Abstract

Scalable Biofabrication and Systematic Characterization of Tissue Spheroids for Directed Tissue Self-Assembly Using Acoustic Waves

Elena Bulanova, Head, 3D Bioprinting Solutions Russia

Tissue spheroids are gaining extensively their place in biofabrication as building blocks. We suggested a straightforward procedure for biofabrication and initial characterization of tissue spheroids with optimal controllable parameters prepared from different cell types employing non-adhesive technology. Applying different immortalized and primary cells we have demonstrated the reproducibility and scalability of spheroid generation, the strong dependency of ultimate spheroid diameter on initial cell seeding density and cell type. Additionally, the spheroids viability was shown to be governed by cell derivation. In our study we suggest a decision procedure to apply for any cell type one starts to work with to prepare a new type of tissue spheroids with predictable controllable optimal features suitable for high quality standards in biofabrication and drug discovery. Proposed protocol includes following necessary criteria: (a) estimation of correlation of tissue spheroids size with initial cell seeding density; (b) estimation of diameter and roundness as a functions of time; (c) estimation of viability as a function of time, (d) estimation of diameter and viability as a function of cell type. We suppose that described protocol could be employed for development of automated image analysis systems of tissue spheroids. Surface acoustic waves could be used for biofabrication of tissue spheroids from the living cells. There are reported attempts of 3D patterning of single cells, cells embedded into hydrogels or plastic beads using acoustic waves. However, acoustic levitation of tissue spheroids has not been reported yet. We hypothesize that tissue spheroids placed in acoustic field could levitate and form desirable 2D and 3D patterns. In order to test this hypothesis an acoustic device generating standing acoustic waves has been designed. Tissue spheroids have been biofabricated from primary sheep chondrocytes and NIH 3T3 fibroblasts. The suspension of tissue spheroids has been exposed to acoustic waves. Ultrasound pressure levels were well below the cavitation threshold, which guaranteed the absence of mechanical damage. It has been demonstrated that tissue spheroids could be acoustically sorted out based on their size and be patterned into chain-like structures and a series of structures arranged in parallel to each other in 3D space. Tissue spheroids have been viable after exposure to acoustic waves. Closely placed by acoustic waves, tissue spheroids were able to undergo tissue fusion process. Taken together these data provide proof of principle that 3D patterning of tissue spheroids by acoustic waves is feasible and opens a realistic opportunity for the development of novel acoustic wave-based 3D bioprinter. However, it remains to be seen how principles of acoustic levitation of tissue spheroids will be translated and implemented into a realistic design of novel acoustic 3D bioprinter.