This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.
This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and fibroblasts are first isolated, counted and mixed at desired cell ratios. They are co-cultured in individual wells in ultra-low attachment 96-well plates. Within 3 days, beating spheroids form. These spheroids are then picked up by a nozzle using vacuum suction and assembled on a needle array using a 3D bioprinter. The spheroids are then allowed to fuse on the needle array. Three days after 3D bioprinting, the spheroids are removed as an intact patch, which is already spontaneously beating. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.
There are many different methods of 3D bioprinting1,2,3. 3D bioprinting is frequently classified by printing technology1, with examples such as inkjet bioprinting, microextrusion bioprinting, laser assisted bioprinting, a combination of methods, or newer approaches. 3D bioprinting can also be classified into scaffold-free or scaffold-dependent methods4. Most methods of 3D bioprinting are scaffold-dependent, where there is a need for biomaterials, e.g. bioinks5 or scaffolds6. However, scaffold-dependent 3D bioprinting face many issues and limitations4,7, such as immunogenicity of scaffolding material, cost of proprietary bioinks, slow speed and toxicity of degradation products.
Scaffold-free cardiac tissue engineering using spheroids has been attempted8, with the potential to overcome these disadvantages of scaffold-dependent tissue engineering. However, as acknowledged by the authors in that paper, it had been difficult to robustly handle and position spheroids in fixed locations, in the process of biofabrication. The concomitant use of 3D bioprinting and spheroid-based tissue engineering has the potential to overcome these difficulties. In this protocol, we describe 3D bioprinting of cardiac tissue without other biomaterials, using only cells in the form of spheroids.
Scaffold-free spheroid-based 3D bioprinters9 have the ability to pick up individual spheroids using vacuum suction and position them on a needle array. The concept of positioning spheroids on a needle array in 3D bioprinting, is inspired from the use of needle arrays (known as "kenzan") in the ancient Japanese art of flower arrangement, ikebana. This system allows spheroids to be precisely positioned in any configuration and results in the individual spheroids fusing together over a short period to create a 3D bioprinted tissue. This method thus allows spheroids to be manipulated with ease, with potential implications for the future of scaffold-free organ biofabrication.
1. Preparation of Cardiomyocytes
2. Preparation of Fibroblasts
3. Preparation of Endothelial Cells
4. Co-culture:
5. 3D Bioprinting of Scaffold-free Cardiac Tissues
6. Removal of 3D Bioprinted Patch from the Needle Array and Patch Maturation
At the end of step 4.4 (co-culture), the cells in each well should aggregate at the bottom of the ultra-low attachment 96-well U-bottom plates to form spheroids by gravity. These spheroids contain hiPSC-CM, HCFs, and HUVECs, and can be visually inspected under light microscopy, where they should appear circular by two-dimensional projection (Figure 1). At the end of step 6.3, the 3D bioprinted cardiac patch should contain tissue voids, due to needle holes created by the needle array (Figure 2, left). At this point, the boundaries between the spheroids should have become indistinct and the patch should have begun to beat spontaneously, exhibiting mechanical integration of spheroids. At the end of step 6.5, the tissue voids should be filled in by surrounding tissue (Figure 2, right), while the 3D bioprinted cardiac patch should continue to beat spontaneously.
Figure 1: Creation of mixed cell multicellular spheroids. hiPSC-CM, HCFs, and HUVECs were mixed at a hiPSC-CM:HCF:HUVEC cell ratio of 70:15:15, and the cell mixture was distributed into ultra-low attachment 96-well U-bottom plates. Within 24 h, mixed cell multicellular spheroids (left, right) formed, one in each well. Scale bar = 500 µm. Please click here to view a larger version of this figure.
Figure 2: Creation of cardiac tissue exhibiting mechanical integration of spheroids. A 3D bioprinted cardiac patch immediately after removal from the needle array with visible needle holes (left). At this time, the boundaries between the spheroids had already become indistinct and the patch had already begun beating spontaneously, thus the spheroids had become mechanically integrated. Three days after removal from the needle array, the tissue voids caused by the needle array were filled in by surrounding tissue (right), and the patch continued beating spontaneously. Scale bar = 1 mm. Please click here to view a larger version of this figure.
It is important to use beating, functional spheroids for 3D bioprinting. If spheroids are not beating, continuing to use them will invariably result in a non-functional 3D bioprinted patch.
One benefit of this approach is the ability to manipulate the cell content of the patch by varying the total number of cells and the percentage of cardiomyocytes, endothelial cells, and fibroblasts in the spheroids. This allows for many different types of cardiac patches to be printed, with varying histological and mechanical properties.
This approach also allows different types of spheroids to be used to make a patch, as well as the 3D bioprinting of cardiac patches with complex 3D designs. This complexity of the final 3D construct is achievable by 3D bioprinting with precise spheroid positioning, but may be difficult to achieve by gentle rotation of floating spheroids7 or simple molding of spheroids into a cylindrical 3D construct16.
The limitations of this approach are the relatively fixed size of the spheroids between 450 to 550 µm due to the fixed distance of 400 µm between the needles in the needle array, as well as possibly reduced cell viability if the spheroids are too large in diameter. The patches are mechanically fragile immediately after removal from the need array; however, the strength and durability are improved with culture for longer durations of time.
Finally, 3D bioprinting of cardiac patches has promising clinical applications17, in cardiac regeneration18,19 and as 3D models of heart disease. These 3D models can be used for applications in predictive pharmacology/toxicology and development of cell and genome directed therapies.
In conclusion, it is feasible to 3D bioprint cardiac tissue without the use of biomaterials. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids. 3D bioprinting of cardiac tissue has promising clinical applications in cardiac tissue regeneration and as 3D models of heart disease.
The authors have nothing to disclose.
The authors acknowledge the following funding sources: Magic That Matters Fund for Cardiovascular Research and the Maryland Stem Cell Research Fund (2016-MSCRFI-2735).
Geltrex | Invitrogen | A1413202 | |
Trypsin/EDTA 0.05% | Thermo Fisher | 15400054 | |
Defined Trypsin inhibitor 0.0125% | Thermo Fisher | R007100 | |
RPMI Cell Media | Invitrogen | 11875-093 | RPMI supplemented with B27 constitutes HIPSC-CM culture media |
B-27 Supplement | Thermo Fisher | 17504044 | RPMI supplemented with B27 constitutes HIPSC-CM culture media |
Countess Automated Cell Counter | Invitrogen | C10227 | |
Human cardiac fibroblasts (adult ventricular type) | Sciencell | 6310 | |
Human umbilical vein endothelial cells | Lonza | CC-2935 | |
PrimeSurface ultra-low attachment 96-well U-bottom plates | Akita Sumitomo Bakelite Co. | MS-9096UZ | |
Regenova Bio 3D Printer | Cyfuse Biomedical K.K. | N/A | www.cyfusebio.com/en/ |
Trypan Blue Solution, 0.4% | Thermo Fisher | 15250061 | |
Troponin T Antibody | Thermo Fisher | 701620 | |
Connexin 43 (Cx43) Antibody | Chemicon | MAB3068 | |
ProLong Gold Antifade Mountant with DAPI | Thermo Fisher | P36935 |