. However, platforms that accurately mimic human hepatic glucose disposition and allow for rapid and scalable quantification of glucose consumption dynamics are lacking. Here, we developed and optimized a colorimetric glucose assay based on the glucose oxidase-peroxidase system and demonstrate that the system can monitor glucose consumption in 3D primary human liver cell cultures over multiple days. The system was highly sensitive (limit of detection of 3.5 µM) and exceptionally accurate (R2  = 0.999) while requiring only nanoliter input volumes (250 nL), enabling longitudinal profiling of individual liver microtissues. By utilizing a novel polymer, off-stoichiometric thiol-ene (OSTE), and click-chemistry based on thiol-Michael additions, we furthermore show that the assay can be covalently bound to custom-build chips, facilitating the integration of the sensor into microfluidic devices. Using this system, we find that glucose uptake of our 3D human liver cultures closely resembles human hepatic glucose uptake in vivo as measured by euglycemic-hyperinsulinemic clamp. By comparing isogenic insulin-resistant and insulin-sensitive liver cultures we furthermore show that insulin and extracellular glucose levels account for 55% and 45% of hepatic glucose consumption, respectively. In conclusion, the presented data show that the integration of accurate and scalable nanoliter glucose sensors with physiologically relevant organotypic human liver models enables longitudinal profiling of hepatic glucose consumption dynamics that will facilitate studies into the biology and pathobiology of glycemic control, as well as antidiabetic drug screening.

Role of the Guanidinium Groups in Ligand-Receptor Binding of Arginine-Containing Short Peptides to the Slow Sodium Channel: Quantitative Approach to Drug Design of Peptide Analgesics.

Several arginine-containing short peptides have been shown by the patch-clamp method to effectively modulate the NaV1.8 channel activation gating system, which makes them promising candidates for the role of a novel analgesic medicinal substance. As demonstrated by the organotypic tissue culture method, all active and inactive peptides studied do not trigger the downstream signaling cascades controlling neurite outgrowth and should not be expected to evoke adverse side effects on the tissue level upon their medicinal administration. The conformational analysis of Ac-RAR-NH2, Ac-RER-NH2, Ac-RAAR-NH2, Ac-REAR-NH2, Ac-RERR-NH2, Ac-REAAR-NH2, Ac-PRERRA-NH2, and Ac-PRARRA-NH2 has made it possible to find the structural parameter, the value of which is correlated with the target physiological effect of arginine-containing short peptides. The distances between the positively charged guanidinium groups of the arginine side chains involved in intermolecular ligand-receptor ion-ion bonds between the attacking peptide molecules and the NaV1.8 channel molecule should fall within a certain range, the lower threshold of which is estimated to be around 9 Å. The distance values have been calculated to be below 9 Å in the inactive peptide molecules, except for Ac-RER-NH2, and in the range of 9-12 Å in the active peptide molecules.

Incorporating NK Cells in a Three-Dimensional Organotypic Culture System for Human Skin Stem Cells: Modeling Skin Diseases and Immune Cell Interplay.

Natural killer (NK) cells are a part of a sophisticated immune system that is necessary for the skin because it is a crucial organ that is continually exposed to environmental influences. Recent studies have shown that NK cell incorporation into three-dimensional (3D) organotypic culture systems for human skin stem cells provides a physiologically relevant environment to study the interactions between immune cells and skin cells, making it a powerful tool for simulating skin diseases and researching these interactions. It has been shown that adding NK cells to 3D organotypic culture systems can improve keratinocyte differentiation and control inflammation in a variety of skin conditions, including psoriasis. In order to increase our knowledge of skin diseases and immune cell interactions, this work intends to propose an optimum approach for adding NK cells to a 3D organotypic culturing system for human skin stem cells. By better comprehending these relationships, researchers hope to develop novel treatments for skin diseases that are more effective and cause fewer side effects than current treatments. To completely understand the mechanisms underlying these interactions and to create new treatments for skin diseases, more research is required. In conclusion, NK cell integration into 3D organotypic culture systems offers a potent tool to investigate immune cell interactions with skin cells in a physiologically appropriate setting, which may result in major improvements in the treatment of skin diseases.

An improved organotypic cell culture system to study tissue-resident macrophages ex vivo.

Tissue-resident macrophages (TRMs) perform organ-specific functions that are dependent on factors such as hematopoietic origin, local environment, and biological influences. A diverse range of in vitro culture systems have been developed to decipher TRM functions, including bone marrow-derived macrophages (BMDMs), induced pluripotent stem cell (iPSC)-derived TRMs, or immortalized cell lines. However, despite the usefulness of such systems, there are notable limitations. Attempts to culture primary macrophages often require purification of cells and lack a high cell yield and consistent phenotype. Here, we aimed to address these limitations by establishing an organotypic primary cell culture protocol. We obtained long-term monocultures of macrophages derived from distinct organs without prior purification using specific growth factors and tissue normoxic conditions that largely conserved a TRM-like identity in vitro. Thus, this organotypic system offers an ideal screening platform for primary macrophages from different organs that can be used for a wide range of assays and readouts.

