Engineered Synthetic Matrices for Human Intestinal Organoid Culture and Therapeutic Delivery.

Human intestinal organoids (HIOs) derived from pluripotent stem cells or adult stem cell biopsies represent a powerful platform to study human development, drug testing, and disease modeling in vitro, and serve as a cell source for tissue regeneration and therapeutic advances in vivo. Synthetic hydrogels can be engineered to serve as analogs of the extracellular matrix to support HIO growth and differentiation. These hydrogels allow for tuning the mechanical and biochemical properties of the matrix, offering an advantage over biologically derived hydrogels such as Matrigel. Human intestinal organoids have been used for repopulating transplantable intestinal grafts and for in vivo delivery to an injured intestinal site. The use of synthetic hydrogels for in vitro culture and for in vivo delivery is expected to significantly increase the relevance of human intestinal organoids for drug screening, disease modeling, and therapeutic applications.

Facile Photopatterning of Perfusable Microchannels in Synthetic Hydrogels to Recreate Microphysiological Environments.

The fabrication of perfusable hydrogels is crucial for recreating in vitro microphysiological environments. Existing strategies to fabricate complex microchannels in hydrogels involve sophisticated equipment/techniques. A cost-effective, facile, versatile, and ultra-fast methodology is reported to fabricate perfusable microchannels of complex shapes in photopolymerizable hydrogels without the need of specialized equipment or sophisticated protocols. The methodology utilizes one-step ultraviolet (UV) light-triggered cross-linking and a photomask printed on inexpensive transparent films to photopattern PEG-norbornene hydrogels. Complex and intricate patterns with high resolution, including perfusable microchannels, can be fabricated in <1 s. The perfusable hydrogel is integrated into a custom-made microfluidic device that permits connection to external pump systems, allowing continuous fluid perfusion into the microchannels. Under dynamic culture, human endothelial cells form a functional and confluent endothelial monolayer that remains viable for at least 7 days and respond to inflammatory stimuli. Finally, approach to photopattern norbornene hyaluronic acid hydrogels is adapted, highlighting the versatility of the technique. This study presents an innovative strategy to simplify and reduce the cost of biofabrication techniques for developing functional in vitro models using perfusable three-dimensional (3D) hydrogels. The approach offers a novel solution to overcome the complexities associated with existing methods, allowing engineering advanced in vitro microphysiological environments.

Engineering multicellular living systems-a Keystone Symposia report.

The ability to engineer complex multicellular systems has enormous potential to inform our understanding of biological processes and disease and alter the drug development process. Engineering living systems to emulate natural processes or to incorporate new functions relies on a detailed understanding of the biochemical, mechanical, and other cues between cells and between cells and their environment that result in the coordinated action of multicellular systems. On April 3-6, 2022, experts in the field met at the Keystone symposium "Engineering Multicellular Living Systems" to discuss recent advances in understanding how cells cooperate within a multicellular system, as well as recent efforts to engineer systems like organ-on-a-chip models, biological robots, and organoids. Given the similarities and common themes, this meeting was held in conjunction with the symposium "Organoids as Tools for Fundamental Discovery and Translation".

Synthetic Matrix Scaffolds Engineer the In Vivo Tumor Immune Microenvironment for Immunotherapy Screening.

Immunotherapy has emerged as one of the most powerful anti-cancer therapies but is stymied by the limits of existing preclinical models with respect to disease latency and reproducibility. Additionally, the influence of differing immune microenvironments within tumors observed clinically and associated with immunotherapeutic resistance cannot be tuned to facilitate drug testing workflows without changing model system or laborious genetic approaches. To address this testing platform gap in the immune oncology drug development pipeline, the authors deploy engineered biomaterials as scaffolds to increase tumor formation rate, decrease disease latency, and diminish variability of immune infiltrates into tumors formed from murine mammary carcinoma cell lines implanted into syngeneic mice. By altering synthetic gel formulations that reshape infiltrating immune cells within the tumor, responsiveness of the same tumor model to varying classes of cancer immunotherapies, including in situ vaccination with a molecular adjuvant and immune checkpoint blockade, diverge. These results demonstrate the significant role the local immune microenvironment plays in immunotherapeutic response. These engineered tumor immune microenvironments therefore improve upon the limitations of current breast tumor models used for immune oncology drug screening to enable immunotherapeutic testing relevant to the variability in tumor immune microenvironments underlying immunotherapeutic resistance seen in human patients.

