Multicomponent bioprinting of heterogeneous hydrogel constructs based on microfluidic printheads

Abstract

Multimaterial bioprinting provides a promising strategy to recapitulate complex heterogeneous architectures of native tissues in artificial tissue analogs in a controlled manner. However, most of the existing multimaterial bioprinting techniques relying on multiple printing nozzles and complicate control program make it difficult to flexibly change the material composition during the printing process. Here, we developed a multicomponent bioprinting strategy to produce heterogeneous constructs using a microfluidic printhead with multiple inlets and one outlet. The composition of the printed filaments can be flexibly changed by adjusting volumetric flow rate ratio. Heterogeneous hydrogel constructs were successfully printed to have predefined spatial gradients of inks or microparticles. A rotary microfluidic printhead was used to maintain the heterogeneous morphology of the printed filaments as the printing path direction changed. Multicellular concentric ring constructs with two kinds of cell types distribution in the printed filaments were fabricated by utilizing coaxial microfluidic printhead and rotary collecting substrate, which significantly improves the printing efficiency for multicomponent concentric structures. The presented approach is simple and promising to potentially print multicomponent heterogeneous constructs for the fabrication of artificial multicellular tissues.

Keywords: bioprinting; heterogeneous constructs; microfluidic printhead; multicomponent printing.

Figure 1

Design of microfluidic printhead for multicomponent bioprinting. (A) Schematic illustration of microfluidic printheads with multiple inlets and an outlet. (B) The assembly of the printhead. (C) Photograph of the fabricated microfluidic printhead. Scale bar=5 mm. (D and E) Schematic and photograph of multicomponent bioprinting platform with microfluidic printhead. Scale bar=5 cm.

Figure 2

Printing of heterogeneous filaments by changing flow rate ration of different solutions in the microfluidic printhead. (A) Schematic for the deposition of heterogeneous filaments from the microfluidic printhead. (B, C, D, E, F) Microscopic images of heterogeneous filaments printed by changing the flow rate ratio of two solutions. Scale bar=200 µm. (G) Quantification of the composition distribution of two inks in the printed filaments. (H, I, J) Fluorescent images of the heterogeneous filaments printed through the three inlets with different flow rate of middle inlet. Scale bar=200 µm. (K) Quantification of the number of green fluorescent particles at different flow rate of the middle inlet.

Figure 3

Flow pattern and printed filaments of alginate solution with different viscosities. (A) Injecting 3% alginate solution (high viscosity) simultaneously and (B) printed heterogeneous filaments. (C) Injecting 3% alginate solution and 1% alginate solution and (D) printed filaments. Scale bars are 200 µm and 1 mm.

Figure 4

Color-coded heterogeneous constructs. (A and B) Dynamically altering the proportion of different inks during printing. (C) Schematic of color-coded gel structure (10 layers) printed under different proportion of flow rate (Q_total=600 µL/h, Q_blue: Q_yellow=0:1, 1:3, 1:1, 3:1, and 1:0) changed per two layers. (D, E, F, G, H) Photograph showing a gradient of colors from yellow to blue during printing. Scale bar=2 mm.

Figure 5

Heterogeneous constructs containing red and green fluorescent microparticles. (A) Schematic of distribution of green/red fluorescent particles in printed constructs. (B) The 3D fluorescence profile of local part of the printed constructs. (C, D, E, F, G) The representative distribution of green/red fluorescence particles at the different heights of the printed construct. Scale bar=100 µm. (H) Quantification of green/red fluorescence particles at different heights in hydrogel structure.

Figure 6

Printing of multicomponent grid structure using rotating microfluidic printhead. (A) Schematic and photograph illustrating printed grid structure without the rotating printhead. (B) Schematic and photograph illustrating printed grid structure with the rotating printhead. (C) Printed triangular pattern with the rotating printhead. (D) Grid structure consists of heterogeneous filaments (red, blue, and purple).

Figure 7

Printing heterogeneous constructs through coaxial microfluidic printheads. (A) Schematic of the coaxial microfluidic printhead. (B) Photograph of the coaxial microfluidic printhead. Scale bar=1 cm. (C) Heterogeneous grid structure (25 layers) printed through using the coaxial microfluidic printhead. Scale bar=2 mm. (D) Schematic of rotating substrate for creating concentric ring “on-the-fly.” (E and F) Fabrication of a heterogeneous concentric ring. Scale bars are 2 mm and 1 mm, respectively. (G) Fabrication of multicellular (H9C2 and HUVEC) concentric rings through coaxial microfluidic printhead. Scale bar=4 mm. (H and I) Fluorescence microscopy image (top view) of multicellular rings. Scale bars are 1 mm and 500 µm, respectively.