Der Mikrokavitäten-Array: Folien-basierte 3D-Zellkultursysteme

Die Geschichte der Zellkultur stützt sich seit jeher auf Systeme, die eine Kultivierung auf planaren Oberflächen, und damit in nur zwei Dimensionen (2D) zuläßt. Zu diesen Systemen gehören Petrischalen, Kulturflaschen und deren Derivate, wie Multiwellplatten. Die über hundertjährige Erfolgsgeschichte der 2D-Systeme ist auf die einfache Handhabbarkeit, die niedrigen Kosten und die Möglichkeit, die Zellen enorm expandieren zu können, zurückzuführen. Sobald jedoch organotypische Leistungen der kultivierten Zellen im Vordergrund stehen, können Versuche in 2D-Systemen nicht oder nur eingeschränkt durchgeführt werden, da häufig insbesondere primäre Zellen ihre organotypischen Leistungen aufgrund dieser sehr gewebsuntypischen Kulturform innerhalb weniger Tage verlieren

Overlooked? Underestimated? Effects of Substrate Curvature on Cell Behavior

In biological systems, form and function are inherently correlated. Despite this strong interdependence, the biological effect of curvature has been largely overlooked or underestimated, and consequently it has rarely been considered in the design of new cell–material interfaces. This review summarizes current understanding of the interplay between the curvature of a cell substrate and the related morphological and functional cellular response. In this context, we also discuss what is currently known about how, in the process of such a response, cells recognize curvature and accordingly reshape their membrane. Beyond this, we highlight state-of-the-art microtechnologies for engineering curved biomaterials at cell-scale, and describe aspects that impair or improve readouts of the pure effect of curvature on cells

Multiscale Microstructure for Investigation of Cell–Cell Communication

A multiscale polydimethylsiloxane (PDMS) chip is presented, which provides an array of mesoscale open wells for cell culturing and, as unique feature, an array of microscale 1 µm deep channels to fluidically connect neighboring wells. As demonstrated with SH‐SY5Y cells, the small dimensions of the channels prevent migration of the cell soma but allow physical contacts established by the outgrowth of protoplasmic protrusions between cells in adjacent wells. Another important feature is the chip\u27s mountability on solid substrates, such as glass. This enables the use of substrates previously patterned with biomolecules, as demonstrated by DNA‐directed immobilization of proteins inside the reactor wells. Given the versatile addressability of cells, whether through surface‐bound or inkjet‐based administration of bioactive substances, it is believed that the reactor could be used for research in cell–cell communication networks, for example, in neurodegenerative diseases such as Alzheimer\u27s disease

Microfluidic gradient generator for drug testing on a colorectal tumor-on-a-chip disease model

Statement of Purpose: Colorectal cancer is the third most common cancer and its incidence increases with ageing. Understanding the mechanisms of tumour growth rely in further advances to unveil cancer-causing agents, drug screening and in the development of personalized therapies. Standard 2D in vitro models and in vivo animal models have undoubtedly contributed to the development of anti-cancer drug candidates. Yet their translation into successful clinical trials is critically low, which reinforces the need of a deeper understanding of tumorigenesis (1). Therefore, 3D models integrating tissue engineering (TE) strategies with microfluidic technology have sparked the expectation on physiologically relevant microfluidic in vitro models (2). The aim of this work is to establish a 3D microfluidic model that enables the reconstitution of physiological functions of microvascular tissue that emulates the human colorectal tumor microenvironment. This model will be established via a microfluidic device with an encapsulating hydrogel compartment comprising a co-culture system of HCT-116 colorectal cancer cells and human intestinal microvascular endothelial cells

Microfluidic collagen patterning for tendon regeneration

We present a microfluidic approach to align collagen fibers for tendon regeneration. Collagen fibers with a specific orientation were patterned in a microfluidic channel by introducing collagen solution through integrated microstructures. The fluid flow in the pillar array was evaluated by computational modeling, and the aligned collagen fibers were analyzed quantitatively. Then, primary rat tenocytes were cultured on oriented and not-oriented collagen micropatterns, and their phenotypical commitment was evaluated. We believe that such a platform would be useful to replicate in vivo microenvironment for the study of regenerative processes

