Search

The coronavirus disease 2019 (COVID-19), caused by the novel coronavirus, SARS-CoV-2, affects tissues from different body systems but mostly the respiratory system, and the damage evoked in the lungs may occasionally result in severe respiratory complications and eventually lead to death. Studies of human respiratory infections have been limited by the scarcity of functional models that mimic in vivo physiology and pathophysiology. In the last decades, organoid models have emerged as potential research tools due to the possibility of reproducing in vivo tissue in culture. Despite being studied for over one year, there is still no effective treatment against COVID-19, and investigations using pulmonary tissue and possible therapeutics are still very limited. Thus, human lung organoids can provide robust support to simulate SARS-CoV-2 infection and replication and aid in a better understanding of their effects in human tissue. The present review describes methodological aspects of different protocols to develop airway and alveoli organoids, which have a promising perspective to further investigate COVID-19.

Objective Cartilage formation is stimulated in mixtures of chondrocytes and human adipose–derived mesenchymal stromal cells (MSCs) both in vitro and in vivo. During coculture, human MSCs perish. The goal of this study is to elucidate the mechanism by which adipose tissue–derived MSC cell death occurs in the presence of chondrocytes. Methods Human primary chondrocytes were cocultured with human MSCs derived from 3 donors. The cells were cultured in monoculture or coculture (20% chondrocytes and 80% MSCs) in pellets (200,000 cells/pellet) for 7 days in chondrocyte proliferation media in hypoxia (2% O2). RNA sequencing was performed to assess for differences in gene expression between monocultures or coculture. Immune fluorescence assays were performed to determine the presence of caspase-3, LC3B, and P62. Results RNA sequencing revealed significant upregulation of >90 genes in the 3 cocultures when compared with monocultures. STRING analysis showed interconnections between >50 of these genes. Remarkably, 75% of these genes play a role in cell death pathways such as apoptosis and autophagy. Immunofluorescence shows a clear upregulation of the autophagic machinery with no substantial activation of the apoptotic pathway. Conclusion In cocultures of human MSCs with primary chondrocytes, autophagy is involved in the disappearance of MSCs. We propose that this sacrificial cell death may contribute to the trophic effects of MSCs on cartilage formation.

Diabetic nephropathy (DN) is mainly regarded as diabetic glomerulopathy, and its progression is tightly correlated with tubular epithelial lesions. However, the underlying molecular mechanisms linking tubular damage and glomerulopathy are poorly understood. Methods: We previously reported that the upregulation of Bim mediated proximal tubular epithelial cell (PTEC) apoptosis and was crucial in the early stages of DN. Herein we modulated Bim expression in PTECs and subsequently determined podocyte (PC) cytoskeletal arrangement by building a Transwell co-culture system in high glucose (HG). Results: Compared to normal glucose, exposure to 40 mM of HG for 48 h induced significant expression of Bim in PTECs and disorganization in the PC cytoskeleton. When cocultured with PTECs in HG, exacerbated filamentous actin (F-actin) rearrangement and reduced synaptopodin levels were detected in PCs. In contrast, gene knockdown of Bim in PTECs was correlated with the absence of PC cytoskeletal disorganization. NFAT2 level and its nuclear translocation in PTECs were decreased by suppressing Bim expression. Upregulating NFAT2 disrupted the beneficial effects on F-actin organization in PCs obtained by inhibiting Bim. LncRNA microarray analysis identified NONHSAT179542.1, which was implicated in Bim-mediated PC cytoskeletal disorder. Conclusion: Our study clarified the functional role of Bim, a pro-apoptotic factor, which is involved in the crosstalk between PTECs and PCs. Bim promotes NFAT2 activation in PTECs, inducing the downregulation of lncRNA NONHSAT179542.1 in PCs, contributing to the cytoskeletal damage. Identification of the role of the Bim/NFAT2 pathway may represent a promising research direction for a better understanding of DN development.

Intercellular communication between monocytes/macrophages and cells involved in tissue regeneration, such as mesenchymal stromal cells (MSCs) and primary tissue cells, is essential for tissue regeneration and recovery of homeostasis. Typically, in the final phase of the inflammation-resolving process, this intercellular communication drives an anti-inflammatory immunomodulatory response. To obtain a safe and effective treatment to counteract the cytokine storm associated with a disproportionate immune response to severe infections, including that associated with COVID-19, by means of naturally balanced immunomodulation, our group has standardized the production under GMP-like conditions of a secretome by coculture of macrophages and MSCs. To characterize this proteome, we determined the expression of molecules related to cellular immune response and tissue regeneration, as well as its possible toxicity and anti-inflammatory potency. The results show a specific molecular pattern of interaction between the two cell types studied, with an anti-inflammatory and regenerative profile. In addition, the secretome is not toxic by itself on human PBMC or on THP-1 monocytes and prevents lipopolysaccharide (LPS)-induced growth effects on those cell types. Finally, PRS CK STORM prevents LPS-induced TNF-A and IL-1B secretion from PBMC and from THP-1 cells at the same level as hydrocortisone, demonstrating its anti-inflammatory potency. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.

The growing application of metagenomics to different ecological and microbiome niches in recent years has enhanced our knowledge of global microbial biodiversity. Among these abundant and widespread microbes, Candidate Phyla Radiation or CPR have been recognised as representing a large proportion of the microbial kingdom (> 26%). CPR are characterised by their obligate symbiotic or exo-parasitic activity with other microbial hosts, mainly bacteria. Currently, isolating CPR is still considered challenging for microbiologists. The idea of this study was to develop an adapted protocol for the co-culture of CPR with a suitable bacterial host. Based on various sputa, we tried to purify CPR (Saccharibacteria members) and to cultivate them with pure hosts. This protocol was monitored by real-time PCR quantification using a specific system for Saccharibacteria designed in this study, as well as by electron microscopy and sequencing. We succeeded in co-culturing and sequencing a complete genome of two new Saccharibacteria species: Candidatus Minimicrobia naudis and Candidatus Minimicrobia vallesae. In addition, we noticed a decrease in the Ct number of Saccharibacteria, and a significant multiplication through their physical association with Schaalia odontolytica strains in the enriched medium that we developed. This work may help bridge gaps in the genomic database by providing new CPR members and, in the future, their currently unknown characteristics may be revealed.IMPORTANCEIn this study, the first real-time PCR system has been developed. This technique is able to quantify specifically Saccharibacteria members in any sample of interest in order to investigate their prevalence. In addition, another easy, specific and sensitive protocol has been developed to maintain the viability of Saccharibacteria cells in an enriched medium with their bacterial host. The use of this protocol subsequently facilitates studying the phenotypic characteristics of CPR and their physical interactions with bacterial species, as well as the sequencing of new genomes to improve the current database.

