Nested Biofabrication: Matryoshka-Inspired Intra-Embedded Bioprinting

Abstract

Engineering functional tissues and organs remains a fundamental pursuit in bio-fabrication. However, the accurate constitution of complex shapes and internal anatomical features of specific organs, including their intricate blood vessels and nerves, remains a significant challenge. Inspired by the Matryoshka doll, here a new method called "Intra-Embedded Bioprinting (IEB)" is introduced building upon existing embedded bioprinting methods. a xanthan gum-based material is used which served a dual role as both a bioprintable ink and a support bath, due to its unique shear-thinning and self-healing properties. IEB's capabilities in organ modeling, creating a miniaturized replica of a pancreas using a photocrosslinkable silicone composite is demonstrated. Further, a head phantom and a Matryoshka doll are 3D printed, exemplifying IEB's capability to manufacture intricate, nested structures. Toward the use case of IEB and employing an innovative coupling strategy between extrusion-based and aspiration-assisted bioprinting, a breast tumor model that included a central channel mimicking a blood vessel, with tumor spheroids bioprinted in proximity is developed. Validation using a clinically-available chemotherapeutic drug illustrated its efficacy in reducing the tumor volume via perfusion over time. This method opens a new way of bioprinting enabling the creation of complex-shaped organs with internal anatomical features.

Keywords: bioprinting; cancer‐on‐a‐chip; intra‐embedded bioprinting; organ modeling.

Figure 1.

A schematic illustrating the step-by-step process of IEB and its applications (created with Biorender.com).

Figure 2.

XaGMA and GelMA hydrogels. A) Synthesis and crosslinking of XaGMA and GelMA. FTIR of B1) GelMA and B2) XaGMA. ¹H NMR of B3) GelMA and B4) XaGMA. SEM images of C1) GelMA, C2) XaGMA, and C3) the XaGMA/GelMA composite.

Figure 3.

Assessment of printability and IEB demonstrations. A) Amplitude sweep test to measure the storage modulus, loss modulus, and yield stress at a strain ranging from 0.1 to 300%, and B) self-healing thixotropy test to validate the material recovery behavior under cyclic high (100 s⁻¹) and low (1 s⁻¹) shear rate (n = 3). C) Bioprinting accuracy of spheroids (n = 10), D) filament width of sacrificial inks in line with the fluid continuity equation, where Q and v represent the flow rate and nozzle speed, respectively (n = 6), and E) filament diameter versus printing speed curves at 15, 81, 21, and 24 kPa pressure levels (n = 6). F) Printing demonstration of PSU design, where “P” was printed with the XaG sacrificial ink (red), “S” with DAPI-stained hMSC spheroids (Blue), and “U” with XaG bioink containing GFP⁺ MDA-MB-231 cells (green), G) printing demonstration of a benzene design with XaG ink (red) and hMSC spheroids (blue and green), and H) IEB of DAPI-stained hMSC spheroids (blue) inside 1.5% XaG loaded with GFP⁺ MDA-MB-231 cells (green) and the XaG ink (red) around it.

Figure 4.

Viability of cells in 1.5% XaG/5% GelMA. A) Representative LIVE/DEAD images of bioprinted ADSCs on Days 3, 7, and 14. B) Viability of ADSCs on cast and bioprinted constructs on Day 3, 7 and 14 (n = 3).

Figure 5.

Printing nested structures. A1) Desing of a pancreatic model with the vasculature and bile ducts, A2) 3D printing in action, and A3) the 3D printed model. B1) The design of a head phantom with anatomical structures including skull, cerebral cortex, and brain stem, and B2) the corresponding four-nested (labelled 1–4) 3D printed structure. C1) The design of a Matryoshka doll, C2) the print in progress, and C3) the corresponding four-nested (labelled 1–4) 3D printed structure.

Figure 6.

Cancer-on-a-chip platform. A) The schematic of the developed cancer-on-a-chip model, B) the components and set-up of the device, C) the bioprinted breast tumor model with D) a magnified image showing the channel and tumors, and E) adipocytes within the bioink. F) Tumor spheroids with 10 and 50 μM DOX perfusion at Days 1 and 3 (spheroids without DOX treatment were used as a control), and G) change in tumor volume post treatment with DOX (n = 3; *p ≤ 0.05).