Biofabrication enables efficient interrogation and optimization of sequential culture of endothelial cells, fibroblasts and cardiomyocytes for formation of vascular cords in cardiac tissue engineering

Abstract

We previously reported that preculture of fibroblasts (FBs) and endothelial cells (ECs) prior to cardiomyocytes (CMs) improved the structural and functional properties of engineered cardiac tissue compared to culture of CMs alone or co-culture of all three cell types. However, these approaches did not result in formation of capillary-like cords, which are precursors to vascularization in vivo. Here we hypothesized that seeding the ECs first on Matrigel and then FBs 24 h later to stabilize the endothelial network (sequential preculture) would enhance cord formation in engineered cardiac organoids. Three sequential preculture groups were tested by seeding ECs (D4T line) at 8%, 15% and 31% of the total cell number on Matrigel-coated microchannels and incubating for 24 h. Cardiac FBs were then seeded (32%, 25% and 9% of the total cell number, respectively) and incubated an additional 24 h. Finally, neonatal rat CMs (60% of the total cell number) were added and the organoids were cultivated for seven days. Within 24 h, the 8% EC group formed elongated cords which eventually developed into beating cylindrical organoids, while the 15% and 31% EC groups proliferated into flat EC monolayers with poor viability. Excitation threshold (ET) in the 8% EC group (3.4 ± 1.2 V cm−1) was comparable to that of the CM group (3.3 ± 1.4 V cm−1). The ET worsened with increasing EC seeding density (15% EC: 4.4 ± 1.5 V cm−1; 31% EC: 4.9 ± 1.5 V cm−1). Thus, sequential preculture promoted vascular cord formation and enhanced architecture and function of engineered heart tissues.

Materials Science and Tissue Engineering: Repairing the Heart

“…Other strategies for inducing rapid vascularization of cardiac tissues include the modular approach 89 and co-culture with endothelial cells and fibroblasts. 19-21, 90 In vivo , angiogenesis involves endothelial cell proliferation and sprouting followed by connection of extended cellular processes and subsequent lumen propagation through vacuole fusion. This process was mimicked by engineering an organized capillary network anchored by an artery and a vein.…”

Vascular Endothelial Growth Factor Secretion by Nonmyocytes Modulates Connexin-43 Levels in Cardiac Organoids

“…The experimental groups investigated to identify the possible sources and targets of VEGF signaling are summarized in Table 1. Three coculture groups were formed: (i) Simultaneous Preculture, as in our earlier work, 4,14 where FBs and ECs were simultaneously seeded and cultured for 2 days prior to CM seeding; (ii) Sequential Preculture, as in our more recent work, 15 where ECs were first seeded for 24 h to allow cord formation, followed by FBs 24 h later to stabilize the cords, and CMs after an additional 24 h; (3) Simultaneous Triculture, 4,13 where all three cell types (ECs, FBs, and CMs) were seeded simultaneously and cultured together. Organoids consisting of ECs, or FBs or Enriched CMs served as monoculture controls, while organoids precultured with ECs (Preculture ECs) or FBs (Preculture FBs) served as coculture controls.…”

Fabrication Techniques for Vascular and Vascularized Tissue Engineering

“…For example, HUVECs cocultured with apical papilla‐derived stem cells in GelMA hydrogels show higher tube formation efficiency with an improved compressive modulus . ECs and supporting cells are frequently cocultured with tissue‐specific types cells such as myofibroblasts, cardiomyocytes, and hepatocytes on scaffolds for the fabrication of functional vascularized tissues. For example, the addition of FBs in EC/myofibroblast coculture system on porous biodegradable PLA or PLGA scaffolds increased the production of VEGF, which improved capillary formation in vitro and in vivo for a tissue‐engineered muscle construct .…”

“…In cocultures, the cell seeding order has been shown to influence capillary formation. For example, sequential seeding on porous scaffolds of ECs, FBs, and cardiomyocytes with 24 h intervals between each cell addition resulted in extensive capillary network formation whereas coculture of these cells when seeded at the same time showed no capillary formation …”