Universal Peptide Hydrogel for Scalable Physiological Formation and Bioprinting of 3D Spheroids from Human Induced Pluripotent Stem Cells

Abstract

Human induced pluripotent stem cells (hiPSCs) are used for drug discoveries, disease modeling and show great potential for human organ regeneration. 3D culture methods have been demonstrated to be an advanced approach compared to the traditional monolayer (2D) method. Here, a self-healing universal peptide hydrogel is reported for manufacturing physiologically formed hiPSC spheroids. With 100 000 hiPSCs encapsulated in 500 µL hydrogel, ≈50 000 spheroids mL −1 (diameter 20-50 µm) are generated in 5 d. The spheroids in the universal peptide hydrogel are viable (85-96%) and show superior pluripotency and differentiation potential based on multiple biomarkers. Cell performance is influenced by the degradability of the hydrogel but not by gel strength. Without postprinting crosslinking aided by UV or visible lights or chemicals, various patterns are easily extruded from a simple star to a kidney-like organ shape using the universal peptide hydrogel bioink showing acceptable printability. A 20.0 × 20.0 × 0.75 mm 3 sheet is finally printed with the universal peptide hydrogel bioink encapsulating hiPSCs and cultured for multiple days, and the hiPSC spheroids are physiologically formed and well maintained.

Injectable Peptide Hydrogel Encapsulation of Mesenchymal Stem Cells Improved Viability, Stemness, Anti-Inflammatory Effects, and Early Stage Wound Healing

“…However, these hydrogels are still relatively hard to handle, which hinders their application in clinical settings. We developed a shear-thinning peptide hydrogel (PGmatrix) [ 48 , 65 ] and used it successfully for virus delivery [ 49 ] and 3D culture of hiPSCs [ 47 ]. The main peptide in PGmatrix was made of 19 amino acids rationally designed by utilizing human muscle and spider silk protein domains [ 47 ] and it provided mainly mechanical support for cells without significant chemical or biological cues [ 47 ].…”

“…We developed a shear-thinning peptide hydrogel (PGmatrix) [ 48 , 65 ] and used it successfully for virus delivery [ 49 ] and 3D culture of hiPSCs [ 47 ]. The main peptide in PGmatrix was made of 19 amino acids rationally designed by utilizing human muscle and spider silk protein domains [ 47 ] and it provided mainly mechanical support for cells without significant chemical or biological cues [ 47 ]. PGmatrix was also identified to have minimal cell damage from mechanical stress through pipetting or extrusion printing nozzle [ 47 ] and high in vivo injection biocompatibility [ 50 ].…”

“…Several peptide hydrogels have been utilized in life science research, including EAK16, RADA16 (commercial name PuraMatrix, BD Bioscience), Fmoc-FF, Fmoc-RGD, and h9e (commercial name PGmatrix, PepGel LLC). Among these peptide hydrogels, PGmatrix has shown significant advantages in 3D cell cultures [ 42 , 45 , 46 , 47 ] and in vivo virus delivery by injection [ 48 , 49 ]. PGmatrix possesses tunable nanoweb pore size, gel strength and indefinite sol–gel recovery properties [ 47 ], and superior biocompatibility, without causing inflammatory effects in subcutaneous injection [ 50 ].…”

“…Among these peptide hydrogels, PGmatrix has shown significant advantages in 3D cell cultures [ 42 , 45 , 46 , 47 ] and in vivo virus delivery by injection [ 48 , 49 ]. PGmatrix possesses tunable nanoweb pore size, gel strength and indefinite sol–gel recovery properties [ 47 ], and superior biocompatibility, without causing inflammatory effects in subcutaneous injection [ 50 ]. We hypothesized that PGmatrix would provide improved functional cyto-properties of MSCs in 3D encapsulation, as well as in vivo delivery.…”

Receptor‐Level Proximity and Fastening of Ligands Modulates Stem Cell Differentiation

“…This assembly is known to further mediate mechanotransduction pathways and stem cell differentiation. [ 9 ] For instance, arrangements [ 10 ] and tethering [ 11 ] of cell‐adhesive ECM proteins are known to modulate the mechanotransduction [ 12 ] and differentiation of stem cells [ 13 ] that mediate tissue repair. [ 14 ] By leveraging these findings, tunable arrangements of RGD sites present in cell‐adhesive ECM proteins at the receptor‐level proximity can efficiently regulate the integrin ligation that triggers the mechanotransduction and differentiation of stem cells.…”