Parth Chansoria

Engineered cardiac and skeletal muscle tissues suspended between flexible posts support disease modeling and pharmacology, yet their stimulation, longitudinal imaging and quantitative analysis often remain fragmented and labor-intensive. Here we present Stimulatrix, an open-source platform integrating automated video acquisition within a cell culture incubator, synchronized electrical stimulation and deep learning for longitudinal assessment of contractile tissues in multiwell plates. The platform quantifies tissue compaction, force generation and contraction kinetics with optional cloud processing reducing dependence on local GPU hardware. We demonstrate the workflow in cardiac tissues comprising human induced pluripotent stem cell-derived cardiomyocytes and cardiac fibroblasts, and in primary human skeletal muscle constructs. Force and kinetic measurements were benchmarked against manual annotations. Longitudinal profiling resolved responses to matrix composition and pacing regimens and tracked doxorubicin-associated loss of cardiac contractile force. Stimulatrix provides an accessible workflow for automated functional phenotyping of engineered muscle tissues.

The prospects of fabricating human tissue grafts or models using cell‐laden bioresins in space have garnered significant interest in recent years. While there is tremendous progress in extrusion or light‐based bioprinting in microgravity conditions, printing of aligned tissues (e.g., muscle, tendon, cardiac, etc.) remains a challenge. Furthermore, current photoresin formulations do not allow long‐term cell encapsulation and are difficult to handle in microgravity conditions. In this study, a new gravity‐independent filamented light (G‐FLight) biofabrication system, which can create viable muscle constructs within seconds, is demonstrated. New photoresin formulations based on gelatin methacrylate (GelMA) for encapsulation of primary cells (murine myoblasts) and storage in printing cuvettes for at least a week at 4 °C or ‐80 °C are also demonstrated. The tissues printed in microgravity based on the new formulations exhibit higher cell viability, number of proliferating cells, and higher numbers of myotubes and fusion index compared to control formulations (i.e., GelMA dissolved in phosphate‐buffered saline). Importantly, the microgravity‐printed tissues also featured similar myotube density and fusion index to those printed using the same resins on‐ground. The G‐Flight printing concept, together with the new resins enabling refrigeration or cryopreservation with encapsulated cells, offers a promising solution for biofabrication in space.

Deep vat printing (DVP) techniques, including tomographic and filamented light (Flight) printing have transformed the tissue engineering landscape by enabling layer‐free bioprinting at high resolution and speeds. Importantly, collagen is one of the most widely used materials in tissue biofabrication, but DVP using collagen‐based photoresins has been under‐explored. In this work, photoclickable collagen‐based resins are demonstrated which are compatible with DVP, enabling prints with up to 50 µm print resolution and speeds less than 20 s per cm ³ . These resins contain iodixanol, which acts as a refractive index matching agent to reduce optical inhomogeneities by added cells and improves the stability of the collagen‐based resins under neutralized conditions. As a potential application, multi‐material tomographic and FLight printing is demonstrated to create intricate tissue constructs featuring muscle and connective tissue interfaces. While tomographic printing allows greater complexity over the overall shape of the constructs, FLight constructs demonstrate enhanced biomimicry through generation of aligned multinucleated myotubes, transition zones between the two cell types, and regularly spaced sarcomere structure (≈2.6 µm average sarcomere length) which facilitates contractility in the muscle regions. The demonstration of photoclickable, isotonic and neutral collagen‐based bioresins offers a new solution for DVP of biomimetic complex tissues and grafts.

The prospects of fabricating human tissue grafts or models using cell-laden bioresins in space has garnered significant interest in recent years. While there has been tremendous progress in extrusion or light-based bioprinting in microgravity conditions, printing of aligned tissues, such as those featuring anisotropic organization of cells and extracellular matrices (e.g., muscle, tendon, cardiac, etc.), remains a challenge. Furthermore, current photoresin formulations do not allow long-term cell encapsulation and are difficult to perform in microgravity. In this study, we demonstrate a new gravity-independent filamented light (G-FLight) biofabrication system with in-built refrigeration and heating units, which can create viable muscle constructs within seconds. We developed new photoresin formulations based on gelatin methacrylate (GelMA) for encapsulation of primary cells (murine myoblasts) and storage in printing cuvettes for at least a week at 4°C or -80°C. The tissues printed in microgravity based on the new formulations exhibited higher cell viability, number of proliferating cells and after maturation higher numbers of myotubes and fusion index compared to control formulations (i.e., GelMA dissolved in phosphate buffered saline). The microgravity-printed tissues also featured similar myotube density and fusion index to those printed using the same resins on-ground. The G-Flight printing concept, together with the new resins enabling refrigeration or cryopreservation with encapsulated cells, offers a promising solution for biofabrication in space.

