Layer-By-Layer Fabrication of Large and Thick Human Cardiac Muscle Patch Constructs With Superior Electrophysiological Properties

Abstract

Engineered cardiac tissues fabricated from human induced pluripotent stem cells (hiPSCs) show promise for ameliorating damage from myocardial infarction, while also restoring function to the damaged left ventricular (LV) myocardium. For these constructs to reach their clinical potential, they need to be of a clinically relevant volume and thickness, and capable of generating synchronous and forceful contraction to assist the pumping action of the recipient heart. Design prerequisites include a structure thickness sufficient to produce a beneficial contractile force, prevascularization to overcome diffusion limitations and sufficient structural development to allow for maximal cell communication. Previous attempts to meet these prerequisites have been hindered by lack of oxygen and nutrient transport due to diffusion limits (100–200 μm) resulting in necrosis. This study employs a layer-by-layer (LbL) fabrication method to produce cardiac tissue constructs that meet these design prerequisites and mimic normal myocardium in form and function. Thick (>2 mm) cardiac tissues created from hiPSC-derived cardiomyocytes, -endothelial cells (ECs) and -fibroblasts (FBs) were assessed, in vitro, over a 4-week period for viability (<6% necrotic cells), cell morphology and functionality. Functional performance assessment showed enhanced t-tubule network development, gap junction communication as well as previously unseen, physiologically relevant conduction velocities (CVs) (>30 cm/s). These results demonstrate that LbL fabrication can be utilized successfully to create prevascularized, functional cardiac tissue constructs from hiPSCs for potential therapeutic applications.

Layer-By-Layer Fabrication of Thicker and Larger Human Cardiac Muscle Patches for Cardiac Repair in Mice

“…hCMPs with thicknesses exceeding 2 mm have also been produced by depositing 2–3 layers of cell-containing fibrin solution into a single mold, and allowing each layer to polymerize before adding the subsequent layer ( 12 ). However, the thickness of an hCMP is limited primarily by the diffusion of oxygen and nutrients from the culture medium.…”

“…The Japanese health ministry has approved studies of hiPSC-derived tissues in a small number of patients ( 10 ), but the hCMPs produced via most manufacturing techniques are typically just a few hundred micrometers thick, which has impeded the translation of this technology to the clinic ( 11 ). Previously, we have generated hCMPs with thicknesses exceeding 2 mm via a novel layer-by-layer (LBL) method of hCMP assembly ( 12 ): hiPSC-derived cardiac cells were suspended in fibrinogen solution, and the solution was mixed with thrombin, deposited into a mold, and allowed to solidify before the procedure was repeated to produce two additional cell layers. In theory, an LBL manufacturing protocol could be used to produce hCMPs of any desired thickness by simply stacking the required number of cell layers ( 13 ); however, as hCMP thickness increases, cell viability tends to decline, because the diffusion of oxygen and nutrients from the media to interior of the hCMP is impaired.…”

Engineering Complex Anisotropic Scaffolds beyond Simply Uniaxial Alignment for Tissue Engineering

“…A compelling challenge is the size limitation of the current engineered anisotropic scaffolds at one of the dimensions, such as the diameter of tissue‐engineered skeletal muscle [ 215 ] and the thickness of cardiac muscle patch, [ 216 ] which restricts their applications in modeling in vitro and regeneration in vivo. Cells are difficult to survive beyond a diffusion limitation of oxygen and nutrient supply, about 100–200 µm away from capillaries or culture medium.…”

Engineering of thick human functional myocardium via static stretching and electrical stimulation

“…hCMPs were assembled in three layers over a three-day period (one layer per day). Each layer was generated by depositing 600 μL of an hiPSC-CM–containing fibrin solution ( Gao et al., 2018 ; Pretorius et al., 2020 , 2021 ) (10 × 10 6 cells/mL) into the frame ( Pretorius et al., 2020 , 2021 ); then, after fibrin polymerization, the layer was cultured in STEMdiff Cardiomyocyte Support Medium with 2 mg/mL ε-aminocaproic acid overnight at 37 °C and 5% CO 2 ; the procedure was repeated twice to form the second and third layers. After the third cell layer was generated, the frame containing the engineered tissue was lifted off of the petri dish and placed on a custom-cut polydimethylsiloxane (PDMS) platform; then, the hCMP was cultured in fresh culture medium consisting of 2% fetal bovine serum (FBS), 2% B27 + (Gibco), and 2 mg/mL ε-aminocaproic acid in RPMI (Gibco) for one week before the maturation protocols were initiated.…”