3D Adipose Tissue Culture Links the Organotypic Microenvironment to Improved Adipogenesis.

Obesity and type 2 diabetes are strongly associated with adipose tissue dysfunction and impaired adipogenesis. Understanding the molecular underpinnings that control adipogenesis is thus of fundamental importance for the development of novel therapeutics against metabolic disorders. However, translational approaches are hampered as current models do not accurately recapitulate adipogenesis. Here, a scaffold-free versatile 3D adipocyte culture platform with chemically defined conditions is presented in which primary human preadipocytes accurately recapitulate adipogenesis. Following differentiation, multi-omics profiling and functional tests demonstrate that 3D adipocyte cultures feature mature molecular and cellular phenotypes similar to freshly isolated mature adipocytes. Spheroids exhibit physiologically relevant gene expression signatures with 4704 differentially expressed genes compared to conventional 2D cultures (false discovery rate < 0.05), including the concerted expression of factors shaping the adipogenic niche. Furthermore, lipid profiles of >1000 lipid species closely resemble patterns of the corresponding isogenic mature adipocytes in vivo (R2 = 0.97). Integration of multi-omics signatures with analyses of the activity profiles of 503 transcription factors using global promoter motif inference reveals a complex signaling network, involving YAP, Hedgehog, and TGFß signaling, that links the organotypic microenvironment in 3D culture to the activation and reinforcement of PPARγ and CEBP activity resulting in improved adipogenesis.

Phenotypic markers of oral keratinocytes seeded on two distinct 3D oral mucosa models.

This study validates the use of a full-thickness oral mucosa model for in vitro studies with a collagen type I matrix, by comparison of this model with two other 3D oral mucosa models: human-sourced and porcine acellular dermal matrices (AlloDerm®/Strattice®, respectively). For the collagen matrix model, gingival fibroblasts were seeded either onto the dermal side of the AlloDerm® and Strattice® matrices or within the collagen matrices in complete culture medium (DMEM). For all scaffolds, DMEM was replaced every 24 h up to 72 h. For the full-thickness oral mucosa models, 72 h after fibroblast seeding, oral keratinocytes were seeded on the epidermal sides of AlloDerm® and Strattice® matrices or collagen matrices. All matrices and models were subjected to histological analysis, complementing phenotypic characterization by evaluation of glucose consumption, cell proliferation, gene expression and synthesis of growth factors. A higher fibroblast ratio was observed for the collagen matrix, in which the distribution of gingival fibroblasts was also more homogeneous. Metabolism, proliferation, and gene expression and synthesis of VEGF of these cells were also increased for the collagen matrix. All matrices provided a suitable substrate for oral keratinocytes adhesion, proliferation, and phenotypic expression; however, higher proliferation, stratification, and differentiation were noted when oral keratinocytes were seeded on the dermal matrices.

Development of an oral mucosa equivalent using a porcine dermal matrix.

We evaluated the suitability of a porcine acellular dermal matrix for the development of a 3-dimensional oral mucosal equivalent using an ex vivo-produced oral mucosal equivalent (EVPOME). Oral keratinocytes were seeded in a submerged model, and then in an air-liquid interphase model, using Transwell® inserts. EVPOME showed good cell viability and increased glucose consumption over time. Histological evaluation showed that stratified differentiated epithelium had formed in all matrices.

Altered expression of epithelial-to-mesenchymal transition proteins in extraprostatic prostate cancer.

Epithelial to mesenchymal transition (EMT) of cancer cells involves loss of epithelial polarity and adhesiveness, and gain of invasive and migratory mesenchymal behaviours. EMT occurs in prostate cancer (PCa) but it is unknown whether this is in specific areas of primary tumours. We examined whether any of eleven EMT-related proteins have altered expression or subcellular localisation within the extraprostatic extension component of locally advanced PCa compared with other localisations, and whether similar changes may occur in in vitro organotypic PCa cell cultures and in vivo PCa models. Expression profiles of three proteins (E-cadherin, Snail, and α-smooth muscle actin) were significantly different in extraprostatic extension PCa compared with intra-prostatic tumour, and 18/27 cases had an expression change of at least one of these three proteins. Of the three significantly altered EMT proteins in pT3 samples, one showed similar significantly altered expression patterns in in vitro organotypic culture models, and two in in vivo Pten-/- model samples. These results suggest that changes in EMT protein expression can be observed in the extraprostatic extension component of locally invasive PCa. The biology of some of these changes in protein expression may be studied in certain in vitro and in vivo PCa models.