The human breast is remarkably plastic and remodels with each birth to produce milk optimally suited for the changing demands of the newborn. This dynamic nature of lactation makes it challenging to study under controlled conditions. Given the health benefits of human milk, models of secretory mammary tissue would offer opportunities to study factors that influence this important food source. First, 3D models of the mammary duct/alveoli (D/A) were designed inspired by shapes found in vivo. Photoresins based on mammary decellularized extracellular matrix (dECM) were optimized to match the mechanical properties of native breast tissue. Next, these D/A models were printed with a volumetric printer and seeded with human milk-derived mammary epithelial cells (MECs). MECs formed stable epithelial layers on the printed surfaces and secreted the β-casein and milk fat globules. This model offers exciting avenues to explore hormonal, nutritional, and mechanobiological factors involved in lactation, thereby improving understanding of lactation for the benefit of infants and their mothers.

Light‐based biofabrication techniques have revolutionized the field of tissue engineering and regenerative medicine. Specifically, the projection of structured light, where the spatial distribution of light is controlled at both macro and microscale, has enabled precise fabrication of complex three dimensional structures with high resolution and speed. However, despite tremendous progress, biofabrication processes are mostly limited to benchtop devices which limit the flexibility in terms of where the fabrication can occur. Here, a Fiber‐assisted Structured Light (FaSt‐Light) projection apparatus for rapid in situ crosslinking of photoresins is demonstrated. This approach uses image‐guide fiber bundles which can project bespoke images at multiple wavelengths, enabling flexibility and spatial control of different photoinitiation systems and crosslinking chemistries and also the location of fabrication. Coupling of different sizes of fibers and different lenses attached to the fibers to project small (several mm) or large (several cm) images for material crosslinking is demonstrated. FaSt‐Light allows control over the cross‐section of the crosslinked resins and enables the introduction of microfilaments which can further guide cellular infiltration, differentiation, and anisotropic matrix production. The proposed approach can lead to a new range of in situ biofabrication techniques which improve the translational potential of photofabricated tissues and grafts.

The human breast is remarkably plastic and remodels with each birth to produce milk optimally suited for the changing demands of the newborn. This dynamic nature of lactation makes it challenging to study under controlled conditions. Given the health benefits of human milk, models of secretory mammary tissue would offer new opportunities to study factors that influence this important food source. First, 3D models of the mammary duct/alveoli (D/A) were designed based on shapes found in vivo . Photoresins based on mammary decellularized extracellular matrix (dECM) were optimized to match the mechanical properties of native breast tissue. Next, these dECM-based D/A models were printed with a volumetric printer and seeded with human milk-derived mammary epithelial cells (MECs). MECs formed stable epithelial layers on the printed surfaces and secreted the beta-casein and milk-fat-globules. This novel model offers exciting avenues to explore hormonal, nutritional, and mechanobiological factors involved in lactation, thereby improving understanding of lactation for the benefit of infant and their mothers.

In patients with sensory nerve loss, such as those experiencing optic nerve damage that leads to vision loss, the thalamus no longer receives the corresponding sensory input. To restore functional sensory input, it is necessary to bypass the damaged circuits, which can be achieved by directly stimulating the appropriate sensory thalamic nuclei. However, available deep brain stimulation electrodes do not provide the resolution required for effective sensory restoration. Therefore, this work develops an implantable biohybrid neural interface aimed at innervating and synaptically stimulating deep brain targets. The interface combines a stretchable stimulation array with an aligned microfluidic axon guidance system seeded with neural spheroids to facilitate the development of a 3 mm long nerve‐like structure. A bioresorbable hydrogel nerve conduit is used as a bridge between the tissue and the biohybrid implant. Stimulation of the spheroids within the biohybrid structure in vitro and use of high‐density CMOS microelectrode arrays show faithful activity conduction across the device. Although functional in vivo innervation and synapse formation has not yet been achieved in this study, implantation of the biohybrid nerve onto the mouse cortex shows that neural spheroids grow axons in vivo and remain functionally active for more than 22 days post‐implantation.

The plasticity and regenerative capacity of skeletal muscle arise from quiescent stem cells activated upon overload, injury, or inflammation. Developing in vitro muscle models to study these properties could advance muscle disease modeling and pre-clinical evaluation. Here, we leverage Filamented Light (FLight) bioprinting as a high-throughput approach for producing mini-muscle tissues. Using Pax7-nGFP myoblasts, we bioprinted mini-muscles from pristine collagen-fibrinogen. The FLight hydrogel consisted of aligned microstructures which guided the formation of aligned myotubes. Mini-muscles demonstrated in vivo -like tissue organization, including highly aligned myotubes and a Pax7 ⁺ cell pool embedded in newly deposited laminin. Both spontaneous and electrically stimulated contractions were observed. Collagen-fibrinogen matrix was promising for maintenance of the Pax7 ⁺ cell pool. Damage from cardiotoxin-induced injury of the mini-muscles led to a massive proliferation of Pax7 ⁺ cells and restoration of the contractile properties. Notably, small molecules such as Repsox could enhance regeneration. FLight printed mini-muscles have potential for applications in muscle biology, exercise/atrophy, disease models, and drug screening.