International audience; To move towards clinical applications, tissue engineering (TE) should be validated with human primary cells and offer easy connection to the native vascularisation. Based on a sheet-like bone substitute developed previously, we investigated a mesenchymal stem cells/endothelial cells (MSCs/ECs) coculture to enhance pre-vascularisation. Using MSCs from six independent donors whose differentiation potential was assessed towards two lineages, we focused on donor variability and cell crosstalk regarding bone differentiation. Coculture was performed on calcium phosphate granules in a specific chamber during 1 month. MSCs were seeded first then ECs were added after 2 weeks, with respective monocultures as control groups. Cell viability and organisation (fluorescence, electronic microscopy), differentiation (ALP staining/activity, RT-qPCR) and mechanical cohesion were analysed. Adaptation of the protocol to coculture was validated (high cell viability and proliferation). Activity and differentiation showed strong trends towards synergistic effects between cell types. MSCs reached early mineralisation stage of maturation. The delayed addition of ECs allowed for their attachment on developed MSCs’ matrix. The main impact of donor variability could be here the lack of cell proliferation potential with some donors, leading to low differentiation and mechanical cohesion and therefore absence of sheet-like shape successfully obtained with others. We suggest therefore adapting protocols to cell proliferation potentials from one batch of cells to the other in a patient-specific approach.

Airway epithelial cells and macrophages participate in inflammatory responses to external noxious stimuli, which can cause epithelial injury. Upon injury, epithelial cells and macrophages act in concert to ensure rapid restoration of epithelial integrity. The nature of the interactions between these cell types during epithelial repair is incompletely understood. We used an in vitro human coculture model of primary bronchial epithelial cells cultured at the air-liquid interface (ALI-PBEC) and polarized primary monocyte-derived macrophages. Using this coculture, we studied the contribution of macrophages to epithelial innate immunity, wound healing capacity, and epithelial exposure to whole cigarette smoke (WCS). Coculture of ALI-PBEC with lipopolysaccharide (LPS)-activated M(GM-CSF) macrophages increased the expression of DEFB4A, CXCL8, and IL6 at 24 h in the ALI-PBEC, whereas LPS-activated M(M-CSF) macrophages only increased epithelial IL6 expression. Furthermore, wound repair was accelerated by coculture with both activated M(GM-CSF) and M(M-CSF) macrophages, also following WCS exposure. Coculture of ALI-PBEC and M(GM-CSF) macrophages resulted in increased CAMP expression in M(GM-CSF) macrophages, which was absent in M(M-CSF) macrophages. CAMP encodes LL-37, an antimicrobial peptide with immune-modulating and repair-enhancing activities. In conclusion, dynamic crosstalk between ALI-PBEC and macrophages enhances epithelial innate immunity and wound repair, even upon concomitant cigarette smoke exposure.

Legionellosis was diagnosed in an immunocompromised 3-year-old girl in Canada. We traced the source of the bacterium through co-culture with an ameba collected from a hot tub in her home. We identified Legionella pneumophila serogroup 6, sequence type 185, and used whole-genome sequencing to confirm the environmental and clinical isolates were of common origin.

Vascular tissue engineering seeks to develop functional blood vessels that comprise of both endothelial cells and pericytes for translational medicine and is often faced with numerous challenges such as nutrients and wastes diffusion problem in the centre of the scaffolds. Various strategies have been adopted to solve the diffusion problem in thick engineered scaffolds. Typically, microchannels or dissolvable microspheres are introduced into three-dimensional (3D) scaffolds as an alternative way to improve the infiltration of scaffolds and endothelial cells are usually incorporated into the biomaterials. While some research groups now focus on finding supporting cells to build further vascularized structures in the scaffolds. In this study, a bioinspired 3D gelatin-methacrylate (Gel-MA) hydrogel with dissolvable microspheres was created to encapsulate human bone marrow stromal cells (HMSCs) and human umbilical vein endothelial cells (HUVECs) which was used to investigate whether HMSCs could play a pericytes-like role and enhance vascularization within the engineered scaffolds. The results showed co-culture of HMSCs and HUVECs demonstrated significantly improved vascularization when compared to either HUVECs or HMSCs monoculture. Angiogenic genes were expressed significantly higher in co-culture group. Moreover, when implanting the pre-vascularized scaffolds in vivo, co-culture system integrated more successfully with host tissue and showed higher host tissue invasion than any other groups. More importantly, both the qPCR and immunofluorescence results indicated MSCs differentiated towards pericytes to enhance vascularization in this study. This paper highlights the enhanced capability of 3D micro-cavitary Gel-MA hydrogel for co-culturing HUVECs and HMSCs to promote vascularization which presents a potential strategy for future tissue repair and regeneration. Ministry of Education (MOE) We acknowledge the funding support from Grant AcRF Tier 1 and 2 Academic Research Fund (2018-T1-001-085 and 2016-T2-1-138 (S)), AcRF, Ministry of Education - Singapore, Singapore and Start-up Grant for Professor (SGP 9380099 to Dong-An Wang), City University of Hong Kong.

Inflammation is one of the factors that may increase the sensitivity of hepatic cells to acetaminophen (APAP) induced toxicity. To investigate the mechanisms, we exposed 3-dimensional (3D) Human Liver Microtissues, a co-culture of primary human hepatocytes (PHH) and Kupffer cells (KCs), to 0, 0.5 (low), 5 (median) and 10 mM (high dose) APAP for 24 h, with/without lipopolysaccharide (LPS). Microarray-technology was used to evaluate the transcriptome changes. In the presence of LPS, the median-dose of APAP is sufficient to inhibit the expression of respiratory chain-and antioxidant-related genes, suggesting the involvement of reactive oxygen species (ROS) and oxidative stress. Furthermore, the median- and high-dose of APAP inhibited the expression of Fc fragment receptor (Fc gamma R)-coding genes, regardless of the presence of LPS. The toll-like receptor 4 (TLR4) expression, however, was continuously elevated after the LPS/APAP co-exposures, which may result in reduced KC-phagocytosis and unbalanced cytokine patterns. Compared to the treatment with LPS only, LPS/APAP co-exposures induced the production of interleukin (IL)-8, a pro-inflammatory cytokine, but suppressed the secretion of IL-6, a cytokine regulating hepatic regeneration, along with the increase in APAP dosages. In addition to the disrupted mitochondrial functions, the presence of LPS exacerbated APAP toxicity. These findings suggest that 3D Microtissues are a suitable model for the mechanistic exploration of inflammation-associated drug toxicity.

