Bioprinting Approaches to Engineering Vascularized 3D Cardiac Tissues

Abstract

Purpose of review: 3D bioprinting technologies hold significant promise for the generation of engineered cardiac tissue and translational applications in medicine. To generate a clinically relevant sized tissue, the provisioning of a perfusable vascular network that provides nutrients to cells in the tissue is a major challenge. This review summarizes the recent vascularization strategies for engineering 3D cardiac tissues.

Recent findings: Considerable steps towards the generation of macroscopic sizes for engineered cardiac tissue with efficient vascular networks have been made within the past few years. Achieving a compact tissue with enough cardiomyocytes to provide functionality remains a challenging task. Achieving perfusion in engineered constructs with media that contain oxygen and nutrients at a clinically relevant tissue sizes remains the next frontier in tissue engineering. The provisioning of a functional vasculature is necessary for maintaining a high cell viability and functionality in engineered cardiac tissues. Several recent studies have shown the ability to generate tissues up to a centimeter scale with a perfusable vascular network. Future challenges include improving cell density and tissue size. This requires the close collaboration of a multidisciplinary teams of investigators to overcome complex challenges in order to achieve success.

Keywords: 3D printing; Bioprinting; Cardiac engineered tissue; Cardiomyocyte; Cardiovascular tissue; Vascularization.

Figure 1:

Three-dimensional vascularized tissues remain stable during long-term perfusion. (A)Schematic depicting a single HUVEC-lined vascular channel supporting a fibroblast cell-laden matrix and housed within a 3D perfusion chip. (B and C) Confocal microscopy image of the vascular network after 42 d, CD-31 (red), vWF (blue), and VE-Cadherin (magenta). (Scale bars: 100 μm.) (D) Long-term perfusion of HUVEC-lined (red) vascular network supporting HNDF- laden (green) matrix shown by top-down (Left) and cross-sectional confocal microscopy at 45 d (Right). (Scale bar: 100 μm.) (E) Quantification of barrier properties imparted by endothelial lining of channels, demonstrated by reduced diffusional permeability of FITC-dextran. (F) GFP-HNDF distribution within the 3D matrix shown by fluorescent intensity as a function of distance from vasculature. Reproduced with permission from Kolesky et al. PNAS 2016

Figure 2:

Multi-cellular 3D bioprinted cardiac tissue constructs. Representative images showing TNNI (red) and Cx43 (green) expressions in CMs and vWF (green) labelling in HUVEC, after 7 days of culture, printed in three different spatial geometries. Janus constructs contained the two different cell lineages within the each laid fiber; 4:2:4 and 2:2:2:2:2 structures were printed altering two layers of HUVEC with two or four layers of CM. Scale bars represent 50 μm. Reproduced with permission from Maiullari et al. Scientific Reports 2018.

Figure 3:

In vitro anastomosis of human embryonic stem cell-derived endothelial cells (hESC-ECs) in engineered microvessels (μVs). a Schematic of in vitro culture device for μV+SA constructs: mTm-hESC-EC μVs formed via perfusion and attachment with bulk-seeded GFP-hESC-ECs in the surrounding collagen gel. b Maximum intensity projection of stitched large image confocal z-stack of μV+SA construct cultured for 4 days and stained for DsRed (red) and GFP (green) to detect mTm- and GFP-expressing hESC-ECs, respectively. Scale bar, 500 μm. c Outlined region (white box) in b stained for DsRed (red, top left), GFP (green, top right), and VE-cadherin (white, bottom left). Merged image, bottom right. Scale bar, 200 μm. d High magnification images of GFP-hESC-ECs (green) integrated with mTm-hESC-EC (red) patterned vessel in μV+SA constructs. Scale bar, 50 μm. e Quantitation of sprouts from patterned μVs by sprout density (no. of sprouts per vessel surface area), sprout length, and sprout diameter in μV only (blue circles) and μV+SA (green circles) constructs after 4 days and 7 days of culture. N = 6, 7, 4, and 3 biologically independent samples for D4 μV only, D4 μV+SA, D7 μV only, and D7 μV+SA, respectively. p = 0.011 for length and p = 0.007 for diameter for D4 μV only and D7 μV only, p > 0.05 for all others (two-tailed t test). f 3D view of GFP+ de novo lumen integrated with mTm+ microvascular sprout (white arrowheads) stained for CD31 (red) and GFP (green). Scale bar, 100 μm. Representative images for b–d, f from seven biologically independent samples of D4 μV+SA, with similar results. Hoechst-stained nuclei, blue. Error bars, mean ± SEM. *p < 0.05 determined using two-tailed t test. D4 after 4 days of culture, D7 after 7 days of culture. Reproduced with permission from Redd et al. Nature communications 2019