A Cell Pre-Wrapping Seeding Technique for Hydrogel-Based Tubular Organ-On-A-Chip

Abstract

Organ-on-a-chip (OOC) models based on microfluidic technology are increasingly used to obtain mechanistic insight into (patho)physiological processes in humans, and they hold great promise for application in drug development and regenerative medicine. Despite significant progress in OOC development, several limitations of conventional microfluidic devices pose challenges. First, most microfluidic systems have rectangular cross sections and flat walls, and therefore tubular/ curved structures, like blood vessels and nephrons, are not well represented. Second, polymers used as base materials for microfluidic devices are much stiffer than in vivo extracellular matrix (ECM). Finally, in current cell seeding methods, challenges exist regarding precise control over cell seeding location, unreachable spaces due to flow resistances, and restricted dimensions/geometries. To address these limitations, an alternative cell seeding technique and a corresponding workflow is introduced to create circular cross-sectioned tubular OOC models by pre-wrapping cells around sacrificial fiber templates. As a proof of concept, a perfusable renal proximal tubule-on-a-chip is demonstrated with a diameter as small as 50 µm, cellular tubular structures with branches and curvature, and a preliminary vascular-renal tubule interaction model. The cell pre-wrapping seeding technique promises to enable the construction of diverse physiological/pathological models, providing tubular OOC systems for mechanistic investigations and drug development.

Keywords: cell pre‐wrapping seeding; hydrogel; renal proximal tubule; sacrificial template; tubular organ‐on‐a‐chip.

Figure 1

From 3D printed sugar fiber to tubular OOC models.

Figure 2

3D sugar printing to create luminal structures in hydrogel: effect of printing parameters on dimensions, and images of printed fiber and luminal geometries. A) Dependency of printed sugar (sucrose–glucose mixture) fiber diameter on nozzle translation speed and extrusion temperature at an extrusion pressure of 1 bar. B) Dependency of printed sugar (maltitol) fiber diameter on nozzle translation speed and extrusion pressure at an extrusion temperature of 135 °C. Images of printed sugar fiber structures with curved (Ci), bifurcating (Di), and parallel (Ei, Fi) geometries; the sugar fiber structures are suspended in a sugar frame at a height of 2 mm above the glass substrate. Perspective and detailed images of the GelMA casts around the fiber structures after sugar dissolution and injection of blue ink showing curved (Cii, Ciii), bifurcating (Dii, Diii), and parallel (Eii, Eiii, Fii, Fiii) luminal geometries.

Figure 3

Key points of cell pre‐wrapping seeding. A) Schematic image of cells suspended in simple cell medium. B) Microscopy images of cells floating in cell medium (i), most of them being flushed away (ii). C) Schematic image presenting preparation of cell‐laden fibrin. D) Microscopy images of cells encapsulated in fibrin and fixed at an initial location (i), and released from fibrin through its degradation (ii). E) Microscopic display of the contact angle of a fibrin drop on the PDLGA coated sugar surface. F) Microscopic display of the contact angle of a fibrin drop on the F‐127 coated surface. G) Microscopic display of the fibrin droplets placed on PDLGA coated sugar fiber. H) Microscopic display of the fibrin droplets placed on F‐127 coated sugar fiber. I) Fluorescence micrograph of F‐actin/nuclei markers of the RPTEC monolayer formed through a cell‐carrying fibrin droplet placed on an F‐127 coated sugar fiber, after 7 days of culture. J) Schematic images illustrating the stamping seeding technique, including: i) extrusion of the cell‐laden fibrin solution; ii) adhesion of the cell suspension on the coated sugar fiber; iii) retraction of the remaining cell suspension. Ki,ii) Microscopic display of the stamping seeding process. Kiii) Microscopic display of the stamping seeding result. L) Schematic illustrations of sugar fiber bound with hydrophilic groups and stamped with cell‐laden fibrin, in laterial view, as well as cross‐section image showing the full wrapping of cell‐laden fibrin all around the fiber. M) Image showing the seeding result of RPTECs on sugar fiber with a diameter of 400 µm. N) Schematic image showing the cross section of the hydrogel lumen covered with epithelium. O) Microscopy morphology of the cross section of the RPTEC monolayer. P) Fluorescence micrograph of F‐actin/nuclei markers of the RPTEC monolayer formed by stamping seeding.

Figure 4

Complete workflow for engineering perfusable microfluidic tubular OOC models by pre‐wrapping seeding cells around 3D printed sacrificial sugar templates, applied to creating a renal proximal tubule‐on‐a‐chip. 1) 3D printing of a suspended sugar fiber structure as a sacrificial template. 2) PDLGA coating to protect the sugar fibers from early dissolution. 3) F‐127 coating to improve surface hydrophilicity for uniform cell seeding. 4) Pre‐wrapping seeding of cell‐laden fibrin around the sugar fiber; the cells are RPTECs for emulating the renal proximal tubule. 5) GelMA casting around the sugar fiber structure and subsequent UV crosslinking. 6) Sugar dissolution in cell medium. 7) Culture under perfusion after connection with a pump system.

Figure 5

Characterization of the renal proximal tubule‐on‐a‐chip model. Fluorescence micrographs of the renal proximal tubule epithelium inside lumens with a diameter of 400 µm (A), 200 µm (B), and 50 µm (C) after 7 days of culture showing of F‐actin and nuclei markers. The cells are RPTECs and the matrix is GelMA. D) Fluorescence micrographs of the renal proximal tubule epithelium inside lumens with branching structures. E) Fluorescence micrographs of the renal proximal tubule epithelium inside lumens with curved shapes. F) Viability characterization of the RPTECs in the renal proximal tubule‐on‐a‐chip model compared to a control group. G) Proliferation characterization of the RPTECs in the renal proximal tubule‐on‐a‐chip model compared to a control group. H–K) Fluorescence micrographs (different magnifications) of the renal proximal tubule epithelium along a 400 µm lumen showing α‐tubulin, F‐actin, and nuclei markers, after 96 h of culture under different flow conditions: H) no flow (static), I) FSS = 0.13 dyne cm⁻²(physiological flow), J) FSS = 0.26 dyne cm⁻², K) FSS = 0.52 dyne cm⁻². L) Overall comparison of cell covered area and tubulin protein expression after 96 h of culture under different flow conditions.

Figure 6

Demonstration of preliminary vascular‐renal tubule interaction model constructed by the cell pre‐wrapping seeding technique. Ai) Schematic of the nephron: the renal tubule is surrounded by vasculature. (Aii) Schematic diagram of the simplified adjacent parallel channel model. B) Fluorescence micrographs (different magnifications) of the engineered vascular‐renal tubule interaction model; HUVEC transfected with GFP: green, F‐actin: red, nuclei: blue. C) Time‐lapse confocal images of the permeating patterns of FITC‐dextran through the endothelialized lumen; HUVEC transfected with GFP: green. D) Profiles of the fluorescence intensity across the endothelialized lumen at different time points. E) Time‐lapse confocal images of the permeating patterns of FITC‐dextran through the blank lumen. F) Profiles of the fluorescence intensity across the blank lumen at different time points. G) Comparison of the permeability of FITC‐dextran through blank and endothelialized lumens. H) Fluorescence micrograph of nuclei (blue) and VE‐cadherin (green) markers of the endothelium. I) Fluorescence micrographs of the endothelium inside lumens with a branching structure.