Mesenchymal stromal cells (MSCs) represent a promising treatment for immune-related diseases due to their diverse immunomodulatory paracrine functions. However, progress of culture-expanded MSCs is hindered by inconsistent cell function, poor localization, and insufficient retention when administered as suspended cell injections, thus placing spatiotemporal dosing constraints on therapeutic functions. To address these limitations, we introduce the combination of in vitro interferon-gamma (IFN-γ) priming, a key stimulator of MSC immunosuppressive potency, and thermoresponsive cultureware to harvest cultured MSCs as directly transplantable scaffold-free immunosuppressive cell sheets. Here, we demonstrate that MSC sheets produced with IFN-γ priming upregulate expression of immunosuppressive factors indoleamine 2,3-dioxygenase (IDO-1), interleukin-10 (IL-10), programmed death ligand-1 (PD-L1), and prostaglandin E2 (PGE2) in both dose- and duration-dependent manners. In addition, IFN-γ primed MSC sheets showed increased ability to inhibit T-cell proliferation via indirect and direct contact, specifically related to increased IDO-1 and PGE2 concentrations. Furthermore, this study’s use of human clinical-grade single-cell-derived clonal bone marrow-derived MSCs, contributes to the future translatability and clinical relevancy of the produced sheets. Ultimately, these results present the combination of IFN-γ priming and MSC sheets as a new strategy to improve MSC-mediated treatment of localized inflammatory diseases.

Objective:Significant eosinophil infiltration and tissue remodeling are common characteristics of conditions associated with chronic airway inflammation, such as chronic rhinosinusitis with nasal polyp and bronchial asthma. This study was designed to elucidate the role of eosinophil-fibroblast interactions in tissue remodeling during chronic airway inflammation., Methods:Peripheral blood eosinophils or EoL-1 eosinophilic leukemia cells were cocultured with nasal polyp fibroblasts (NPFs). Coculture-induced release of exosomes, major components of extracellular vesicles (EVs), and a profibrotic cytokine, vascular endothelial growth factor (VEGF), were evaluated by enzyme-linked immunosorbent assay., Results:Eosinophil-NPF interactions stimulated the release of exosomes and VEGF into culture supernatants. Coculture-induced release of exosomes was stimulated earlier than VEGF release, at 3 h of incubation. The average size of the EVs released by NPFs was 133 ± 3.6 nm. NPF-derived EVs (exosome concentration: 25 pg/mL) significantly stimulated VEGF release from EoL-1 cells. Pretreatment of NPFs with exosome inhibitor, GW4869 or DMA attenuated the release of exosomes and VEGF from cocultured EoL-1 cells and NPFs., Conclusion:The results of this study indicate that eosinophil-fibroblast interactions are important in the pathophysiology of tissue remodeling in eosinophil-predominant airway inflammation and that NPF-derived exosomes play a crucial role in the release of VEGF.

Many human infections result from the action of multispecies bacterial communities. Within these communities, bacteria have been proposed to directly interact via physical and chemical means, resulting in increased disease and antimicrobial tolerance., Pseudomonas aeruginosa and Staphylococcus aureus are two of the most common coinfecting bacteria in human infections, including the cystic fibrosis (CF) lung. There is emerging evidence that coinfection with these microbes enhances disease severity and antimicrobial tolerance through direct interactions. However, one of the challenges to studying microbial interactions relevant to human infection is the lack of experimental models with the versatility to investigate complex interaction dynamics while maintaining biological relevance. Here, we developed a model based on an in vitro medium that mimics human CF lung secretions (synthetic CF sputum medium [SCFM2]) and allows time-resolved assessment of fitness and community spatial structure at the micrometer scale. Our results reveal that P. aeruginosa and S. aureus coexist as spatially structured communities in SCFM2 under static growth conditions, with S. aureus enriched at a distance of 3.5 μm from P. aeruginosa. Multispecies aggregates were rare, and aggregate (biofilm) sizes resembled those in human CF sputum. Elimination of P. aeruginosa’s ability to produce the antistaphylococcal small molecule HQNO (2-heptyl-4-hydroxyquinoline N-oxide) had no effect on bacterial fitness but altered the spatial structure of the community by increasing the distance of S. aureus from P. aeruginosa to 7.6 μm. Lastly, we show that coculture with P. aeruginosa sensitizes S. aureus to killing by the antibiotic tobramycin compared to monoculture growth despite HQNO enhancing tolerance during coculture. Our findings reveal that SCFM2 is a powerful model for studying P. aeruginosa and S. aureus and that HQNO alters S. aureus biogeography and antibiotic susceptibility without affecting fitness.

Mesenchymal stem cells from bone marrow have powerful immunomodulatory capabilities. The interactions between jaw periosteal cells (JPCs) and macrophages are not only relevant for the application of JPCs in regenerative medicine, but this understanding could also help treating diseases like osteonecrosis of the jaw. In previous studies, we analyzed, for the first time, immunomodulatory features of 2D- and 3D-cultured JPCs. In the present work, the effects of JPCs on the polarization state of macrophages in contact coculture were analyzed. To improve the macrophage polarization study, different concentrations of PMA (5 nM, 25 nM, and 150 nM) or different medium supplementations (10% FBS, 10% hPL and 5% hPL) were compared. Further, in order to analyze the effects of JPCs on macrophage polarization, JPCs and PMA-stimulated THP-1 cells were cocultured under LPS/IFN-γ or IL-4/IL-13 stimulatory conditions. Surface marker expression of M1 and M2 macrophages were analyzed under the different culture supplementations in order to investigate the immunomodulatory properties of JPCs. Our results showed that 5 nM PMA can conduct an effective macrophage polarization. The analyses of morphological parameters and surface marker expression showed more distinct M1/M2 phenotypes over FBS supplementation when using 5% hPL during macrophage polarization. In the coculture, immunomodulatory properties of JPCs improved significantly under 5% hPL supplementation compared to other supplementations. We concluded that, under the culture condition with 5% hPL, JPCs were able to effectively induce THP-1-derived macrophage polarization.

Growing cells as 3D structures need not be difficult. Often, it is not necessary to change cell type, additives or growth media used. All that needs to be changed is the geometry: cells (whether primary, induced pluripotent, transformed or immortal) simply have to be grown in conditions that promote cell–cell adhesion while allowing gas, nutrient, signal, and metabolite exchange. Downstream analysis can become more complicated because many assays (like phase contrast microscopy) cannot be used, but their replacements have been in use for many years. Most importantly, there is a huge gain in value in obtaining data that is more representative of the organism in vivo. It is the goal of the protocols presented here to make the transition to a new dimension as painless as possible. Grown optimally, most biopsy derived organoids will retain patient phenotypes, while cell (both stem cell, induced or otherwise or immortalized) derived organoids or spheroids will recover tissue functionality.

Rheumatoid arthritis (RA) is a chronic, inflammatory, and systemic autoimmune disease that affects the connective tissue and primarily the joints. If not treated, RA ultimately leads to progressive cartilage and bone degeneration. The etiology of the pathogenesis of RA is unknown, demonstrating heterogeneity in its clinical presentation, and is associated with autoantibodies directed against modified self-epitopes. Although many models already exist for RA for preclinical research, many current model systems of arthritis have limited predictive value because they are either based on animals of phylogenetically distant origin or suffer from overly simplified in vitro culture conditions. These limitations pose considerable challenges for preclinical research and therefore clinical translation. Thus, a sophisticated experimental human-based in vitro approach mimicking RA is essential to (i) investigate key mechanisms in the pathogenesis of human RA, (ii) identify targets for new therapeutic approaches, (iii) test these approaches, (iv) facilitate the clinical transferability of results, and (v) reduce the use of laboratory animals. Here, we summarize the most commonly used in vitro models of RA and discuss their experimental feasibility and physiological proximity to the pathophysiology of human RA to highlight new human-based avenues in RA research to increase our knowledge on human pathophysiology and develop effective targeted therapies.

