Printing three-dimensional tissue analogues with decellularized extracellular matrix bioink

Abstract

The ability to print and pattern all the components that make up a tissue (cells and matrix materials) in three dimensions to generate structures similar to tissues is an exciting prospect of bioprinting. However, the majority of the matrix materials used so far for bioprinting cannot represent the complexity of natural extracellular matrix (ECM) and thus are unable to reconstitute the intrinsic cellular morphologies and functions. Here, we develop a method for the bioprinting of cell-laden constructs with novel decellularized extracellular matrix (dECM) bioink capable of providing an optimized microenvironment conducive to the growth of three-dimensional structured tissue. We show the versatility and flexibility of the developed bioprinting process using tissue-specific dECM bioinks, including adipose, cartilage and heart tissues, capable of providing crucial cues for cells engraftment, survival and long-term function. We achieve high cell viability and functionality of the printed dECM structures using our bioprinting method.

Figure 1. Schematic elucidating the tissue printing process using dECM bioink.

Respective tissues were decellularized after harvesting with a combination of physical, chemical and enzymatic processes, solubilized in acidic condition, and adjusted to physiological pH. Tissue printing was performed with the dECM bioink encapsulating living stem cells via a layer-by-layer approach followed by gelation at 37 °C. The 3D cell-printed structure has applications in various border areas including tissue engineering, in vitro drug screening and tissue/cancer model.

Figure 2. Decellularization of the native tissues and their biochemical analysis.

Optical and microscopic images of native and decellularized (a) cartilage tissue (scale bar, 50 μm), (b) heart tissue (scale bar, 100 μm), and (c) adipose tissue (scale bar, 100 μm). ECM components (Collagen and GAGs) and DNA contents of native and decellularized (d) cartilage (cdECM), (e) heart (hdECM) and (f) adipose (adECM) tissue. All experiments were performed in triplicate. Error bars represent s.d. (*P<0.05; NS, no significance).

Figure 3. Rheological behaviour of the dECM pre-gels.

Sol to gel transition (a) of the dECM pre-gels prepared from cartilage dECM (cdECM), heart dECM (hdECM) and adipose dECM (adECM). Rheological properties of the dECM pre-gels (b) viscosity at 15 °C, (c) gelation kinetics from 4 °C to 37 °C. (Initial temperature: 4 °C, increment of 5 °C min⁻¹ with holding at 15 °C for 5 min, reaching to 37 °C, and maintaining 37 °C for 40 min), and (d) dynamic modulus at varying frequency at 37 °C. All experiments were performed in triplicate. Error bars represent s.d.

Figure 4. Printing process of particular tissue constructs with dECM bioink.

(a) Heart tissue construct was printed with only heart dECM (hdECM). Cartilage and adipose tissues were printed with cartilage dECM (cdECM) and adipose dECM (adECM), respectively, and in combination with PCL framework (scale bar, 5 mm). (b) Representative microscopic images of hdECM construct (scale bar, 400 μm), (c) s.e.m. images of hybrid structure of cdECM with PCL framework (scale bar, 400 μm) and (d) microscopic images of cell-printed structure of adECM with PCL framework (scale bar, 400 μm).

Figure 5. Long-term stability and cellular compatibility of the printed tissue constructs.

Representative images of the whole constructs prepared with hdECM (a) and adECM (b) at progressing days of cell culture (scale bar, 2 mm). Confocal image of the whole construct prepared with adECM at day 14 of culture (c) showing live cells (green dots) and dead cells (red dots) (scale bar, 2 mm). An image of the whole construct was reconstructed from ~32 images taken at different positions. Representative images of apoptosis through TUNEL (d) and Live/Dead (e) assays (scale bar, 100 μm). TUNEL assay displays very minimal apoptotic cells, which suggests that the generated stress at 2 s⁻¹ shear rate at the nozzle wall did not cause deleterious effect on the encapsulated cells and resulted similar apoptosis to the non-printed gel (0 s⁻¹). Cell viability was >95% at day 1 and >90% at both day 7 and 14.

Figure 6. Evaluation of differentiation into tissue-specific lineages and structural maturation of cells.

Comparative gene expression analysis for (a) chondrogenic (SOX9 and COl2A1), (b) cardiogenic (Myh6 and Actn1) and (c) adipogenic (PPARγ and LPL) in COL and particular dECM (cdECM or hdECM or adECM) at day 14. Immunofluorescence images showing chondrogenic differentiation of hTMSCs in (d) COL and (e) cdECM constructs showing COL type II staining (COLII, red), cell nuclei (DAPI, blue) and F-actin (green) (scale bar, 50 μm). Structural maturation of myoblasts in (f) COL and (g) hdECM construct showing Myh7 (red) and cell nuclei (DAPI, blue) (scale bar, 200 μm). Adipogenic differentiation of hASCs in (h) COL and (i) adECM constructs showing PPARγ (red), COL IV (green) and cell nuclei (DAPI, blue) (scale bar, 50 μm). All experiments were performed in triplicate. Error bars represent s.d. (*P<0.05).