Towards safe Stem Cell-Based Regenerative Medicine: Implementation of 3D Bioprinting and Microfluidic Systems

Background and Aim: Stem cells (SCs) are the frontline regenerativemedicine source. Efforts are being made to found stem cell-basedregenerative medicine onto tissue engineering technologies; however,the safety and immunotolerance of the engineered implants remaincritical issues for clinical applications. Ideally the engineered tissuegrafts should not only support cell adhesion/ingrowths but also eliminateharmful dividing SCs, be nonimmunogenic and support angiogenesis.This presentation attempts to highlight the significance of Fail-Safe andimmuno-cloaking systems combined with microfluidic systems and3D-bioprinting technologies to address these issues.Methods: Based on the protocol developed in the Nagy’s Lab (Liang Q,et al. A solution for cell therapy safety. Nature. In press), the Ganciclovir(GVC)-inducible suicide system was generated for human embryonicSCs (hESCs). The Fail-Safe system was evaluated using the teratomaassay in NSG mice. The Fail-Safe hESCs were genetically manipulatedto become immune-cloaked through the modulation of eight genesinvolved in immunoresponse. The Fail-Safe immuno-cloaked hESCswere differentiated into functioning cardiomyocytes and endothelialcells (ECs). Relying on microfluidic tools, a microvasculature-on-achipwas developed to create a perfused network of endothelializedmicrochannels connected to a blood flow mimicking medium containinghuman peripheral blood mononuclear cells (PBMCs). Integrated withtwo-photon confocal microscopy imaging, the microfluidic deviceenabled the real-time assessment of cloaking behavior of ECs. The ECswere bioprinted into 3D hybrid nano-reinforced hydrogel scaffolds madeof methacrylated collagen and alginate followed by characterizing thescaffold physical and biological characterization. Also, the cloakedcardiomyocytes functionality on the bioprintable hydrogel was assessed.Results: The GCV treatment of the Fail-Safe derived teratomas in NSG miceresulted in stabilizing teratoma size. The cloaked ESCs were successfullydifferentiated into cloaked ECs (and cardiomyocytes) confirmed byflow cytometry analyses, immunohistochemistry and functional assays.The microchannels of the PDMS-based microfluidic device with thevasculature-like network were coated with ECM components whichpromoted EC attachment and maturation to form perfused lumens in-situ.The real-time qualitative/quantitative evaluation of the endothelializedmicrofluidic device revealed a significantly lower PBMC attachment andEC death for cloaked ECs. The differentiated ECs and cardiomyocytesexhibited positive tube formation and contractility when grown onmethacrylated collagen hydrogel. The cloaked cells were successfullybioprinted into a hybrid scaffold consisting of methacrylated collagenand alginate. Electrical and mechanical properties of the 3D scaffoldwere improved by modulation of bioprinting pattern and incorporationof functionalized carbon nanotube into the hydrogel.Conclusion: The hESC-based Fail-Safe system enabled us to arrestdividing stem cells in NSD mice. Our real-time assessment of theinteraction between human PBMCs and cloaked cells in the microfluidicdevice showed that the cloaked ECs and cardiomyocytes exhibitedimmunotolerance behavior. The successful implementation of bioprintingof cloaked cells offers great promise to create 3D hydrogel scaffoldswhich take advantage of the allogenic tolerance technology for a saferegenerative therapy.

کلیدواژه ها:

Bioprinting ، Cardiomyocyte ، Endothelial ، Immune tolerance ، Microfluidic system ، Stem cell therapy