Osteosarcoma is among the most common cancers in young patients and is responsible for one-tenth of all cancer-related deaths in children. Surgery often leads to bone defects in excised tissue, while residual cancer cells may remain. Degradable magnesium alloys get increasing attention as orthopedic implants, and some studies have reported potential antitumor activity. However, most of the studies do not take the complex interaction between malignant cells and their surrounding stroma into account. Here, we applied a coculture model consisting of green fluorescent osteosarcoma cells and red fluorescent fibroblasts on extruded Mg and Mg&ndash, 6Ag with a tailored degradation rate. In contrast to non-degrading Ti-based material, both Mg-based materials reduced relative tumor cell numbers. Comparing the influence of the material on a sparse and dense coculture, relative cell numbers were found to be statistically different, thus relevant, while magnesium alloy degradations were observed as cell density-independent. We concluded that the sparse coculture model is a suitable mechanistic system to further study the antitumor effects of Mg-based material.

Objectives The effect of age on the response of peripheral blood mononuclear cells (PBMCs) to immunosuppression induced by human periodontal ligament stem cells (hPDLSCs) is unclear. The identity of the cytokines most effective in inducing the PBMC immune response remains unknown. This study investigated the effects of age on immunophenotype, proliferation, activation, and cytokine secretion capacities of PBMCs following co-culture with hPDLSCs. Methods PBMCs were collected from younger (16–19 years) and older (45–55 years) donors, then co-cultured with confirmed hPDLSCs for various lengths of time. T lymphocyte proliferation and cell surface marker expression were analyzed by flow cytometry. Cytokine expression levels were measured by quantitative polymerase chain reaction assays and enzyme-linked immunosorbent assays. Results CD28 expression by T lymphocytes decreased with age, indicating reduced proliferation; CD95 expression increased with age, indicating enhanced apoptosis. Moreover, hPDLSCs inhibited T lymphocyte proliferation in both age groups; this inhibition was stronger in cells from older donors than in cells from younger donors. Age reduced the secretion of interleukin-2 and interferon-γ, whereas it increased the secretion of tumor necrosis factor-β by PBMCs cultured with hPDLSCs. Conclusions Aging may have a robust effect on the response of PBMCs towards hPDLSC-induced immunosuppression.

For culture models of primary cells of the human nasal mucosa, monocultures with epithelial cells (ECs) are used as well as cocultures with ECs and fibroblasts (FBs). Well-differentiated models of the respiratory nasal epithelium can be used for ecogenotoxicological assessments, for experiments on host/pathogen interactions, or tissue engineering. However, long-term cultivation and repeated passaging may induce a loss of DNA integrity or cell functionality. The aim of this study was to evaluate these parameters in test systems created from primary nasal mucosa cells. Enzymatic and sequential cell isolation from nasal tissue was performed. EC monocultures and compartment-separated EC-FB cocultures were cultivated over three passages under air/liquid interface conditions. DNA stability and regenerative capacity at the DNA and chromosomal level as well as proliferation and cell differentiation were examined. Both methods showed equivalent levels of DNA stability and regenerative capacity over all passages. Sequential growth of the coculture provided higher cell purity, while enzymatic cell harvest was associated with FB contamination in EC culture. Mucociliary differentiation was verified with electron microscopy in both methods. Functionality measured by lipopolysaccharide stimulation of interleukins was constant over long-term cultivation. Our data confirm DNA stability in long-term cell cultivation as well as functional integrity in both culture methods. Sequential cell isolation should be favored over enzymatic isolation due to higher culture purity. Impact statement Cell culture models are frequently used for ecogenotoxicological assessments, for experiments on host/pathogen interactions, or tissue engineering. However, DNA stability and functional integrity after long-term cultivation in such tissue models have not been investigated, yet. This study is the first showing systematic and evident data on DNA damage and functional aspects in primary human cell culture models of nasal epithelium.

Resorbable synthetic scaffolds designed to regenerate living tissues and organs inside the body have emerged as a clinically attractive technology to replace diseased blood vessels. However, mismatches between scaffold design and in vivo hemodynamic loading (i.e., cyclic stretch and shear stress) can result in aberrant inflammation and adverse tissue remodeling, leading to premature graft failure. Yet, the underlying mechanisms remain elusive. Here, a human in vitro model is presented that mimics the transient local inflammatory and biomechanical environments that drive scaffold-guided tissue regeneration. The model is based on the coculture of human (myo)fibroblasts and macrophages in a bioreactor platform that decouples cyclic stretch and shear stress. Using a resorbable supramolecular elastomer as the scaffold material, it is revealed that cyclic stretch initially reduces proinflammatory cytokine secretion and, especially when combined with shear stress, stimulates IL-10 secretion. Moreover, cyclic stretch stimulates downstream (myo)fibroblast proliferation and matrix deposition. In turn, shear stress attenuates cyclic-stretch-induced matrix growth by enhancing MMP-1/TIMP-1-mediated collagen remodeling, and synergistically alters (myo)fibroblast phenotype when combined with cyclic stretch. The findings suggest that shear stress acts as a stabilizing factor in cyclic stretch-induced tissue formation and highlight the distinct roles of hemodynamic loads in the design of resorbable vascular grafts.

Alongside in vivo models, a simpler and more mechanistic approach is required to study the effects of myostatin on skeletal muscle because myostatin is an important negative regulator of muscle size. In this study, myostatin was administered to murine (C2C12) and human (CHQ) myoblasts and myotubes. Canonical and noncanonical signaling downstream to myostatin, related ligands, and their receptor were analyzed. The effects of tumorkines were analyzed after coculture of C2C12 and colon cancer-C26 cells. The effects of myostatin on canonical and noncanonical signaling were strongly reduced in C2C12 cells after differentiation. This may be explained by increased follistatin, an endogenous blocker of myostatin and altered expression of activin receptor ligands. In contrast, CHQ cells were equally responsive to myostatin, and follistatin remained unaltered. Both myostatin administration and the coculture stimulated pathways associated with inflammation, especially in C2C12 cells. In conclusion, the effects of myostatin on intracellular signaling may be cell line- or organism-specific, and C2C12 myotubes seem to be a nonoptimal in vitro model for investigating the effects of myostatin on canonical and noncanonical signaling in skeletal muscle. This may be due to altered expression of activin receptor ligands and their regulators during muscle cell differentiation.

Carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), a transmembrane glycoprotein, has multiple functions. In tongue squamous cell carcinoma (TSCC), CEACAM1 overexpression is correlated with neutrophil infiltration, and both are associated with poor clinical outcomes. However, the mechanism underlying CEACAM1's effect on neutrophil function in TSCC remains unclear. We cocultured tongue carcinoma cells overexpressing CEACAM1‐4L, CEACAM1‐4S and differentiated HL‐60 cells. This significantly upregulated the expression of MMP‐9, interleukin 8, and VEGF‐A in the differentiated HL‐60 cells and downregulated the expression of TNF‐α, relative to vector and blank control groups (P

The obligate intracellular bacterium, Chlamydia pneumoniae, has been identified as a risk factor for several chronic inflammatory diseases in addition to respiratory tract infections. The dissemination of C. pneumoniae from respiratory tract to secondary sites of infection occurs via infected monocyte / macrophage line cells, in which C. pneumoniae can persist as an antibiotic-refractory phenotype. To allow more detailed studies on the epithelium-monocyte/macrophage transition of the infection, new in vitro bioassays are needed. To this end, a coculture system with human continuous cell lines was established. Respiratory epithelial HL cells were infected with C. pneumoniae and THP-1 monocytes were added into the cultures at 67 h post infection. After a 5 h coculture, THP-1 cells were collected with a biotinylated HLA antibody and streptavidin-coated magnetic beads and C. pneumoniae genome copy numbers in THP-1 determined by quantitative PCR. The assay was optimized for cell densities, incubation time, THP-1 separation technique and buffer composition, and its robustness was demonstrated by a Z' value of 0.6. The mitogen-activated protein kinase (MAPK) inhibitors: SP600125 (JNK inhibitor), SB203580 (p38 inhibitor) and FR180204 (ERK inhibitor) suppressed the transfer of C. pneumoniae from HL to THP-1 cells, making them suitable positive controls for the assay. Based on analysis of separate steps of the process, the MAPK inhibitors suppress the bacterial entry to THP-1 cells. The transfer of C. pneumoniae from epithelium to phagocytes represents a crucial step in the establishment of persistent infections by this pathogen, and the presented methods enables future studies to block this process by therapeutic means.

15-lipoxygenase (15-LOX) is a critical enzyme that allows the direction of arachidonic acid metabolism to change from inflammation into the resolution. This study aims to reveal how the immunomodulation properties of mesenchymal stem cells (MSC) alter by the 15-LOX overexpression. For this purpose, peripheral blood mononuclear cells (PBMCs) isolated from seven healthy volunteers, and both MSCs and 15-LOX overexpressing MSCs (15-LOXMSCs) were co-cultured at different cell ratios (1/1, 1/5 and 1/10). Alterations of CD4+Tbet+, CD4+Gata3+, CD4+RoRC2+, and CD4+FoxP3+ lymphocyte frequencies were detected by flow cytometry, and IFN-?, IL-4, IL-6, IL-10, IL-17a, TGF-? and LXA4 levels of medium supernatants were measured by ELISA method. According to our findings, MSC and 15-LOXMSCs have a suppressive effect on PHA activated PBMCs. However, as the ratio of PBMCs increased, the effects of 15-LOXMSCs increased significantly, while the effects of MSCs decreased. The most notable effect of the 15-LOX modification was the significant reduction in IL-6, IL-10 and IL-17a expression and the accompanying increase in TGF-? and LXA4 levels. We also observed a similar situation between CD4+RoRC2+ and CD4+FoxP3+ cell frequencies. These data suggested that the effects of MSCs on the balance of Th17 / Treg could change by the 15-LOX overexpression, and this might be in favor of the Treg cells. © 2021, 16-TIP-041, The Scientific Research Projects Commission of Ege University financially supported the project for this study, with the reference number 16-TIP-041 .

The cardiac autonomic nervous system (cANS) regulates cardiac function by innervating cardiac tissue with axons, and cardiomyocytes (CMs) and neurons undergo comaturation during the heart innervation in embryogenesis. As cANS is essential for cardiac function, its dysfunctions might be fatal; therefore, cardiac innervation models for studying embryogenesis, cardiac diseases, and drug screening are needed. However, previously reported neuron-cardiomyocyte (CM) coculture chips lack studies of functional neuron–CM interactions with completely human-based cell models. Here, we present a novel completely human cell-based and electrophysiologically functional cardiac innervation on a chip in which a compartmentalized microfluidic device, a 3D3C chip, was used to coculture human induced pluripotent stem cell (hiPSC)-derived neurons and CMs. The 3D3C chip enabled the coculture of both cell types with their respective culture media in their own compartments while allowing the neuronal axons to traverse between the compartments via microtunnels connecting the compartments. Furthermore, the 3D3C chip allowed the use of diverse analysis methods, including immunocytochemistry, RT-qPCR and video microscopy. This system resembled the in vivo axon-mediated neuron–CM interaction. In this study, the evaluation of the CM beating response during chemical stimulation of neurons showed that hiPSC-neurons and hiPSC-CMs formed electrophysiologically functional axon-mediated interactions. publishedVersion

The current study used an ex vivo [embryonic day (E)18] chick femur defect model to examine the bone regenerative capacity of implanted 3-dimensional (3D) skeletal-endothelial cell constructs. Human bone marrow stromal cell (HBMSC) and HUVEC spheroids were implanted within a bone defect site to determine the osteogenic potential of the skeletal-endothelial cell unit. Cells were pelleted as co- or monocell spheroids and placed within 1-mm-drill defects in the mid-diaphysis of E18 chick femurs and cultured organotypically for 10 d. Micro-computed tomography analysis revealed significantly ( P = 0.0001) increased levels of bone volume (BV) and BV/tissue volume ratio in all cell-pellet groups compared with the sham defect group. The highest increase was seen in BV in femurs containing the HUVEC and HBMSC monocell constructs. Type II collagen expression was particularly pronounced within the cell spheres containing HBMSCs and HUVECs, and CD31-positive cell clusters were prominent within HUVEC-implanted defects. These studies demonstrate the importance of the 3D osteogenic-endothelial niche interaction in bone regeneration. Elucidating the component cell interactions in the osteogenic-vascular niche and the role of exogenous factors in driving these osteogenic processes will aid the development of better bone reparative strategies.-Inglis, S., Kanczler, J. M., Oreffo, R. O. C. 3D human bone marrow stromal and endothelial cell spheres promote bone healing in an osteogenic niche.

