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Using Spheroids for 3D Bioprinting

Updated: Jun 10, 2022

There are many different methods of 3D bioprinting, such as microextrusion, inkjet, laser-assisted bioprinting, and combinations of different techniques [1]. When it comes to the use of support materials for cell attachment and growth, the 3D bioprinting methods can be classified into scaffold-free or scaffold-dependent. However, scaffolding material’s immunogenicity, slow speed of degradation and/or toxicity of its products are issues that scaffold-dependent 3D bioprinting faces [1,2]. Scaffold-free tissue engineering using spheroids has the potential to overcome these limitations.

Spheroids are multicellular aggregates formed by either spontaneous self-assembly or forced cell-cell adhesion without scaffolds. They can usually maintain the native extracellular matrix composition by secreting their own ECM instead of requiring other foreign ECM-mimicking materials [3]. This technology’s primary mechanism is called spheroid fusion, which is guided by the minimization of energy, the same biophysical mechanism that happens in spheroid formation [4]. Cells aggregate in spherical shapes to maximize intracellular adhesion and minimize intracellular energy [5].

Figure 1: The processes involved in spheroid formation [5].

There are four distinct approaches to enable spheroid cultures. The first one is based on the use of low-adhesion plates to promote the self-aggregation of cells into spheroids [6,7]. The second approach uses hanging drop plates (HDPs) to promote the formation of multicellular spheroids [6,8]. The use of a bioreactor, a spinner flask, or a microgravity reactor to drive cells to self-aggregate into spheroids under dynamic culture conditions, also allowing large-scale production, are other ways to enable these cultures [6,9]. Another approach is to use micro-/nano-patterned surfaces as scaffolds to control cell adhesion and migration [6,10].

A study described a 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells, and fibroblasts were isolated, counted, and mixed at desired cell ratios. After three days, these cells turned into spheroids. Following this process, these spheroids were picked up by a nozzle using vacuum suction, they were assembled on a needle array using a 3D bioprinter, and then allowed to fuse. Three days after 3D bioprinting, the spheroids were removed as an intact patch, exhibiting a mechanical integration of component spheroids and showing a high promise in cardiac tissue regeneration and as 3D models of heart disease [1].

In another study, the potential use of human gingiva-derived mesenchymal stem cells (GMSCs) as the only cellular component in 3D bioprinted scaffold-free neural constructs was assessed. The constructs were developed to be used in a surgical procedure to bridge facial nerve defects in rats. The study demonstrated that GMSCs have the propensity to aggregate into compact 3D-spheroids that could produce their own matrix. The nerve constructs were printed from GMSC spheroids and allowed to mature in a bioreactor. In vivo transplantation of the GMSC-laden nerve constructs promoted regeneration and functional recovery when used to bridge segmental defects in the facial nerves of rats. The results of this study suggested that the use of spheroids is very promising for the repair and regeneration of peripheral nerve defects [11].

Figure 2: Steps involved in the fabrication of 3D bioprinted scaffold-free nerve constructs from GMSC spheroids; and clinical photographs showing the regenerated facial nerve tissues at 12 weeks after transplantation with silicon tube, facial nerve autograft, or 3D bioprinted nerve constructs [11].


1 - Ong CS, Fukunishi T, Nashed A, et al. Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting. J Vis Exp. 2017;(125):55438.

2 - Moldovan NI, Hibino N, Nakayama K. Principles of the Kenzan Method for Robotic Cell Spheroid-Based Three-Dimensional Bioprinting. Tissue Eng Part B Rev. 2017;23(3):237-244.

3- Zhuang P, Sun AX, An J, Chua CK, Chew SY. 3D neural tissue models: From spheroids to bioprinting. Biomaterials. 2018;154:113-133.

4- Sego TJ, Kasacheuski U, Hauersperger D, Tovar A, Moldovan NI. A heuristic computational model of basic cellular processes and oxygenation during spheroid-dependent biofabrication. Biofabrication. 2017;9(2):024104.

5- Zhou Y. The Application of Ultrasound in 3D Bioprinting. Molecules. 2016;21(5):590. Published 2016 May 5.

6- Fang Y, Eglen RM. Three-Dimensional Cell Cultures in Drug Discovery and Development. SLAS Discovery. 2017;22(5):456-472.

7- Vinci M, Gowan S, Boxall F, et al. Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation. BMC Biol. 2012;10:29.

8- Tung YC, Hsiao AY, Allen SG, Torisawa YS, Ho M, Takayama S. High-throughput 3D spheroid culture and drug testing using a 384 hanging drop array. Analyst. 2011;136(3):473-478.

9- Youn BS, Sen A, Behie LA, Girgis-Gabardo A, Hassell JA. Scale-up of breast cancer stem cell aggregate cultures to suspension bioreactors. Biotechnol Prog. 2006;22(3):801-810.

10- Yoshii Y, Waki A, Yoshida K, et al. The use of nanoimprinted scaffolds as 3D culture models to facilitate spontaneous tumor cell migration and well-regulated spheroid formation. Biomaterials. 2011;32(26):6052-6058.

11- Zhang, Q., Nguyen, P.D., Shi, S. et al. 3D bio-printed scaffold-free nerve constructs with human gingiva-derived mesenchymal stem cells promote rat facial nerve regeneration. Sci Rep 8, 6634 (2018).

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