Advanced technologies to control stem-cell based organogenesis

Organoids represent the first in vitro cell culture systems that closely resemble native tissues in terms of cellular composition, architecture and key aspects of physiology. Through the discovery of these cultures, researchers could demonstrate that stem cells retain in vitro their innate tendency to self-organize, thus giving rise to complex structures resembling functioning tissues. Until now, most organoid systems have been obtained relying on ill-defined and clinically irrelevant 3D matrices, in which these organoids are homogeneously exposed to biomolecules that induce in vitro development. However, tissue growth and specification in vivo is a tightly orchestrated process, where a multitude of effectors are presented to the developing tissue spatiotemporally. Thus, even though stem and progenitor cells are able to self-organize to striking extents, there is an imminent need to establish new technologies to control the growth of these self-organizing, organ-mimicking structures. The few already described dynamic culture systems, such as gradient generators, will have to be considerably revisited to accommodate the final size and the extended culture time of these developing in vitro tissues. Considering these limitations, we introduce two innovative technologies that provide the expected flexibility to adapt to organoid cultures and that offer control on morphogenesis in vitro. First, we established a unique method to generate microfluidic networks inside naturally derived and synthetic hydrogels. Using short-pulsed lasers, perfusable microchannels can be fabricated in any transparent matrix. This technique has several advantages over conventional microfabrication approaches, as microchannels can be easily fabricated a posteriori in 3D cell-laden hydrogels. In addition, the resulting microfluidic network can be varied on demand depending on cellular growth and morphogenetic events, without impairing cell viability. Next, in order to control organoids better, a versatile hydrogel-based microwell platform harboring U-shaped microcavities of any size or shape was developed. A wide range of cell types can be readily aggregated into highly homogenous cellular clusters. The differentiation of retinal organoids can be improved by optimizing the culture substrate in combination with a specific medium formulation, giving rise to more photoreceptors. Furthermore, the novel technology was used to reduce the variability and improve the traceability of current organoid cultures grown in 3D matrices. For example, aggregation of low numbers of intestinal stem cells instead of single cells resulted in more homogenous organoid formation. The improved homogeneity allows for non-biased analyses at single organoid levels. Finally, new methods were explored to facilitate the application of these next-generation organoid cultures in pharmacological screenings. Using intestinal organoids derived from a murine disease model of cystic fibrosis, a new label-free method was introduced to precisely read out transepithelial fluid exchanges. By measuring changes in the local molecular density within the organoid lumen, key functional metrics representing transepithelial fluxes could be measured. Taken together, this thesis introduces cutting-edge technologies that offer the possibility to exert control over in vitro organoid development and should therefore facilitate the translation of organoid technology towards pharmaceutical and clinical applications

Bioengineered embryoids mimic post-implantation development in vitro.

The difficulty of studying post-implantation development in mammals has sparked a flurry of activity to develop in vitro models, termed embryoids, based on self-organizing pluripotent stem cells. Previous approaches to derive embryoids either lack the physiological morphology and signaling interactions, or are unconducive to model post-gastrulation development. Here, we report a bioengineering-inspired approach aimed at addressing this gap. We employ a high-throughput cell aggregation approach to simultaneously coax mouse embryonic stem cells into hundreds of uniform epiblast-like aggregates in a solid matrix-free manner. When co-cultured with mouse trophoblast stem cell aggregates, the resulting hybrid structures initiate gastrulation-like events and undergo axial morphogenesis to yield structures, termed EpiTS embryoids, with a pronounced anterior development, including brain-like regions. We identify the presence of an epithelium in EPI aggregates as the major determinant for the axial morphogenesis and anterior development seen in EpiTS embryoids. Our results demonstrate the potential of EpiTS embryoids to study peri-gastrulation development in vitro

