3D bioprinted human iPSC-derived somatosensory constructs with functional and highly purified sensory neuron networks

Abstract

Engineering three-dimensional (3D) sensible tissue constructs, along with the complex microarchitecture wiring of the sensory nervous system, has been an ongoing challenge in the tissue engineering field. By combining 3D bioprinting and human pluripotent stem cell (hPSC) technologies, sensible tissue constructs could be engineered in a rapid, precise, and controllable manner to replicate 3D microarchitectures and mechanosensory functionalities of the native sensory tissue (e.g. response to external stimuli). Here, we introduce a biofabrication approach to create complex 3D microarchitecture wirings. We develop an hPSC-sensory neuron (SN) laden bioink using highly purified and functional SN populations to 3D bioprint microarchitecture wirings that demonstrate responsiveness to warm/cold sense-inducing chemicals and mechanical stress. Specifically, we tailor a conventional differentiation strategy to our purification method by utilizing p75 cell surface marker and DAPT treatment along with neuronal growth factors in order to selectively differentiate neural crest cells into SNs. To create spatial resolution in 3D architectures and grow SNs in custom patterns and directions, an induced pluripotent stem cell (iPSC)-SN-laden gelatin bioink was printed on laminin-coated substrates using extrusion-based bioprinting technique. Then the printed constructs were covered with a collagen matrix that guided SNs growing in the printed micropattern. Using a sacrificial bioprinting technique, the iPSC-SNs were seeded into the hollow microchannels created by sacrificial gelatin ink printed in the gelatin methacryloyl supporting bath, thereby demonstrating controllability over axon guidance in curved lines up to several tens of centimeters in length on 2D substrates and in straight microchannels in 3D matrices. Therefore, this biofabrication approach could be amenable to incorporate sensible SN networks into the engineered skin equivalents, regenerative skin implants, and augmented somatosensory neuro-prosthetics that have the potential to regenerate sensible functions by connecting host neuron systems in injured areas.

Tunable and Compartmentalized Multimaterial Bioprinting for Complex Living Tissue Constructs

“…To evaluate cellular behaviors in the GCG support gels, gelatin and GelMA were mixed in different ratios, such as 1:3 (25:75 GCG), 2:2 (50:50 GCG), and 0:1 (0:100 GCG), and then cross-linked by UV light to make stable hydrogels in biological conditions. In our previous study, bulk GelMA prepolymer solution as a thermal healing supporting bath was used as an ECM-mimic for fabricating neurospheroid brain-like constructs. Here, we emphasize the ECM-mimic function of the GCG support baths compared with the bulk GelMA hydrogel.…”

Towards 3D Bioprinted Spinal Cord Organoids

“…Results from previous studies indicate that gelatin can be a suitable biomatrix for 3D bioprinting of stem cells [ 33 , 34 , 35 , 36 , 37 , 38 ] and may even in itself promote their neural differentiation [ 39 , 40 , 41 ]. The results suggest that culturing BC in gelatin-based hydrogels, which are enzymatically cross-linked with mTG, do not have a negative impact on viability compared to BC cultures on pre-cross-linked gelatin and reference cultures.…”

Functional bioengineered models of the central nervous system

“…A CNS–peripheral nervous system model, generated with specialized dorsal horn sensory neurons responsive to µ-opioid receptor activators and inhibitors, can be applied to study ascending spinal sensory pathways 206 . Similarly, neuro-mesodermal assembloids and 3D-bioprinted somatosensory constructs respond to capsaicin and menthol, recapitulating the receptive features of several pathways governing pain, temperature and taste 207 , 208 .…”