PSC-derived intestinal organoids with apical-out orientation as a tool to study nutrient uptake, drug absorption and metabolism

Intestinal organoids recapitulate many features of the in vivo gastrointestinal tract and have revolutionized in vitro studies of intestinal function and disease. However, the restricted accessibility of the apical surface of the organoids facing the central lumen (apical-in) limits studies related to nutrient uptake and drug absorption and metabolism. Here, we demonstrate that pluripotent stem cell (PSC)-derived intestinal organoids with reversed epithelial polarity (apical-out) can successfully recapitulate tissue-specific functions. In particular, these apical-out organoids show strong epithelial barrier formation with all the major junctional complexes, nutrient transport and active lipid metabolism. Furthermore, the organoids express drug-metabolizing enzymes and relevant apical and basolateral transporters. The scalable and robust generation of functional, apical-out intestinal organoids lays the foundation for a completely new range of organoid-based high-throughput/high-content in vitro applications in the fields of nutrition, metabolism and drug discovery

The Galapagos Chip Platform for High-Throughput Screening of Cell Adhesive Chemical Micropatterns

In vivo cells reside in a complex extracellular matrix (ECM) that presents spatially distributed biochemical and ‑physical cues at the nano- to micrometer scales. Chemical micropatterning is successfully used to generate adhesive islands to control where and how cells attach and restore cues of the ECM in vitro. Although chemical micropatterning has become a powerful tool to study cell–material interactions, only a fraction of the possible micropattern designs was covered so far, leaving many other possible designs still unexplored. Here, a high-throughput screening platform called “Galapagos chip” is developed. It contains a library of 2176 distinct subcellular chemical patterns created using mathematical algorithms and a straightforward UV-induced two-step surface modification. This approach enables the immobilization of ligands in geometrically defined regions onto cell culture substrates. To validate the system, binary RGD/polyethylene glycol patterns are prepared on which human mesenchymal stem cells are cultured, and the authors observe how different patterns affect cell and organelle morphology. As proof of concept, the cells are stained for the mechanosensitive YAP protein, and, using a machine-learning algorithm, it is demonstrated that cell shape and YAP nuclear translocation correlate. It is concluded that the Galapagos chip is a versatile platform to screen geometrical aspects of cell–ECM interaction

Investigation of the effects of phthalates on in vitro thyroid models with RNA-Seq and ATAC-Seq

IntroductionPhthalates are a class of endocrine-disrupting chemicals that have been shown to negatively correlate with thyroid hormone serum levels in humans and to cause a state of hyperactivity in the thyroid. However, their mechanism of action is not well described at the molecular level.MethodsWe analyzed the response of mouse thyroid organoids to the exposure to a biologically relevant dose range of the phthalates bis(2-ethylhexyl) phthalate (DEHP), di-iso-decylphthalate (DIDP), di-iso-nonylphthalate (DINP), and di-n-octylphthalate (DnOP) for 24 h and simultaneously analyzed mRNA and miRNA expression via RNA sequencing. Using the expression data, we performed differential expression and gene set enrichment analysis. We also exposed the human thyroid follicular epithelial cell line Nthy-ori 3-1 to 1 µM of DEHP or DINP for 5 days and analyzed changes in chromatin accessibility via ATAC-Seq.ResultsDose-series analysis showed how the expression of several genes increased or decreased at the highest dose tested. As expected with the low dosing scheme, the compounds induced a modest response on the transcriptome, as we identified changes in only mmu-miR-143-3p after DINP treatment and very few differentially expressed genes. No effect was observed on thyroid markers. Ing5, a component of histones H3 and H4 acetylation complexes, was consistently upregulated in three out of four conditions compared to control, and we observed a partial overlap among the genes differentially expressed by the treatments. Gene set enrichment analysis showed enrichment in the treatment samples of the fatty acid metabolism pathway and in the control of pathways related to the receptor signalling and extracellular matrix organization. ATAC-Seq analysis showed a general increase in accessibility compared to the control, however we did not identify significant changes in accessibility in the identified regions.DiscussionWith this work, we showed that despite having only a few differentially expressed genes, diverse analysis methods could be applied to retrieve relevant information on phthalates, showing the potential of in vitro thyroid-relevant systems for the analysis of endocrine disruptors

