Advanced Online Monitoring of In Vitro Human 3D Full-Thickness Skin Equivalents.

Skin equivalents and skin explants are widely used for dermal penetration studies in the pharmacological development of drugs. Environmental parameters, such as the incubation and culture conditions affect cellular responses and thus the relevance of the experimental outcome. However, available systems such as the Franz diffusion chamber, only measure in the receiving culture medium, rather than assessing the actual conditions for cells in the tissue. We developed a sampling design that combines open flow microperfusion (OFM) sampling technology for continuous concentration measurements directly in the tissue with microfluidic biosensors for online monitoring of culture parameters. We tested our design with real-time measurements of oxygen, glucose, lactate, and pH in full-thickness skin equivalent and skin explants. Furthermore, we compared dermal penetration for acyclovir, lidocaine, and diclofenac in skin equivalents and skin explants. We observed differences in oxygen, glucose, and drug concentrations in skin equivalents compared to the respective culture medium and to skin explants.

Heterogeneity of mesenchymal stem cell-derived extracellular vesicles is highly impacted by the tissue/cell source and culture conditions.

Extracellular vesicles (EVs) are cell-derived membrane structures exerting major effects in physiological as well as pathological processes by functioning as vehicles for the delivery of biomolecules to their target cells. An increasing number of effects previously attributed to cell-based therapies have been recognized to be actually mediated by EVs derived from the respective cells, suggesting the administration of purified EVs instead of living cells for cell-based therapies. In this review, we focus on the heterogeneity of EVs derived from mesenchymal stem/stromal cells (MSC) and summarize upstream process parameters that crucially affect the resulting therapeutic properties and biological functions. Hereby, we discuss the effects of the cell source, medium composition, 3D culture, bioreactor culture and hypoxia. Furthermore, aspects of the isolation and storage strategies influences EVs are described. Conclusively, optimization of upstream process parameters should focus on controlling MSC-derived EV heterogeneity for specific therapeutic applications.

Innovative Platform for the Advanced Online Monitoring of Three-Dimensional Cells and Tissue Cultures.

The use of 3D cell cultures has gained increasing importance in medical and pharmaceutical research. However, the analysis of the culture medium is hardly representative for the culture conditions within a 3D model which hinders the standardization of 3D cultures and translation of results. Therefore, we developed a modular monitoring platform combining a perfusion bioreactor with an integrated minimally invasive sampling system and implemented sensors that enables the online monitoring of culture parameters and medium compounds within 3D cultures. As a proof-of-concept, primary cells as well as cell lines were cultured on a collagen or gelatin methacryloyl (GelMA) hydrogel matrix, while monitoring relevant culture parameters and analytes. Comparing the interstitial fluid of the 3D models versus the corresponding culture medium, we found considerable differences in the concentrations of several analytes. These results clearly demonstrate that analyses of the culture medium only are not relevant for the development of standardized 3D culture processes. The presented bioreactor with an integrated sampling and sensor platform opens new horizons for the development, optimization, and standardization of 3D cultures. Furthermore, this technology holds the potential to reduce animal studies and improve the transferability of pharmaceutical in vitro studies by gaining more relevant results, bridging the gap towards clinical translation.

Towards More Predictive, Physiological and Animal-free In Vitro Models: Advances in Cell and Tissue Culture 2020 Conference Proceedings.

Experimental systems that faithfully replicate human physiology at cellular, tissue and organ level are crucial to the development of efficacious and safe therapies with high success rates and low cost. The development of such systems is challenging and requires skills, expertise and inputs from a diverse range of experts, such as biologists, physicists, engineers, clinicians and regulatory bodies. Kirkstall Limited, a biotechnology company based in York, UK, organised the annual conference, Advances in Cell and Tissue Culture (ACTC), which brought together people having a variety of expertise and interests, to present and discuss the latest developments in the field of cell and tissue culture and in vitro modelling. The conference has also been influential in engaging animal welfare organisations in the promotion of research, collaborative projects and funding opportunities. This report describes the proceedings of the latest ACTC conference, which was held virtually on 30th September and 1st October 2020, and included sessions on in vitro models in the following areas: advanced skin and respiratory models, neurological disease, cancer research, advanced models including 3-D, fluid flow and co-cultures, diabetes and other age-related disorders, and animal-free research. The roundtable session on the second day was very interactive and drew huge interest, with intriguing discussion taking place among all participants on the theme of replacement of animal models of disease.

