simple-lithography-free-single-cell-micropatterning-using-laser-cut

Simple Lithography-Free Single Cell Micropatterning using Laser-Cut Stencils

Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single ...

Stanford University School of Medicine

Stanford University

Smooth muscle tissue is characterized by aligned structures, which is critical for its contractile functions. Smooth muscle injury is common and can be caused by various diseases and degenerative processes, and there remains a strong need to develop effective therapies for smooth muscle tissue regeneration with restored structures. To guide cell alignment, previously cells were cultured on 2D nano/microgrooved substrates, but such method is limited to fabricating 2D aligned cell sheets only. Alternatively, aligned electrospun nanofiber has been employed as 3D scaffold for cell alignment, but cells can only be seeded post fabrication, and nanoporosity of electrospun fiber meshes often leads to poor cell distribution. To overcome these limitations, we report aligned gelatin-based microribbons (µRBs) as macroporous hydrogels for guiding smooth muscle alignment in 3D. We developed aligned µRB-like hydrogels using wet spinning, which allows easy fabrication of tissue-scale (cm) macroporous matrices with alignment cues and supports direct cell encapsulation. The macroporosity within µRB-based hydrogels facilitated cell proliferation, new matrix deposition, and nutrient diffusion. In aligned µRB scaffold, smooth muscle cells showed high viability, rapid adhesion, and alignment following µRB direction. Aligned µRB scaffolds supported retention of smooth muscle contractile phenotype, and accelerated uniaxial deposition of new matrix (collagen I/IV) along the µRB. In contrast, cells encapsulated in conventional gelatin hydrogels remained round with matrix deposition limited to pericellular regions only. We envision such aligned µRB scaffold can be broadly applicable in growing other anisotropic tissues including tendon, nerves and blood vessel.

Winner of the Young Investigator Award of the Society for Biomaterials (USA) for 2016, 10th World Biomaterials Congress, May 17-22, 2016, Montreal QC, Canada: Aligned microribbon-like hydrogels for guiding three-dimensional smooth muscle tissue regeneration.

The creation of physiologically-relevant human cardiac tissue with defined cell structure and function is essential for a wide variety of therapeutic, diagnostic, and drug screening applications. Here we report a new scalable method using Faraday waves to enable rapid aggregation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) into predefined 3D constructs. At packing densities that approximate native myocardium (10-10 cells/ml), these hiPSC-CM-derived 3D tissues demonstrate significantly improved cell viability, metabolic activity, and intercellular connection when compared to constructs with random cell distribution. Moreover, the patterned hiPSC-CMs within the constructs exhibit significantly greater levels of contractile stress, beat frequency, and contraction-relaxation rates, suggesting their improved maturation. Our results demonstrate a novel application of Faraday waves to create stem cell-derived 3D cardiac tissue that resembles the cellular architecture of a native heart tissue for diverse basic research and clinical applications.

Bioacoustic-enabled patterning of human iPSC-derived cardiomyocytes into 3D cardiac tissue.

Engineering 3D human cardiac tissues is of great importance for therapeutic and pharmaceutical applications. As cardiac tissue substitutes, extracellular matrix-derived hydrogels have been widely explored. However, they exhibit premature degradation and their stiffness is often orders of magnitude lower than that of native cardiac tissue. There are no reports on establishing interconnected cardiomyocytes in 3D hydrogels at physiologically-relevant cell density and matrix stiffness. Here we bioengineer human cardiac microtissues by encapsulating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in chemically-crosslinked gelatin hydrogels (1.25 × 10/mL) with tunable stiffness and degradation. In comparison to the cells in high stiffness (16 kPa)/slow degrading hydrogels, hiPSC-CMs in low stiffness (2 kPa)/fast degrading and intermediate stiffness (9 kPa)/intermediate degrading hydrogels exhibit increased intercellular network formation, α-actinin and connexin-43 expression, and contraction velocity. Only the 9 kPa microtissues exhibit organized sarcomeric structure and significantly increased contractile stress. This demonstrates that muscle-mimicking stiffness together with robust cellular interconnection contributes to enhancement in sarcomeric organization and contractile function of the engineered cardiac tissue. This study highlights the importance of intercellular connectivity, physiologically-relevant cell density, and matrix stiffness to best support 3D cardiac tissue engineering.

Contractile force generation by 3D hiPSC-derived cardiac tissues is enhanced by rapid establishment of cellular interconnection in matrix with muscle-mimicking stiffness.

Hypertrophic cardiomyopathy (HCM) is frequently caused by mutations in myosin-binding protein C3 ( MYBPC3) resulting in a premature termination codon (PTC). The underlying mechanisms of how PTC mutations in MYBPC3 lead to the onset and progression of HCM are poorly understood. This study's aim was to investigate the molecular mechanisms underlying the pathogenesis of HCM associated with MYBPC3 PTC mutations by utilizing human isogenic induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs).

A Premature Termination Codon Mutation in MYBPC3 Causes Hypertrophic Cardiomyopathy via Chronic Activation of Nonsense-Mediated Decay.

Different tissue types are characterized by varying stiffness and biochemical ligands. Increasing substrate stiffness has been shown to trigger Yes-associated protein (YAP) translocation from the cytoplasm to the nucleus, yet the role of ligand density in modulating mechanotransduction and stem cell fate remains largely unexplored. Using polyacrylamide hydrogels coated with fibronectin as a model platform, we showed that stiffness-induced YAP translocation occurs only at intermediate ligand densities. At low or high ligand densities, YAP localization is dominated by ligand density independent of substrate stiffness. We further showed that ligand density-induced YAP translocation requires cytoskeleton tension and αVβ3-integrin binding. Finally, we demonstrate that increasing ligand density alone can enhance osteogenic differentiation regardless of matrix stiffness. Together, the findings from the present study establish ligand density as an important parameter for modulating stem cell mechanotransduction and differentiation, which is mediated by integrin clustering, focal adhesion, and cytoskeletal tension.

Biochemical Ligand Density Regulates Yes-Associated Protein Translocation in Stem Cells through Cytoskeletal Tension and Integrins.

3D bioprinting technologies hold significant promise for the generation of engineered cardiac tissue and translational applications in medicine. To generate a clinically relevant sized tissue, the provisioning of a perfusable vascular network that provides nutrients to cells in the tissue is a major challenge. This review summarizes the recent vascularization strategies for engineering 3D cardiac tissues.

Soah Lee

Stanford Cardiovascular Institute

Stanford University School of Medicine

Simple Lithography-Free Single Cell Micropatterning using Laser-Cut Stencils