3D Printing of Personalized Thick and Perfusable Cardiac Patches and Hearts

Abstract

Generation of thick vascularized tissues that fully match the patient still remains an unmet challenge in cardiac tissue engineering. Here, a simple approach to 3D-print thick, vascularized, and perfusable cardiac patches that completely match the immunological, cellular, biochemical, and anatomical properties of the patient is reported. To this end, a biopsy of an omental tissue is taken from patients. While the cells are reprogrammed to become pluripotent stem cells, and differentiated to cardiomyocytes and endothelial cells, the extracellular matrix is processed into a personalized hydrogel. Following, the two cell types are separately combined with hydrogels to form bioinks for the parenchymal cardiac tissue and blood vessels. The ability to print functional vascularized patches according to the patient's anatomy is demonstrated. Blood vessel architecture is further improved by mathematical modeling of oxygen transfer. The structure and function of the patches are studied in vitro, and cardiac cell morphology is assessed after transplantation, revealing elongated cardiomyocytes with massive actinin striation. Finally, as a proof of concept, cellularized human hearts with a natural architecture are printed. These results demonstrate the potential of the approach for engineering personalized tissues and organs, or for drug screening in an appropriate anatomical structure and patient-specific biochemical microenvironment.

Keywords: 3D printing; hearts; hydrogels; induced pluripotent stem cells (iPSCs); tissue and organ engineering.

Figure 1

Concept schematic. An omentum tissue is extracted from the patient and while the cells are separated from the matrix, the latter is processed into a personalized thermoresponsive hydrogel. The cells are reprogrammed to become pluripotent and are then differentiated to cardiomyocytes and endothelial cells, followed by encapsulation within the hydrogel to generate the bioinks used for printing. The bioinks are then printed to engineer vascularized patches and complex cellularized structures. The resulting autologous engineered tissue can be transplanted back into the patient, to repair or replace injured/diseased organs with low risk of rejection.

Figure 2

Bioinks characterization. A human omentum a) before and b) after decellularization. c) A personalized hydrogel at room temperature (left) and after gelation at 37 °C (right). d) A SEM image of the personalized hydrogel ultrastructural morphology, and e) a histogram of the fibers diameter. f) Rheology measurements of 1% w/v and 2.5% w/v omentum hydrogels, showing the gelation process upon incubation at 37 °C. g) Stromal cells originated from human omental tissues were reprogrammed to become pluripotent stem cells (red: OCT4, green: Ki67 and blue: nuclei). h) Differentiation to ECs as determined by CD31 (green) and vimentin staining (red). Differentiation to cardiac lineage: i) staining for sarcomeric actinin (red), j) staining for NKX2‐5 (red), and TNNT2 (green). Scale bars: (e) = 10 µm, (g,i,j) = 50 µm, (h) = 25 µm.

Figure 3

Imaging of the heart and patch modeling. CT image of a) a human heart and b) left ventricle coronary arteries. c) A model of oxygen concentration profile in an engineered patch. d) Replanning of the model showed better oxygen diffusion, sufficient to support cell viability.

Figure 4

3D printing of personalized cardiac patches. a) A 3D model of the cardiac patch. b) A side view of the printing concept and the distinct cellular bioinks. c) A printed vascularized cardiac patch. d) Cell viability after printing. e) A printed blood vessel, continuously layered with GFP‐expressing ECs. f) A printed iPSCs‐derived cardiac patch where the blood vessels (CD31 in green) are seen in‐between the cardiac tissue (actinin in pink). g,h) Cross‐sections of a single lumen, showing the interactions of GFP‐expressing ECs and RFP‐expressing fibroblasts. i–k) Calcium imaging within a printed vascularized cardiac patch (separate regions of interest are represented in different colors. The white arrow represents signal direction). i) The lumen of a blood vessel can be easily observed in‐between the cardiac cells. j,k) Quantification of calcium transients across a lumen of the vascularized cardiac patch. l) Transplantation of the printed patch in between two layers of rat omentum. Dashed, white line highlights the borders of the patch. m–o) Sarcomeric actinin (red) and nuclei (blue) staining of sections from the explanted patch (panel (o) represents a high magnification of the marked area in (n). Scale bars: (c) = 1 cm, (e,g,h,l,m) = 100 µm, (f) = 500 µm, (n) = 50 µm, (o) = 25 µm.

Figure 5

Printing the personalized hydrogel in a supporting medium. a) A scheme of the 3D printing concept. The construct is free‐formed printed inside the support followed by incubation at 37 °C to crosslink the personalized hydrogel. Then, the structure can be safely extracted by an enzymatic or chemical degradation process of the support material and transferred into growth medium for culturing. b) A multilayered crisscross construct printed inside the support bath, and c) after its extraction. d) Cell viability before and after printing and after extraction. e) 3D confocal image of a double layered construct, printed in the support medium. f) A single strand of the personalized hydrogel within the support. g,h) Accurate, high resolution thick structures printed from the personalized hydrogel. Scale bars: (b) = 0.5 cm, (e) = 1 mm, (f) = 100 µm, (g,h) = 1 cm.

Figure 6

Printing thick vascularized tissues. a) A top view of a lumen entrance (CD31; green) in a thick cardiac tissue (actinin; pink). b) A model of a tripod blood vessel within a thick engineered cardiac tissue (coordinates in mm), and c) the corresponding lumens in each indicated section of the printed structure. d) Tissue perfusion visualized from dual viewpoints. e–k) A printed small‐scaled, cellularized, human heart. e) The human heart CAD model. f,g) A printed heart within a support bath. h) After extraction, the left and right ventricles were injected with red and blue dyes, respectively, in order to demonstrate hollow chambers and the septum in‐between them. i) 3D confocal image of the printed heart (CMs in pink, ECs in orange). j,k) Cross‐sections of the heart immunostained against sarcomeric actinin (green). Scale bars: (a,c,h, i,j) = 1 mm, (g) = 0.5 cm, (k) = 50 µm.