Towards Physiologic Culture Approaches to Improve Standard Cultivation of Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are of great interest for their use in cell-based therapies due to their multipotent differentiation and immunomodulatory capacities. In consequence of limited numbers following their isolation from the donor tissue, MSCs require extensive expansion performed in traditional 2D cell culture setups to reach adequate amounts for therapeutic use. However, prolonged culture of MSCs in vitro has been shown to decrease their differentiation potential and alter their immunomodulatory properties. For that reason, preservation of these physiological characteristics of MSCs throughout their in vitro culture is essential for improving the efficiency of therapeutic and in vitro modeling applications. With this objective in mind, many studies already investigated certain parameters for enhancing current standard MSC culture protocols with regard to the effects of specific culture media components or culture conditions. Although there is a lot of diversity in the final therapeutic uses of the cells, the primary stage of standard isolation and expansion is imperative. Therefore, we want to review on approaches for optimizing standard MSC culture protocols during this essential primary step of in vitro expansion. The reviewed studies investigate and suggest improvements focused on culture media components (amino acids, ascorbic acid, glucose level, growth factors, lipids, platelet lysate, trace elements, serum, and xenogeneic components) as well as culture conditions and processes (hypoxia, cell seeding, and dissociation during passaging), in order to preserve the MSC phenotype and functionality during the primary phase of in vitro culture.

3D Printing of Cell Culture Devices: Assessment and Prevention of the Cytotoxicity of Photopolymers for Stereolithography.

3D printing is increasingly important for the rapid prototyping of advanced and tailor-made cell culture devices. In this context, stereolithography represents a method for the rapid generation of prototypes from photocurable polymers. However, the biocompatibility of commercially available photopolymers is largely unknown. Therefore, we evaluated the cytotoxicity of six polymers, two of them certified as biocompatible according to ISO 10993-5:2009, and we evaluated, if coating with Parylene, an inert polymer widely used in medical applications, might shield cells from the cytotoxic effects of a toxic polymer. In addition, we evaluated the processability, reliability, and consistency of the details printed. Human mesenchymal stem cells (MSCs) were used for cytotoxicity testing as they are widely used and promising for numerous applications in regenerative medicine. MSCs were incubated together with printed photopolymers, and the cytotoxicity was assessed. All photopolymers significantly reduced the viability of MSCs while the officially biocompatible resins displayed minor toxic effects. Further, coating with Parylene completely protected MSCs from toxic effects. In conclusion, none of the tested polymers can be fully recommended for rapid prototyping of cell culture devices. However, coating with Parylene can shield cells from toxic effects and thus might represent a viable option until more compatible materials are available.

Matrix decoded - A pancreatic extracellular matrix with organ specific cues guiding human iPSC differentiation.

The extracellular matrix represents a dynamic microenvironment regulating essential cell functions in vivo. Tissue engineering approaches aim to recreate the native niche in vitro using biological scaffolds generated by organ decellularization. So far, the organ specific origin of such scaffolds was less considered and potential consequences for in vitro cell culture remain largely elusive. Here, we show that organ specific cues of biological scaffolds affect cellular behavior. In detail, we report on the generation of a well-preserved pancreatic bioscaffold and introduce a scoring system allowing standardized inter-study quality assessment. Using multiple analysis tools for in-depth-characterization of the biological scaffold, we reveal unique compositional, physico-structural, and biophysical properties. Finally, we prove the functional relevance of the biological origin by demonstrating a regulatory effect of the matrix on multi-lineage differentiation of human induced pluripotent stem cells emphasizing the significance of matrix specificity for cellular behavior in artificial microenvironments.

Human Pluripotent Stem Cell-Derived Multipotent Vascular Progenitors of the Mesothelium Lineage Have Utility in Tissue Engineering and Repair.

In this report we describe a human pluripotent stem cell-derived vascular progenitor (MesoT) cell of the mesothelium lineage. MesoT cells are multipotent and generate smooth muscle cells, endothelial cells, and pericytes and self-assemble into vessel-like networks in vitro. MesoT cells transplanted into mechanically damaged neonatal mouse heart migrate into the injured tissue and contribute to nascent coronary vessels in the repair zone. When seeded onto decellularized vascular scaffolds, MesoT cells differentiate into the major vascular lineages and self-assemble into vasculature capable of supporting peripheral blood flow following transplantation. These findings demonstrate in vivo functionality and the potential utility of MesoT cells in vascular engineering applications.

Evaluation of a Miniaturized Biologically Vascularized Scaffold in vitro and in vivo.

