3D Bioprinting the Cardiac Purkinje System Using Human Adipogenic Mesenchymal Stem Cell Derived Purkinje Cells.

PURPOSE: The objective of this study was to reprogram human adipogenic mesenchymal stem cells (hADMSCs) to form Purkinje cells and to use the reprogrammed Purkinje cells to bioprint Purkinje networks. METHODS: hADMSCs were reprogrammed to form Purkinje cells using a multi-step process using transcription factors ETS2 and MESP1 to first form cardiac progenitor stem cells followed by SHOX2 and TBX3 to form Purkinje cells. A novel bioprinting method was developed based on Pluronic acid as the sacrificial material and type I collagen as the structural material. The reprogrammed Purkinje cells were used in conjunction with the novel bioprinting method to bioprint Purkinje networks. Printed constructs were evaluated for retention of functional protein connexin 40 (Cx40) and ability to undergo membrane potential changes in response to physiologic stimulus. RESULTS: hADMSCs were successfully reprogrammed to form Purkinje cells based on the expression pattern of IRX3, IRX5, SEMA and SCN10. Reprogrammed purkinje cells were incorporated into a collagen type-1 bioink and the left ventricular Purkinje network was printed using anatomical images of the bovine Purkinje system as reference. Optimization studies demonstrated that 1.8 mg/mL type-I collagen at a seeding density of 300,000 cells per 200 µL resulted in the most functional bioprinted Purkinje networks. Furthermore, bioprinted Purkinje networks formed continuous syncytium, retained expression of vital functional gap junction protein Cx40 post-print, and exhibited membrane potential changes in response to electric stimulation and acetylcholine evaluated by DiBAC4(5), an electrically responsive dye. CONCLUSION: Based on the results of this study, hADMSCs were successfully reprogrammed to form Purkinje cells and bioprinted to form Purkinje networks.

Formation of Adipose Stromal Vascular Fraction Cell-Laden Spheroids Using a Three-Dimensional Bioprinter and Superhydrophobic Surfaces.

The therapeutic infusion of adipose-derived stromal vascular fraction (SVF) cells for the treatment of multiple diseases, has progressed to numerous human clinical trials; however, the often poor retention of the cells following implantation remains a common drawback of direct cell injection. One solution to cellular retention at the injection site has been the use of biogels to encapsulate cells within a microenvironment before and upon implantation. The current study utilized three-dimensional bioprinting technology to evaluate the ability to form SVF cell-laden spheroids with collagen I as a gel-forming biomatrix. A superhydrophobic surface was created to maintain the bioprinted structures in a spheroid shape. A hydrophilic disc was printed onto the hydrophobic surface to immobilize the spheroids during the gelation process. Conditions for the automated rapid formation of SVF cell-laden spheroids were explored, including time/pressure relationships for spheroid extrusion during bioprinting. The formed spheroids maintain SVF viability in both static culture and dynamic spinner culture. Spheroids also undergo a time-dependent contraction with the retention of angiogenic sprout phenotype over the 14-day culture period. The use of a biphilic surface exhibiting both superhydrophobicity to maintain the spheroid shape and a hydrophilicity to immobilize the spheroid during gel formation produces SVF cell-laden spheroids that can be immediately transplanted for therapeutic applications.