Microfabricated dynamic brain organoid cocultures to assess the effects of surface geometry on assembloid formation.

Organoids have emerged as valuable tools for the study of development and disease. Assembloids are formed by integrating multiple organoid types to create more complex models. However, the process by which organoids integrate to form assembloids remains unclear and may play an important role in the resulting organoid structure. Here, a microfluidic platform is developed that allows separate culture of distinct organoid types and provides the capacity to partially control the geometry of the resulting organoid surfaces. Removal of a microfabricated barrier then allows the shaped and positioned organoids to interact and form an assembloid. When midbrain and unguided brain organoids were allowed to assemble with a defined spacing between them, axonal projections from midbrain organoids and cell migration out of unguided organoids were observed and quantitatively measured as the two types of organoids fused together. Axonal projection directions were statistically biased toward other midbrain organoids, and unguided organoid surface geometry was found to affect cell invasion. This platform provides a tool to observe cellular interactions between organoid surfaces that are spaced apart in a controlled manner, and may ultimately have value in exploring neuronal migration, axon targeting, and assembloid formation mechanisms.

Compressive molding of engineered tissues via thermoresponsive hydrogel devices.

Biofabrication of tissues requires sourcing appropriate combinations of cells, and then arranging those cells into a functionally-useful construct. Recently, organoids with diverse cell populations have shown great promise as building blocks from which to assemble more complex structures. However, organoids typically adopt spherical or uncontrolled morphologies, which intrinsically limit the tissue structures that can be produced using this bioassembly technique. Here, we develop microfabricated smart hydrogel platforms in thermoresponsive poly(N-isopropylacrylamide) to compressively mold microtissues such as spheroids or organoids into customized forms, on demand. These Compressive Hydrogel Molders (CHyMs) compact at cell culture temperatures to force loaded tissues into a new shape, and then expand to release the tissues for downstream applications. As a first demonstration, breast cancer spheroids were biaxially compacted in cylindrical cavities, and uniaxially compacted in rectangular ones. Spheroid shape changes persisted after the tissues were released from the CHyMs. We then demonstrate long-term molding of spherical brain organoids in ring-shaped CHyMs over one week. Fused bridges formed only when brain organoids were encased in Matrigel, and the resulting ring-shaped organoids expressed tissue markers that correspond with expected differentiation profiles. These results demonstrate that tissues differentiate appropriately even during long-term molding in a CHyM. This platform hence provides a new tool to shape pre-made tissues as desired, via temporary compression and release, allowing an exploration of alternative organoid geometries as building blocks for bioassembly applications.

Hydrogel Mechanics Influence the Growth and Development of Embedded Brain Organoids.

Brain organoids are three-dimensional, tissue-engineered neural models derived from induced pluripotent stem cells that enable studies of neurodevelopmental and disease processes. Mechanical properties of the microenvironment are known to be critical parameters in tissue engineering, but the mechanical consequences of the encapsulating matrix on brain organoid growth and development remain undefined. Here, Matrigel was modified with an interpenetrating network (IPN) of alginate, to tune the mechanical properties of the encapsulating matrix. Brain organoids grown in IPNs were viable, with the characteristic formation of neuroepithelial buds. However, organoid growth was significantly restricted in the stiffest matrix tested. Moreover, stiffer matrixes skewed cell populations toward mature neuronal phenotypes, with fewer and smaller neural rosettes. These findings demonstrate that the mechanics of the culture environment are important parameters in brain organoid development and show that the self-organizing capacity and subsequent architecture of brain organoids can be modulated by forces arising from growth-induced compression of the surrounding matrix. This study therefore suggests that carefully designing the mechanical properties of organoid encapsulation materials is a potential strategy to direct organoid growth and maturation toward desired structures.

Applying hiPSCs and Biomaterials Towards an Understanding and Treatment of Traumatic Brain Injury.

Traumatic brain injury (TBI) is the leading cause of disability and mortality in children and young adults and has a profound impact on the socio-economic wellbeing of patients and their families. Initially, brain damage is caused by mechanical stress-induced axonal injury and vascular dysfunction, which can include hemorrhage, blood-brain barrier disruption, and ischemia. Subsequent neuronal degeneration, chronic inflammation, demyelination, oxidative stress, and the spread of excitotoxicity can further aggravate disease pathology. Thus, TBI treatment requires prompt intervention to protect against neuronal and vascular degeneration. Rapid advances in the field of stem cells (SCs) have revolutionized the prospect of repairing brain function following TBI. However, more than that, SCs can contribute substantially to our knowledge of this multifaced pathology. Research, based on human induced pluripotent SCs (hiPSCs) can help decode the molecular pathways of degeneration and recovery of neuronal and glial function, which makes these cells valuable tools for drug screening. Additionally, experimental approaches that include hiPSC-derived engineered tissues (brain organoids and bio-printed constructs) and biomaterials represent a step forward for the field of regenerative medicine since they provide a more suitable microenvironment that enhances cell survival and grafting success. In this review, we highlight the important role of hiPSCs in better understanding the molecular pathways of TBI-related pathology and in developing novel therapeutic approaches, building on where we are at present. We summarize some of the most relevant findings for regenerative therapies using biomaterials and outline key challenges for TBI treatments that remain to be addressed.