In tissue engineering, the generation and functional maintenance of dense voluminous tissues is mainly restricted due to insufficient nutrient supply. Larger three-dimensional constructs, which exceed the nutrient diffusion limit become necrotic and/or apoptotic in long-term culture if not provided with an appropriate vascularization. Here, we established protocols for the generation of a pre-vascularized biological scaffold with intact arterio-venous capillary loops from rat intestine, which is decellularized under preservation of the feeding and draining vascular tree. Vessel integrity was proven by marker expression, media/blood reflow and endothelial LDL uptake. In vitro maintenance persisted up to 7 weeks in a bioreactor system allowing a stepwise reconstruction of fully vascularized human tissues and successful in vivo implantation for up to 4 weeks, although with time-dependent decrease of cell viability. The vascularization of the construct lead to a 1.5× increase in cellular drug release compared to a conventional static culture in vitro. For the first time, we performed proof-of-concept studies demonstrating that 3D tissues can be maintained within a miniaturized vascularized scaffold in vitro and successfully implanted after re-anastomosis to the intrinsic blood circulation in vivo. We hypothesize that this technology could serve as a powerful platform technology in tissue engineering and regenerative medicine.

Cellular re- and de-programming by microenvironmental memory: why short TGF-ß1 pulses can have long effects.

BACKGROUND: Fibrosis poses a substantial setback in regenerative medicine. Histopathologically, fibrosis is an excessive accumulation of collagen affected by myofibroblasts and this can occur in any tissue that is exposed to chronic injury or insult. Transforming growth factor (TGF)-ß1, a crucial mediator of fibrosis, drives differentiation of fibroblasts into myofibroblasts. These cells exhibit α-smooth muscle actin (α-SMA) and synthesize high amounts of collagen I, the major extracellular matrix (ECM) component of fibrosis. While hormones stimulate cells in a pulsatile manner, little is known about cellular response kinetics upon growth factor impact. We therefore studied the effects of short TGF-ß1 pulses in terms of the induction and maintenance of the myofibroblast phenotype. RESULTS: Twenty-four hours after a single 30 min TGF-ß1 pulse, transcription of fibrogenic genes was upregulated, but subsided 7 days later. In parallel, collagen I secretion rate and α-SMA presence were elevated for 7 days. A second pulse 24 h later extended the duration of effects to 14 days. We could not establish epigenetic changes on fibrogenic target genes to explain the long-lasting effects. However, ECM deposited under singly pulsed TGF-ß1 was able to induce myofibroblast features in previously untreated fibroblasts. Dependent on the age of the ECM (1 day versus 7 days' formation time), this property was diminished. Vice versa, myofibroblasts were cultured on fibroblast ECM and cells observed to express reduced (in comparison with myofibroblasts) levels of collagen I. CONCLUSIONS: We demonstrated that short TGF-ß1 pulses can exert long-lasting effects on fibroblasts by changing their microenvironment, thus leaving an imprint and creating a reciprocal feed-back loop. Therefore, the ECM might act as mid-term memory for pathobiochemical events. We would expect this microenvironmental memory to be dependent on matrix turnover and, as such, to be erasable. Our findings contribute to the current understanding of fibroblast induction and maintenance, and have bearing on the development of antifibrotic drugs.

MYC-induced epigenetic activation of GATA4 in lung adenocarcinoma.

Human lung cancer is a disease with high incidence and accounts for most cancer-related deaths in both men and women. Metastasis is a common event in non-small cell lung carcinoma (NSCLC), diminishing the survival chance of the patients with this type of tumor. It has been shown that MYC is involved in the development of metastasis from NSCLC, but the mechanism underlying this switch remained to be identified. Here, we focus on GATA4 as a MYC target in the development of metastasis with origin in lung adenocarcinoma, the most common type of NSCLC. Epigenetic alterations at the GATA4 promoter level were observed after MYC expression in lung adenocarcinoma in vivo and in vitro. Such alterations include site-specific demethylation that accompanies the displacement of the MYC-associated zinc finger protein (MAZ) from the GATA4 promoter, which leads to GATA4 expression. Histone modification analysis of the GATA4 promoter revealed a switch from repressive histone marks to active histone marks after MYC binding, which corresponds to active GATA4 expression. Our results thus identify a novel epigenetic mechanism by which MYC activates GATA4 leading to metastasis in lung adenocarcinoma, suggesting novel potential targets for the development of antimetastatic therapy.