Characterization of printable cellular micro-fluidic channels for tissue engineering

Abstract

Tissue engineering has been a promising field of research, offering hope of bridging the gap between organ shortage and transplantation needs. However, building three-dimensional (3D) vascularized organs remains the main technological barrier to be overcome. One of the major challenges is the inclusion of a vascular network to support cell viability in terms of nutrients and oxygen perfusion. This paper introduces a new approach to the fabrication of vessel-like microfluidic channels that has the potential to be used in thick tissue or organ fabrication in the future. In this research, we investigate the manufacturability of printable micro-fluidic channels, where micro-fluidic channels support mechanical integrity as well as enable fluid transport in 3D. A pressure-assisted solid freeform fabrication platform is developed with a coaxial needle dispenser unit to print hollow hydrogel filaments. The dispensing rheology is studied, and effects of material properties on structural formation of hollow filaments are analyzed. Sample structures are printed through the developed computer-controlled system. In addition, cell viability and gene expression studies are presented in this paper. Cell viability shows that cartilage progenitor cells (CPCs) maintained their viability right after bioprinting and during prolonged in vitro culture. Real-time PCR analysis yielded a relatively higher expression of cartilage-specific genes in alginate hollow filament encapsulating CPCs, compared with monolayer cultured CPCs, which revealed that printable semi-permeable micro-fluidic channels provided an ideal environment for cell growth and function.

Figure 1

The experimental setup: (a) 3D model of the coaxial nozzle, (b) cross-sectional view of coaxial nozzle assembly model with fluid flow paths for hydrogel and crosslinker solutions, (c) the coaxial nozzle system, and (d) the robotic printer platform.

Figure 2

Effect of hydrogel properties on gelation of hollow filaments.

Figure 3. (a) Effect of CaCl₂ and (b) alginate concentration on hollow filament dimensions. (Single asterisk (*) indicates significant differences between groups (p<0.05))

Figure 4. Hollow filament dimensions with different (a) CaCl₂ dispensing rates and (b) alginate dispensing rates, and core-to-filament ratio with different relative speed between crosslinker and alginate flow. (Single asterisk (*) indicates significant differences between groups (p<0.05))

Figure 5

Sample printed structures: (a) a printed filament allowing media transport shown with a yellow food dye, (b) an image of a single filament analyzed under a digital microscope, (c) a printed single-layer micro-fluidic channel, and (d) a printed 8-layer micro-fluidic channels.

Figure 6

(a) Perfusion of oxygenized cell type media, (b) media flow with intentionally generated air bubbles showing flow, and (c) embedding microfluidic channels within multilayer bulk hydrogel.

Figure 7

Multi-scale uniaxial tensile testing of hollow filaments: (a) meso-scale analysis illustrating overall deformation on a 2 cm long filament and (b) micro-scale analysis along the cross-section, and the effect of (c) alginate and (d) crosslinker concentrations on tensile deformation of printed micro-fluidic channels and (e)-(f) corresponding modulus of elasticity values obtained from the literature [16].

Figure 8

Laser confocal imaging of viability staining. CPCs labeled with calcein AM and ethidium homodimer after cell encapsulation in an alginate hollow filament (cell viability assay) and imaged with confocal laser scanning microscope. Live and dead cells were fluorescent green and fluorescent red, respectively. (a-b) No dead cells were visible in Day 1 and Day 4 images and (c) a few dead cells were observed in the Day 7 image

Figure 9

Time course of CPC viability after the bioprinting process. Cell viability was analyzed using Image J. Each symbol represents the average of the results for three z-projections composed of six planes from confocal laser imaging. The error bars indicate standard deviations (n=3).

Figure 10

Confocal images showing hollow property of filaments: (a-f) z-projection from top to center of a cell-encapsulated hollow filament, with 50 μm intervals showing the solid wall of fabricated filaments and the hollow channel in the center.

Figure 11

Real-time PCR showed relatively higher expression of cartilage-specific marker PRG-4, Sox-9, COL-2: all showed over two-fold up-regulation in alginate hollow filament encapsulating CPCs after bioprinting, compared with CPCs in monolayer culture. ACAN showed over 12-fold higher expression level. The error bars indicate standard deviations (n=3). (Single asterisk (*) and double asterisks (**) represent significant differences, P<0.05 and P<0.01, from the monolayer control group, respectively.)