Liquid polystyrene: a room-temperature photocurable soft lithography compatible pour-and-cure-type polystyrene

Materials matter in microfluidics. Since the introduction of soft lithography as a prototyping technique and polydimethylsiloxane (PDMS) as material of choice the microfluidics community has settled with using this material almost exclusively. However{,} for many applications PDMS is not an ideal material given its limited solvent resistance and hydrophobicity which makes it especially disadvantageous for certain cell-based assays. For these applications polystyrene (PS) would be a better choice. PS has been used in biology research and analytics for decades and numerous protocols have been developed and optimized for it. However{,} PS has not found widespread use in microfluidics mainly because{,} being a thermoplastic material{,} it is typically structured using industrial polymer replication techniques. This makes PS unsuitable for prototyping. In this paper{,} we introduce a new structuring method for PS which is compatible with soft lithography prototyping. We develop a liquid PS prepolymer which we term as {"}Liquid Polystyrene{"} (liqPS). liqPS is a viscous free-flowing liquid which can be cured by visible light exposure using soft replication templates{,} e.g.{,} made from PDMS. Using liqPS prototyping microfluidic systems in PS is as easy as prototyping microfluidic systems in PDMS. We demonstrate that cured liqPS is (chemically and physically) identical to commercial PS. Comparative studies on mouse fibroblasts L929 showed that liqPS cannot be distinguished from commercial PS in such experiments. Researchers can develop and optimize microfluidic structures using liqPS and soft lithography. Once the device is to be commercialized it can be manufactured using scalable industrial polymer replication techniques in PS - the material is the same in both cases. Therefore{,} liqPS effectively closes the gap between {"}microfluidic prototyping{"} and {"}industrial microfluidics{"} by providing a common material

Spatially controlled cell adhesion on three-dimensional substrates

The microenvironment of cells in vivo is defined by spatiotemporal patterns of chemical and biophysical cues. Therefore, one important goal of tissue engineering is the generation of scaffolds with defined biofunctionalization in order to control processes like cell adhesion and differentiation. Mimicking extrinsic factors like integrin ligands presented by the extracellular matrix is one of the key elements to study cellular adhesion on biocompatible scaffolds. By using special thermoformable polymer films with anchored biomolecules micro structured scaffolds, e.g. curved and µ-patterned substrates, can be fabricated. Here, we present a novel strategy for the fabrication of µ-patterned scaffolds based on the “Substrate Modification and Replication by Thermoforming” (SMART) technology: The surface of a poly lactic acid membrane, having a low forming temperature of 60°C and being initially very cell attractive, was coated with a photopatterned layer of poly(L-lysine) (PLL) and hyaluronic acid (VAHyal) to gain spatial control over cell adhesion. Subsequently, this modified polymer membrane was thermoformed to create an array of spherical microcavities with diameters of 300 µm for 3D cell culture. Human hepatoma cells (HepG2) and mouse fibroblasts (L929) were used to demonstrate guided cell adhesion. HepG2 cells adhered and aggregated exclusively within these cavities without attaching to the passivated surfaces between the cavities. Also L929 cells adhering very strongly on the pristine substrate polymer were effectively patterned by the cell repellent properties of the hyaluronic acid based hydrogel. This is the first time cell adhesion was controlled by patterned functionalization of a polymeric substrate with UV curable PLL-VAHyal in thermoformed 3D microstructures