Vascularized organoid engineered by modular assembly enables blood perfusion

Abstract

Tissue engineering is one approach to address the donor-organ shortage, but to attain clinically significant viable cell densities in thick tissues, laboratory-constructed tissues must have an internal vascular supply. We have adopted a biomimetic approach and assembled microscale modular components, consisting of submillimeter-sized collagen gel rods seeded with endothelial cells (ECs) into a (micro)vascularized tissue; in some prototypes the gel contained HepG2 cells to illustrate the possibilities. The EC-covered modules then were assembled into a larger tube and perfused with medium or whole blood. The interstitial spaces among the modules formed interconnected channels that enabled this perfusion. Viable cell densities were high, within an order of magnitude of cell densities within tissues, and the percolating nature of the flow through the construct was evident in microcomputed tomography and Doppler ultrasound measurements. Most importantly, the ECs retained their nonthrombogenic phenotype and delayed clotting times and inhibited the loss of platelets associated with perfusion of whole blood through the construct. Unlike the conventional scaffold and cell-seeding paradigm of other tissue-engineering approaches, this modular construct has the potential to be scalable, uniform, and perfusable with whole blood, circumventing the limitations of other approaches.

Fig. 1.

Modular construct design and fabrication. (a) Collagen with or without HepG2 cells is drawn into the lumen of a sterilized PE tube and incubated at 37°C for 30 min to allow gelation. The PE tubing containing the gel is fed through an automated tubing cutter and sectioned into 2-mm lengths, which are collected in a sterile centrifuge tube. Cell culture medium is added, and the tube is vortexed to release the collagen–cell modules from the lumen of the sectioned PE pieces. PE sections float, whereas collagen modules sink. The collagen cylinders with encapsulated HepG2 cells are subsequently seeded with HUVEC. Once complete coverage of the collagen surface with HUVEC has been achieved (typically 2–3 days), the cell-seeded cylinders are assembled into a larger structure (here a tube) to form the construct. Assembly of the modules creates a network of interconnected channels that permeate the construct. Medium or blood is perfused through this network to supply nutrients to the cells within the construct. (b) Light micrograph of a collagen–HepG2 module before HUVEC seeding. (c) Confocal microscopy image of vascular endothelial (VE)-cadherin-stained module indicating a confluent layer of HUVEC over the module surface at 7 days after seeding. (d) Modular construct in the flow circuit being perfused with PBS. (e) Confocal microscope image of a collagen–HepG2–HUVEC module retrieved from a construct after 7 days of medium perfusion with HepG2 cells labeled with Vybrant CFDA SE.

Fig. 2.

Characterization of constant porosity and shear stress on the surface of the modules during construct perfusion. (a) Flow and shear profiles through two collagen modular constructs. Flow rate of PBS through two separate constructs (construct length, 0.5 cm; construct diameter, 0.3 cm) as a function of applied pressure difference (hydrostatic head); open and filled points represent different constructs. Each point is the mean of two flow-rate measurements made at each pressure difference. (Inset) The slope of the fitted line was used to calculate construct porosity by using the Ergun equation (13) from which the shear stress on the surface of the modules was calculated for each construct. (b) Microcomputed tomography image of microfil cast of a poloxamine (22) modular construct (without HUVEC). Poloxamine is a stiffer material, enabling microfil casting. Light-colored regions correspond to the microfil (i.e., the channels), and dark regions correspond to modules, illustrating the interconnectedness of the flow channels that are normally lined with ECs. Porosity based on the number of light pixels was 22.6%. A relatively high pressure was required to fully infiltrate the viscous microfil, and hence this technique was not suitable for assessing porosity in a lower-stiffness HUVEC-coated collagen construct.

Fig. 3.

Characterization of module thrombogenicity using whole-blood studies. (a) Clot formation times. The presence of HUVEC on the modules significantly increased the time to clot formation (P = 1.4 × 10⁻⁵) of slightly heparinized whole blood (0.75 units/ml) in a clotting test. In some cases, clot formation never actually occurred, and the test was terminated between 4,500 and 5,400 s; in these instances, the recorded time was the test termination time. Mean clot time is represented by the thick central line within the box. Open circles and stars represent outliers and extreme outliers, respectively. (b) Fresh whole blood (0.75 units/ml heparin) perfused through a HUVEC-covered modular construct (filled circles) maintains platelet levels no different from those measured in the absence of modules (open circles, flow circuit blank; includes polypropylene mesh required to keep modules in place). Blood perfusion through a control modular construct in which HUVEC have been removed by dispase–collagenase action (open squares), however, results in significant reductions in platelet number, indicating platelet activation and the thrombogenic response that occurs in the absence of HUVEC. Error bars indicate SEM (n = 3, 4, and 7 for background, dispase-treated modular constructs, and HUVEC-covered modular constructs, respectively).