3D Bioprinting Human‐Induced Pluripotent Stem Cells and Drug‐Releasing Microspheres to Produce Responsive Neural Tissues

Abstract

3D bioprinting can produce complex human tissue mimics using stem cells (SCs). Herein, cylindrical constructs containing human‐induced pluripotent stem cell (hiPSC)‐derived neural progenitor cells (NPCs) encapsulated in a fibrin‐based bioink containing polycaprolactone (PCL)–retinoic acid (RA) and purmorphamine (puro)‐releasing microspheres are bioprinted in a layer‐by‐layer fashion using the microfluidic‐based RX1 bioprinter to engineer responsive neural tissues. The differentiated constructs contain neurons expressing ChAT, GABA, and MAP2, astrocytes expressing GFAP, and oligodendrocytes expressing O4 as indicated by immunocytochemistry and flow cytometry analysis on days 30 and 45. The bioprinted tissues also respond to treatment with acetylcholine (Ach) and gamma‐aminobutyric acid (GABA) on days 30 and 45. The use of microsphere‐laden bioinks efficiently promotes neural tissue differentiation and maturation in situ using a lower amount of morphogens in comparison with using soluble drugs. This bioprinting strategy serves as a cost‐effective solution for engineering humanized neural tissues.

Physical and Mechanical Characterization of Fibrin-Based Bioprinted Constructs Containing Drug-Releasing Microspheres for Neural Tissue Engineering Applications

“…The differentiation of NPCs to mature phenotypes in bioprinted constructs depends on several factors, including the stiffness and porosity of the bioink that comprises the microenvironment. Our group has previously established that fibrin-based bioink printed with and without microspheres enables neuronal survival, proliferation, differentiation, and maturation [59,60,62,68,70]. In this study, we have determined the physicomechanical properties of bioprinted constructs as well as manually combined bioink and crosslink for tissue engineering applications.…”

“…Microspheres (1-1000 µm in diameter) are drug-delivery particles that release drugs at a controlled rate to overcome localized drug-delivery issues and enhance the therapeutic efficacy of a drug [52,59,60,68]. Our previous work validated that microspheres provide a protective environment around the cells that reduces damage to cells while printing, which enhances cell viability post-printing; additionally, these bioprinted constructs containing microspheres provide an appropriate 3D microenvironment for cells to grow and differentiate [60].…”

“…Constructs were also bioprinted with varying densities of hiPSC-derived NPCs, such as 1, 2.5, and 5 million cells per 1 mL of bioink, to analyze viscoelastic properties. NPCs were mixed with bioink as in previously described protocols, and bioprinted constructs were fixed with paraformaldehyde before analyzing [59,60,70]. The printing speed was 25 mm/s, and channel pressures consisted of 60 mbar for the bioink, 50 mbar for the crosslinker, and 100 mbar for buffer solution.…”

“…We have designed and validated our fibrin-based bioink incorporated with drugreleasing microspheres for creating functional neural tissues from hiPSC-derived NPCs in our previous works [59,60]. hiPSCs are especially relevant to neural tissue engineering since brain cells from patients are rarely available for research.…”

“…Our group found that drug-releasing microspheres in bioprinted constructs enhanced neuronal differentiation by providing the localized and controlled delivery of morphogens over time. In contrast to procedures that require changing morphogencontaining media frequently, microspheres are a more cost-effective morphogen-delivery method [59,60].…”