Rock inhibitor may compromise human induced pluripotent stem cells for cardiac differentiation in 3D

Cardiomyocytes differentiated from human induced pluripotent stem cells(iPSCs)are valuable for the understanding/treatment of the deadly heart diseases and their drug screening.However,the very much needed homogeneous 3D cardiac differentiation of human iPSCs is still challenging.Here,it is discovered surprisingly that Rock inhibitor(RI),used ubiquitously to improve the survival/yield of human iPSCs,induces early gastrulation-like change to human iPSCs in 3D culture and may cause their heterogeneous differentiation into all the three germ layers(i.e.,ectoderm,mesoderm,and endoderm)at the commonly used concentration(10μM).This greatly compromises the capacity of human iPSCs for homogeneous 3D cardiac differentiation.By reducing the RI to 1μM for 3D culture,the human iPSCs retain high pluripotency/quality in inner cell mass-like solid 3D spheroids.Consequently,the beating efficiency of 3D cardiac differentiation can be improved to more than 95%in~7 days(compared to less than~50%in 14 days for the 10μM RI condition).Furthermore,the outset beating time(OBT)of all resultant cardiac spheroids(CSs)is synchronized within only 1 day and they form a synchronously beating 3D construct after 5-day culture in gelatin methacrylol(GelMA)hydrogel,showing high homogeneity(in terms of the OBT)in functional maturity of the CSs.Moreover,the resultant cardiomyocytes are of high quality with key functional ultrastructures and highly responsive to cardiac drugs.These discoveries may greatly facilitate the utilization of human iPSCs for understanding and treating heart diseases.

Noncovalent reversible binding-enabled facile fabrication of leak-free PDMS microfluidic devices without plasma treatment for convenient cell loading and retrieval

The conventional approach for fabricating polydimethylsiloxane(PDMS)microfluidic devices is a lengthy and inconvenient procedure and may require a clean-room microfabrication facility often not readily available.Furthermore,living cells can’t survive the oxygen-plasma and high-temperature-baking treatments required for covalent bonding to assemble multiple PDMS parts into a leak-free device,and it is difficult to disassemble the devices because of the irreversible covalent bonding.As a result,seeding/loading cells into and retrieving cells from the devices are challenging.Here,we discovered that decreasing the curing agent for crosslinking the PDMS prepolymer increases the noncovalent binding energy of the resultant PDMS surfaces without plasma or any other treatment.This enables convenient fabrication of leak-free microfluidic devices by noncovalent binding for various biomedical applications that require high pressure/flow rates and/or long-term cell culture,by simply hand-pressing the PDMS parts without plasma or any other treatment to bind/assemble.With this method,multiple types of cells can be conveniently loaded into specific areas of the PDMS parts before assembly and due to the reversible nature of the noncovalent bonding,the assembled device can be easily disassembled by hand peeling for retrieving cells.Combining with 3D printers that are widely available for making masters to eliminate the need of photolithography,this facile yet rigorous fabrication approach is much faster and more convenient for making PDMS microfluidic devices than the conventional oxygen plasma-baking-based irreversible covalent bonding method.