Microarrayed human bone marrow organoids for modeling blood stem cell dynamics

In many leukemia patients, a poor prognosis is attributed either to the development of chemotherapy resistance by leukemic stem cells (LSCs) or to the inefficient engraftment of transplanted hematopoietic stem/progenitor cells (HSPCs) into the bone marrow (BM). Here, we build a 3D in vitro model system of bone marrow organoids (BMOs) that recapitulate several structural and cellular components of native BM. These organoids are formed in a high-throughput manner from the aggregation of endothelial and mesenchymal cells within hydrogel microwells. Accordingly, the mesenchymal compartment shows partial maintenance of its self-renewal and multilineage potential, while endothelial cells self-organize into an interconnected vessel-like network. Intriguingly, such an endothelial compartment enhances the recruitment of HSPCs in a chemokine ligand/receptor-dependent manner, reminiscent of HSPC homing behavior in vivo. Additionally, we also model LSC migration and nesting in BMOs, thus highlighting the potential of this system as a well accessible and scalable preclinical model for candidate drug screening and patient-specific assays

Pharmacological induction of a progenitor state for the efficient expansion of primary human hepatocytes

The liver is an organ with strong regenerative capacity, yet primary hepatocytes have a low amplification potential in vitro, a major limitation for the cell-based therapy of liver disorders and for ex vivo biological screens. Induced pluripotent stem cells (iPSCs) may help to circumvent this obstacle but often harbor genetic and epigenetic abnormalities, limiting their potential. Here, we describe the pharmacological induction of proliferative human hepatic progenitor cells (HPCs) through a cocktail of growth factors and small molecules mimicking the signaling events involved in liver regeneration. Human HPCs from healthy donors and pediatric patients proliferated vigorously while maintaining their genomic stability and could be redifferentiated in vitro into metabolically competent cells that supported the replication of hepatitis B and delta viruses. Redifferentiation efficiency was boosted by three-dimensional culture. Finally, transcriptome analysis showed that HPCs were more closely related to mature hepatocytes than iPSC-derived hepatocyte-like cells were. Conclusion: HPC induction holds promise for a variety of applications such as ex vivo disease modeling, personalized drug testing or metabolic studies, and development of a bioartificial liver

Cell bead based three-dimensional (3D) hepatic model toward high throughput drug-induced toxicity screening

For several decades, hepatotoxicity has been a major issue for drug development.Recently, three-dimensional (3D) cell culture has been believed to improve cell drug response accuracy. However important limitations such as slow production, lack of cell density control and lack of cell-extracellular matrix (ECM) interactions are still remaining in the current systems. We believe that cell culture on collagen gel microbeads, developed by Takeuchiʼs group, could overcome these limitations. Thus, in this study, we evaluated the implementation of this cell culture system as a potential model for druginduced toxicity screening. Using HepG2 cell line, we showed that cells grown on collagen microbeads significantly up-regulate drug metabolism specific genes, i.e. CYP enzymes, very soon after seeding, compared to monolayer culture, whereas spheroid cultures show an up-regulation from 48 hours only. Moreover, after having incubated HepG2 cells grown in monolayer, in spheroid, and on collagen microbeads with acetaminophen for 24 hours, we detected higher cell sensitivity to the compound in the collagen microbeads culture condition. Thus, we suggest this culture system to be a promising in-vitro hepatic model for pharmaceutical drug toxicology screening. Finally, as we believe that integrated cell-based microchips are the future of drug screening and development, we will then try to design an arraying system able to combine single cell bead culture and localized drug perfusion to demonstrate cell bead culture systemʼs practical application in high throughput screening

Organoid arrays

The invention provides methods for producing arrays of organoids, the arrays thereof and uses of such arrays

Dialect fieldworkers in nineteenth-century Ireland

The fabrication of microfluidic devices is often still a time-consuming and costly process. Here we introduce a very simple and cheap microfabrication process based on "razor writing", also termed xurography, for the ultra-rapid prototyping of microfluidic devices. Thin poly(dimethylsiloxane) (PDMS) membranes are spin-coated on flexible plastic foil and cut into user-defined shapes with a bench-top cutter plotter. The PDMS membranes can then be assembled into desirable microdevices via plasma bonding. The plastic foil allows manipulation of exceptionally thin (30-300 mu m) PDMS layers and can be readily peeled after fabrication. This versatile technique can be used to produce a wide variety of microfluidic device prototypes within just a few hours