Acute ischaemia causes a significant loss of blood vessels leading to deterioration of organ function. Multiple ischaemic conditions are associated with up‐regulation of activin A, but its effect on endothelial cells (EC) in the context of hypoxia is understudied. This study evaluated the role of activin A in vasculogenesis in hypoxia. An in vitro vasculogenesis model, in which EC were cocultured with adipose stromal cells (ASC), was used. Incubation of cocultures at 0.5% oxygen led to decrease in EC survival and vessel density. Hypoxia up‐regulated inhibin BA (monomer of activin A) mRNA by 4.5‐fold and activin A accumulation in EC‐conditioned media by 10‐fold, but down‐regulated activin A inhibitor follistatin by twofold. Inhibin BA expression was also increased in human EC injected into ischaemic mouse muscles. Activin A secretion was positively modulated by hypoxia mimetics dimethyloxalylglycine and desferrioxamine. Silencing HIF1α or HIF2α expression decreased activin A secretion in EC exposed to hypoxia. Introduction of activin A to cocultures decreased EC number and vascular density by 40%; conversely, blockade of activin A expression in EC or its activity improved vasculogenesis in hypoxia. Activin A affected EC survival directly and by modulating ASC paracrine activity leading to diminished ability of the ASC secretome to support EC survival and vasculogenesis. In conclusion, hypoxia up‐regulates EC secretion of activin A, which, by affecting both EC and adjacent mesenchymal cells, creates a micro‐environment unfavourable for vasculogenesis. This finding suggests that blockade of activin A signalling in ischaemic tissue may improve preservation of the affected tissue.

Malassezia spp. are part of the normal human and animal mycobiota but are also associated with a variety of dermatological diseases. The absence of a transformation system hampered studies to reveal mechanisms underlying the switch from the non-pathogenic to pathogenic life style. Here we describe, a highly efficient Agrobacterium-mediated genetic transformation system for Malassezia furfur and M. pachydermatis. A binary T-DNA vector with the hygromycin B phosphotransferase (hpt) selection marker and the green fluorescent protein gene (gfp) was introduced in M. furfur and M. pachydermatis by combining the transformation protocols of Agaricus bisporus and Cryptococcus neoformans. Optimal temperature and co-cultivation time for transformation were 5 and 7 days at 19 °C and 24 °C, respectively. Transformation efficiency was 0.75–1.5% for M. furfur and 0.6–7.5% for M. pachydermatis. Integration of the hpt resistance cassette and gfp was verified using PCR and fluorescence microscopy, respectively. The T-DNA was mitotically stable in approximately 80% of the transformants after 10 times sub-culturing in the absence of hygromycin. Improving transformation protocols contribute to study the biology and pathophysiology of Malassezia.

Here we present a blood-brain barrier (BBB) model that enables high-resolution imaging of nanoparticle (NP) interactions with endothelial cells and the capture of rare NP translocation events. The enabling technology is an ultrathin silicon nitride (SiN) membrane (0.5 μm pore size, 20% porosity, 400 nm thickness) integrated into a dual-chamber platform that facilitates imaging at low working distances (∼50 μm). The platform, the μSiM-BBB (microfluidic silicon membrane-BBB), features human brain endothelial cells and primary astrocytes grown on opposite sides of the membrane. The human brain endothelial cells form tight junctions on the ultrathin membranes and exhibit a significantly higher resistance to FITC-dextran diffusion than commercial membranes. The enhanced optical properties of the SiN membrane allow high-resolution live-cell imaging of three types of NPs, namely, 40 nm PS-COOH, 100 nm PS-COOH, and apolipoprotein E-conjugated 100 nm SiO2, interacting with the BBB. Despite the excellent barrier properties of the endothelial layer, we are able to document rare NP translocation events of NPs localized to lysosomal compartments of astrocytes on the "brain side" of the device. Although the translocation is always low, our data suggest that size and targeting ligand are important parameters for NP translocation across the BBB. As a platform that enables the detection of rare transmission across tight BBB layers, the μSiM-BBB is an important tool for the design of nanoparticle-based delivery of drugs to the central nervous system. European Commission - Seventh Framework Programme (FP7) Science Foundation Ireland -- replace EuroNanoMed III National Natural Science Foundation Of China SFI-NSFC Partnership Programme NIH Lundbeck Foundation 2021-05-11 JG: PDF replaced with correct version

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory bulb. Throughout adult life, they are key for axonal growing of newly generated olfactory neurons, from the lamina propria to the ONL. Due to their pro-regenerative properties, these cells have been used to foster axonal regeneration in spinal cord or optic nerve injury models. We present an in vitro model to assay and measure OEG neuroregenerative capacity after neural injury. In this model, reversibly immortalized human OEG (ihOEG) is cultured as a monolayer, retinas are extracted from adult rats and retinal ganglion neurons (RGN) are cocultured onto the OEG monolayer. After 96 h, axonal and somatodendritic markers in RGNs are analyzed by immunofluorescence and the number of RGNs with axon and the mean axonal length/neuron are quantified. This protocol has the advantage over other in vitro assays that rely on embryonic or postnatal neurons, that it evaluates OEG neuroregenerative properties in adult tissue. Also, it is not only useful for assessing the neuroregenerative potential of ihOEG but can be extended to different sources of OEG or other glial cells.

Impact statement This work demonstrates an effective method of enhancing osteogenesis of mesenchymal stromal cells on electrospun scaffolds through coculturing with endothelial cells. Furthermore, we provide the optimized conditions for cocultures on electrospun fibrous scaffolds and engineered bone tissues with oriented topography on aligned fibers. This study demonstrates promising findings for growing oriented tissue-engineered cocultures with significant increase in osteogenesis over monoculture conditions.Vascularization is a critical process during bone regeneration. The lack of vascular networks leads to insufficient oxygen and nutrients supply, which compromises the survival of regenerated bone. One strategy for improving the survival and osteogenesis of tissue-engineered bone grafts involves the coculture of endothelial cells (ECs) with mesenchymal stromal cells (MSCs). Moreover, bone regeneration is especially challenging due to its unique structural properties with aligned topographical cues, with which stem cells can interact. Inspired by the aligned fibrillar nanostructures in human cancellous bone, we fabricated polycaprolactone (PCL) electrospun fibers with aligned and random morphology, cocultured human MSCs with human umbilical vein ECs (HUVECs), and finally investigated how these two factors modulate osteogenic differentiation of human MSCs (hMSCs). After optimizing cell ratio, a hMSCs/HUVECs ratio (90:10) was considered to be the best combination for osteogenic differentiation. Coculture results showed that hMSCs and HUVECs adhered to and proliferated well on both scaffolds. The aligned structure of PCL fibers strongly influenced the morphology and orientation of hMSCs and HUVECs; however, fiber alignment was observed to not affect alkaline phosphate (ALP) activity or mineralization of hMSCs compared with random scaffolds. More importantly, cocultured cells on both random and aligned scaffolds had significantly higher ALP activities than monoculture groups, which indicated that coculture with HUVECs provided a larger relative contribution to the osteogenesis of hMSCs compared with fiber alignment. Taken together, we conclude that coculture of hMSCs with ECs is an effective strategy to promote osteogenesis on electrospun scaffolds, and aligned fibers could be introduced to regenerate bone tissues with oriented topography without significant deleterious effects on hMSCs differentiation. This study shows the ability to grow oriented tissue-engineered cocultures with significant increases in osteogenesis over monoculture conditions.

