3D high throughput screening and profiling of embryoid bodies in thermoformed microwell plates

3D organoids using stem cells to study development and disease are now widespread. These models are powerful to mimic in vivo situations but are currently associated with high variability and low throughput. For biomedical research, platforms are thus necessary to increase reproducibility and allow high-throughput screens (HTS). Here, we introduce a microwell platform, integrated in standard culture plates, for functional HTS. Using micro-thermoforming, we form round-bottom microwell arrays from optically clear cyclic olefin polymer films, and assemble them with bottom-less 96-well plates. We show that embryonic stem cells aggregate faster and more reproducibly (centricity, circularity) as compared to a state-of-the-art microwell array. We then run a screen of a chemical library to direct differentiation into primitive endoderm (PrE) and, using on-chip high content imaging (HCI), we identify molecules, including regulators of the cAMP pathway, regulating tissue size, morphology and PrE gene activity. We propose that this platform will benefit to the systematic study of organogenesis in vitro

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

Mechanistic Computational Models of Epithelial Cell Transporters-the Adorned Heroes of Pharmacokinetics

Epithelial membrane transporter kinetics portray an irrefutable role in solute transport in and out of cells. Mechanistic models are used to investigate the transport of solutes at the organ, tissue, cell or membrane scale. Here, we review the recent advancements in using computational models to investigate epithelial transport kinetics on the cell membrane. Various methods have been employed to develop transport phenomena models of solute flux across the epithelial cell membrane. Interestingly, we noted that many models used lumped parameters, such as the Michaelis-Menten kinetics, to simplify the transporter-mediated reaction term. Unfortunately, this assumption neglects transporter numbers or the fact that transport across the membrane may be affected by external cues. In contrast, more recent mechanistic transporter kinetics models account for the transporter number. By creating models closer to reality researchers can investigate the downstream effects of physical or chemical disturbances on the system. Evidently, there is a need to increase the complexity of mechanistic models investigating the solute flux across a membrane to gain more knowledge of transporter-solute interactions by assigning individual parameter values to the transporter kinetics and capturing their dependence on each other. This change results in better pharmacokinetic predictions in larger scale platforms. More reliable and efficient model predictions can be made by creating mechanistic computational models coupled with dedicated in vitro experiments. It is also vital to foster collaborative efforts among transporter kinetics researchers in the modeling, material science and biological fields

Modeling indoxyl sulfate transport in a bioartificial kidney: Two-step binding kinetics or lumped parameters model for uremic toxin clearance?

Toxin removal by the kidney is deficient in a patient suffering from end-stage kidney disease (ESKD), and current dialysis therapies are insufficient in subsidizing this loss. A bioartificial kidney (BAK) aspires to offer ESKD patients a more effective alternative to dialysis. Mathematical models are necessary to support further developments and improve designs for the BAK before clinical trials. The BAK differentiates itself from dialysis by incorporating a living proximal tubule cell monolayer to account for the active transport of protein-bound uremic toxins, namely indoxyl sulfate (IS) in this study. Optimizing such a device is far from trivial due to the non intuitive spatiotemporal dynamics of the IS removal process. This study used mathematical models to compare two types of active transport kinetics. i.e., two-step binding and lumped parameter. The modeling results indicated that the transporter density is the most influential parameter for the IS clearance. Moreover, a uniform distribution of transporters increases the IS clearance, highlighting the need for a high-quality, functional proximal tubule monolayer in the BAK. In summary, this study contributed to an improved understanding of IS transport in the BAK, which can be used along with laboratory experiments to develop promising renal replacement therapies in the future

Thin fluorinated polymer film microcavity arrays for 3D cell culture and label-free automated feature extraction

There is an increasing need for automated label-free morphometric analysis using brightfield microscopy images of 3D cell culture systems. This requires automated feature detection which can be achieved by improving the image contrast, e.g. by reducing the refractive index mismatch in the light path. Here, a novel microcavity platform fabricated using microthermoforming of thin fluorinated ethylene-propylene (FEP) films which match the refractive index of cell culture medium and provide a homogenous background signal intensity is described. FEP is chemically inert, mechanically stable and has been used as a substrate for light sheet microscopy. The microcavities promote formation of mouse embryonic stem cell (mESC) aggregates, which show axial elongation and germ layer specification similar to embryonic development. A label-free feature extraction pipeline based on a machine-learning plugin for FIJI is used to extract morphometric features from time-lapse imaging in a highly robust and reproducible manner. Lastly, the pipeline is utilized for testing the effect of the drug Latrunculin A on the mESC aggregates, highlighting the platform's potential for high-content screening (HCS) in drug discovery. This new microengineered tool is an important step towards label-free imaging of free-floating stem cell aggregates and paves the way for high-content drug testing and translational studies

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

Biodegradable Elastic Sponge from Nanofibrous Biphasic Calcium Phosphate Ceramic as an Advanced Material for Regenerative Medicine

Biodegradable porous calcium phosphate (CaP) ceramics are widely used as synthetic graft substitutes for bone regeneration, owing to their chemical and structural similarity to bone and associated bioactivity in terms of bone-bonding, osteoconductive, and even osteoinductive properties. Nevertheless, the intrinsic brittleness and poor processability of porous CaP ceramics strongly impair their clinical applicability. Herein, a biphasic calcium phosphate (BCP) sponge is developed that consists of a self-supporting network of seamlessly interwoven hydroxyapatite nanowires and β-tricalcium phosphate nanofibers and possesses a highly interconnected porous structure with open cell geometry and ultrahigh porosity. Owing to its unique properties, the ceramic sponge can be easily processed into various shapes and dimensions, such as cylindrical scaffolds and thin, flexible membranes. Moreover, the BCP sponge can be introduced into a bone defect in a compacted or folded state from a syringe and, upon wetting, expand to its original shape, thereby filling the cavity. The nanofibrous sponge gradually degrades in vitro and rapidly mineralizes when immersed in simulated body fluid. Moreover, it adsorbs significantly more proteins than a conventional porous BCP ceramic. Finally, the nanofibrous sponge supports the attachment, proliferation, and osteogenic differentiation of human mesenchymal stromal cells comparable to the conventional porous BCP ceramic

Biomaterials and Microfluidics for Drug Discovery and Development

Microfluidic devices are now one of the most promising tools to mimic in vivo like conditions, either in normal or disease scenarios, such as tumorigenesis or pathogenesis. Together with the potential of biomaterials, its combination with microfluidics represents the ability to more closely mimic cells' natural microenvironment concerning its three-dimensional (3D) nature and continuous perfusion with nutrients and cells' crosstalk. Due to miniaturization and increased experimental throughput, microfluidics have generated significant interest in the drug discovery and development domain. Herein, the most recent advances in the field of microfluidics for drug discovery are overviewed, and the role of biomaterials in 3D in vitro models and the contribution of organ-on-a-chip technologies highlighted

Microwell Scaffolds for Extrahepatic Transplantation of Islets of Langerhans

Diabetes mellitus: pathophysiological changes and therap