Real-time monitoring of human Schwann cells on heparin-collagen coatings reveals enhanced adhesion and growth factor response.

In this work, we evaluate the enhancing effect of six bilayers of heparin/collagen (HEP/COL)6 layer-by-layer coatings on human Schwann cell (hSCs) adhesion and proliferation in the presence or absence of nerve growth factor (NGF). hSCs behavior and in vitro bioactivity were studied during six days of culture using end-point viability and proliferation assays as well as an impedance-based real-time monitoring system. An end-point viability assay revealed that hSCs cultured on the (HEP/COL)6 coatings increased their growth by more than 230% compared to controls. However, an EdU proliferation assay revealed that the proliferation rate of hSCs in all conditions were similar, with 45% of cells proliferating after 18 hours of incubation. Fluorescence microscopy revealed that hSCs spreading was similar between the tissue culture plastic control and the (HEP/COL)6. The presence of NGF in solution resulted in cells with a larger spread area. Real-time monitoring of hSCs seeded on (HEP/COL)6 with and without NGF reveals that initial cell adhesion is improved by the presence of the (HEP/COL)6 coatings, and it is further improved by the presence of NGF. Our results suggest that (HEP/COL)6 coatings enhance Schwann cell behavior and response to NGF. This simple modification could be applied to current nerve regeneration strategies to improve the repair of damaged nerve.

Generation of small intestinal organoids for experimental intestinal physiology.

Human intestinal organoids (HIOs) derived from pluripotent stem cells were first described almost a decade ago as a method to differentiate intestinal tissue containing both epithelium and supporting mesenchymal cells. The original protocol documents a directed differentiation approach to first induce definitive endoderm from pluripotent stem cells, followed by hindgut specification, resulting in the self-organization of 3D hindgut spheroids. These hindgut spheroids are then embedded in a basement membrane extracellular matrix (ECM) such as Matrigel and mature into HIOs over about 4 weeks in culture. Since the initial HIO protocol was published, the methods to generate HIOs have been updated over time including revisions to the directed differentiation protocol and implementation of new culture methods for spheroids such as embedding in alginate or polyethylene glycol hydrogels as defined alternatives to Matrigel. Additionally, HIOs have been utilized for new applications such as co-culture with bacteria. This protocol compiles the most up to date information on HIO generation and presents alternative experimental applications.

Identification of matrix physicochemical properties required for renal epithelial cell tubulogenesis by using synthetic hydrogels.

Synthetic hydrogels with controlled physicochemical matrix properties serve as powerful in vitro tools to dissect cell-extracellular matrix (ECM) interactions that regulate epithelial morphogenesis in 3D microenvironments. In addition, these fully defined matrices overcome the lot-to-lot variability of naturally derived materials and have provided insights into the formation of rudimentary epithelial organs. Therefore, we engineered a fully defined synthetic hydrogel with independent control over proteolytic degradation, mechanical properties, and adhesive ligand type and density to study the impact of ECM properties on epithelial tubulogenesis for inner medullary collecting duct (IMCD) cells. Protease sensitivity of the synthetic material for membrane-type matrix metalloproteinase-1 (MT1-MMP, also known as MMP14) was required for tubulogenesis. Additionally, a defined range of matrix elasticity and presentation of RGD adhesive peptide at a threshold level of 2 mM ligand density were required for epithelial tubulogenesis. Finally, we demonstrated that the engineered hydrogel supported organization of epithelial tubules with a lumen and secreted laminin. This synthetic hydrogel serves as a platform that supports epithelial tubular morphogenetic programs and can be tuned to identify ECM biophysical and biochemical properties required for epithelial tubulogenesis.