Vascular pericytes are an important cellular component in the tumor microenvironment, however, their role in supporting cancer invasion is poorly understood. We hypothesized that PDGF-BB could be involved in the transition of human retinal pericytes (HRPC) in cancer-activated fibroblasts (CAF), induced by the 92.1 uveal melanoma (UM) cell line. In our model system, HRPC were conditioned by co-culturing with 92.1UM for 6 days (cHRPC), in the presence or absence of imatinib, to block PDGF receptor-&beta, (PDGFR&beta, ). The effects of the treatments were tested by wound healing assay, proliferation assay, RT-PCR, high-content screening, Western blot analysis, and invasion assay. Results showed profound changes in cHRPC shape, with increased proliferation and motility, reduction of NG2 and increase of TGF-&beta, 1, &alpha, SMA, vimentin, and FSP-1 protein levels, modulation of PDGF isoform mRNA levels, phospho-PDGFR&beta, and PDGFR&beta, as well as phospho-STAT3 increases. A reduction of IL-1&beta, and IFN&gamma, and an increase in TNF&alpha, IL10, and TGF-&beta, 1, CXCL11, CCL18, and VEGF mRNA in cHRPC were found. Imatinib was effective in preventing all the 92.1UM-induced changes. Moreover, cHRPC elicited a significant increase of 92.1UM cell invasion and active MMP9 protein levels. Our data suggest that retinal microvascular pericytes could promote 92.1UM growth through the acquisition of the CAF phenotype.

Transcription factor (TF)‐based reprogramming of somatic tissues holds great promise for regenerative medicine. Previously, we demonstrated that the TFs GATA2, GFI1B, and FOS convert mouse and human fibroblasts to hemogenic endothelial‐like precursors that generate hematopoietic stem progenitor (HSPC)‐like cells over time. This conversion is lacking in robustness both in yield and biological function. Herein, we show that inclusion of GFI1 to the reprogramming cocktail significantly expands the HSPC‐like population. AFT024 coculture imparts functional potential to these cells and allows quantification of stem cell frequency. Altogether, we demonstrate an improved human hemogenic induction protocol that could provide a valuable human in vitro model of hematopoiesis for disease modeling and a platform for cell‐based therapeutics., FEBS Letters, 593 (23), ISSN:0014-5793, ISSN:1873-3468

Human dental pulp stem cells (hDPSCs) present several advantages, including their ability to be non‑invasively harvested without ethical concern. The secretome of hDPSCs can promote the functional recovery of various tissue injuries. However, the protective effects on hypoxia‑induced skeletal muscle injury remain to be explored. The present study demonstrated that C2C12 myoblast coculture with hDPSCs attenuated CoCl2‑induced hypoxic injury compared with C2C12 alone. The hDPSC secretome increased cell viability and differentiation and decreased G2/M cell cycle arrest under hypoxic conditions. These results were further verified using hDPSC‑conditioned medium (hDPSC‑CM). The present data revealed that the protective effects of hDPSC‑CM depend on the concentration ratio of the CM. In terms of the underlying molecular mechanism, hDPSC‑CM activated the Wnt/β‑catenin pathway, which increased the protein levels of Wnt1, phosphorylated‑glycogen synthase kinase‑3β and β‑catenin and the mRNA levels of Wnt target genes. By contrast, an inhibitor (XAV939) of Wnt/β‑catenin diminished the protective effects of hDPSC‑CM. Taken together, the findings of the present study demonstrated that the hDPSC secretome alleviated the hypoxia‑induced myoblast injury potentially through regulating the Wnt/β‑catenin pathway. These findings may provide new insight into a therapeutic alternative using the hDPSC secretome in skeletal muscle hypoxia‑related diseases.

The last decade has been marked by two eminent discoveries that have changed our perception of the virology field: The discovery of giant viruses and a distinct new class of viral agents that parasitize their viral factories, the virophages. Coculture and metagenomics have actively contributed to the expansion of the virophage family by isolating dozens of new members. This increase in the body of data on virophage not only revealed the diversity of the virophage group, but also the relevant ecological impact of these small viruses and their potential role in the dynamics of the microbial network. In addition, the isolation of virophages has led us to discover previously unknown features displayed by their host viruses and cells. In this review, we present an update of all the knowledge on the isolation, biology, genomics, and morphological features of the virophages, a decade after the discovery of their first member, the Sputnik virophage. We discuss their parasitic lifestyle as bona fide viruses of the giant virus factories, genetic parasites of their genomes, and then their role as a key component or target for some host defense mechanisms during the tripartite virophage−giant virus−host cell interaction. We also present the latest advances regarding their origin, classification, and definition that have been widely discussed.

Using proteomics analysis, we previously compared MCF7 breast cancer cells grown as 3D tumor spheres, with the same cell line grown as monolayers. Our results indicated that during 3D anchorage-independent growth, the cellular machinery associated with i) mitochondrial biogenesis and ii) ribosomal biogenesis, were both significantly increased. Here, for simplicity, we refer to these two new oncogenic hallmarks as "mito-stemness" and "ribo-stemness" features. We have now applied this same type of strategy to begin to understand how fibroblasts and MCF7 breast cancer cells change their molecular phenotype, when they are co-cultured together. We have previously shown that MCF7-fibroblast co-cultures are a valuable model of resistance to apoptosis induced by hormonal therapies, such as Tamoxifen and Fulvestrant. Here, we directly show that these mixed co-cultures demonstrate the induction of mito-stemness and ribo-stemness features, likely reflecting a mechanism for cancer cells to increase their capacity for accumulating biomass. In accordance with the onset of a stem-like phenotype, KRT19 (keratin 19) was induced by ~6-fold during co-culture. KRT19 is a well-established epithelial CSC marker that is used clinically to identify metastatic breast cancer cells in sentinel lymph node biopsies. The potential molecular therapeutic targets that we identified by label-free proteomics of MCF7-fibroblast co-cultures were then independently validated using a bioinformatics approach. More specifically, we employed publically-available transcriptional profiling data derived from primary tumor samples from breast cancer patients, which were previously subjected to laser-capture micro-dissection, to physically separate breast cancer cells from adjacent tumor stroma. This allowed us to directly validate that the proteins up-regulated in this co-culture model were also transcriptionally elevated in patient-derived breast cancer cells in vivo. This powerful approach for target identification and translational validation, including the use of patient outcome data, can now be applied to other tumor types as well, to validate new therapeutic targets that are more clinically relevant, for patient benefit. Moreover, we discuss the therapeutic implications of these findings for new drug development, drug repurposing and Tamoxifen-resistance, to effectively target mito-stemness and ribo-stemness features in breast cancer patients. We also discuss the broad implications of this "organelle biogenesis" approach to cancer therapy.

The liver possesses a unique microenvironment with a complex internal vascular system and cell-cell interactions. Nonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease, and although much effort has been dedicated to building models to target NAFLD, most in vitro systems rely on simple models failing to recapitulate complex liver functions. Here, an in vitro system is presented to study NAFLD (steatosis) by coculturing human hepatocellular carcinoma (HepG2) cells and umbilical vein endothelial cells (HUVECs) into spheroids. Analysis of colocalization of HepG2-HUVECs along with the level of steatosis reveals that the NAFLD pathogenesis could be better modeled when 20% of HUVECs are presented in HepG2 spheroids. Spheroids with fat supplements progressed to the steatosis stage on day 2, which could be maintained for more than a week without being harmful for cells. Transferring spheroids onto a chip system with an array of interconnected hexagonal microwells proves helpful for monitoring functionality through increased albumin secretions with HepG2-HUVEC interactions and elevated production of reactive oxygen species for steatotic spheroids. The reversibility of steatosis is demonstrated by simply stopping fat-based diet or by antisteatotic drug administration, the latter showing a faster return of intracellular lipid levels to the basal level.

In vitro digestions are essential for determining the bioavailability of compounds, such as nutrients. We have developed a cell-free, miniaturized enzymatic digestive system, employing three micromixers connected in series to mimic the digestive functions of the mouth, stomach and small intestine. This system continuously processes samples, e.g. containing nutrients, to provide a constant flow of digested materials which may be presented to a subsequent gut-on-a-chip absorption module, containing living human intestinal cells. Our system incorporates three-compartment enzymatic digestion, one of the key functions of the gastrointestinal tract. In each of these compartments, we modify the chemical environment, including pH, buffer, and mineral composition, to closely mimic the local physiological environment and create optimal conditions for digestive processes to take place. It will therefore provide an excellent addition to existing gut-on-a-chip systems, providing the next step in determining the bio-availability of orally administered compounds in a fast and continuous-flow ex vivo system. In this paper, we demonstrate enzymatic digestion in each separate compartment using compounds, starch and casein, as model nutrients. The use of transparent, microfluidic micromixers based on chaotic advection, which can be probed directly with a microscope, enabled enzyme kinetics to be monitored from the very start of a reaction. Furthermore, we have digested lactoferrin in our system, demonstrating complete digestion of this milk protein in much shorter times than achievable with standard in vitro digestions using batch reactors.

Background The use of in vitro cell cultures is a powerful tool for obtaining key insights into the behaviour and response of cells to interventions in normal and disease situations. Unlike in vivo settings, in vitro experiments allow a fine-tuned control of a range of microenvironmental elements independently within an isolated setting. The recent expansion in the use of three-dimensional (3D) in vitro assays has created a number of representative tools to study cell behaviour in a more physiologically 3D relevant microenvironment. Complex 3D in vitro models that can recapitulate human tissue biology are essential for understanding the pathophysiology of disease. Aim The development of the 3D coculture collagen contraction and invasion assay, the ?organotypic assay,? has been widely adopted as a powerful approach to bridge the gap between standard two-dimensional tissue culture and in vivo mouse models. In the cancer setting, these assays can then be used to dissect how stromal cells, such as cancer-associated fibroblasts (CAFs), drive extracellular matrix (ECM) remodelling to alter cancer cell behaviour and response to intervention. However, to date, many of the published organotypic protocols are low-throughput, time-consuming (up to several weeks), and work-intensive with often limited scalability. Our aim was to develop a fast, high-throughput, scalable 3D organotypic assay for use in oncology screening and drug development. Methods and results Here, we describe a modified 96-well organotypic assay, the ?Mini-Organo,? which can be easily completed within 5 days. We demonstrate its application in a wide range of mouse and human cancer biology approaches including evaluation of stromal cell 3D ECM remodelling, 3D cancer cell invasion, and the assessment of efficacy of potential anticancer therapeutic targets. Furthermore, the organotypic assay described is highly amenable to customisation using different cell types under diverse experimental conditions. Conclusions The Mini-Organo high-throughput 3D organotypic assay allows the rapid screening of potential cancer therapeutics in human and mouse models in a time-efficient manner. (Less)

Rationale: Cardiovascular diseases often cause substantial heart damage and even heart failure due to the limited regenerative capacity of adult cardiomyocytes. The direct cardiac reprogramming of fibroblasts could be a promising therapeutic option for these patients. Although exogenous transcriptional factors can induce direct cardiac reprogramming, the reprogramming efficiency is too low to be used clinically. Herein, we introduce a cardiac-mimetic cell-culture system that resembles the microenvironment in the heart and provides interactions with cardiomyocytes and electrical cues to the cultured fibroblasts for direct cardiac reprogramming. Methods: Nano-thin and nano-porous membranes and heart like electric stimulus were used in the cardiac-mimetic cell-culture system. The human neonatal dermal fibroblasts containing cardiac transcription factors were plated on the membrane and cultured with the murine cardiomyocyte in the presence of the electric stimulus. The reprogramming efficiency was evaluated by qRT-PCR and immunocytochemistry. Results: Nano-thin and nano-porous membranes in the culture system facilitated interactions between fibroblasts and cardiomyocytes in coculture. The cellular interactions and electric stimulation supplied by the culture system dramatically enhanced the cardiac reprogramming efficiency of cardiac-specific transcriptional factor-transfected fibroblasts. Conclusion: The cardiac-mimetic culture system may serve as an effective tool for producing a feasible number of reprogrammed cardiomyocytes from fibroblasts.

Coculture systems employing adipose tissue-derived mesenchymal stromal/stem cells (ASC) and endothelial cells (EC) represent a widely used technique to model vascularization. Within this system, cell-cell communication is crucial for the achievement of functional vascular network formation. Extracellular vesicles (EVs) have recently emerged as key players in cell communication by transferring bioactive molecules between cells. In this study we aimed to address the role of EVs in ASC/EC cocultures by discriminating between cells, which have received functional EV cargo from cells that have not. Therefore, we employed the Cre-loxP system, which is based on donor cells expressing the Cre recombinase, whose mRNA was previously shown to be packaged into EVs and reporter cells containing a construct of floxed dsRed upstream of the eGFP coding sequence. The evaluation of Cre induced color switch in the reporter system via EVs indicated that there is no EV-mediated RNA transmission either between EC themselves or EC and ASC. However, since Cre mRNA was not found present in EVs, it remains unclear if Cre mRNA is generally not packaged into EVs or if EVs are not taken up by the utilized cell types. Our data indicate that this technique may not be applicable to evaluate EV-mediated cell-to-cell communication in an in vitro setting using EC and ASC. Further investigations will require a functional system showing efficient and specific loading of Cre mRNA or protein into EVs. This research was partially funded by the Austrian Research promotion agency. Severin Mühleder was funded by the Austrian Science Fund (FWF). Sí