Poster S18 - Automated Positioning of Tumor Spheroids into Hydrogel-Based Microfluidic Chips for High-Throughput Screening Applications

Introduction: Bioprinting has emerged as a transformative technology, enabling the fabrication of complex, three-dimensional (3D) structures that closely mimic native biological tissues. It has been used to model tumor microenvironment (TME), a complex system of multiple cell types in the native extracellular matrix (ECM) [1]. In this perspective, integrating cellular components, such as tumor spheroids, into bioprinted constructs with high precision and reproducibility has remained a significant challenge. Tumor spheroids, as cell clusters, serve as pivotal models for studying tumor behavior, cancer metastasis, and drug efficacy due to their resemblance to native tumors regarding cellular heterogeneity and microenvironmental cues. We hypothesize that precise positioning control of tumor spheroids within hydrogel structures can improve the reproducibility of experiments and ensure the functionality of the printed biological structures. Recognizing the need for precise spatial control of tumor spheroids within bioprinted matrices, our research introduces a novel adaptation to the conventional extrusion- and inkjet-based bioprinting platforms.

Materials and

Methods: Spheroids of HT1080 ranging from 500 to 25,000 cells/well were formed in ultra-low attachment 96-well plates over three days. Gelatin methacryloyl (GelMA) was synthesized, and a 7% w/v GelMA precursor was prepared based on a well-established protocol [2]. A Polyethylene Glycol Diacrylate (PEGDA) microfluidic chip, based on our previously published work [3], was printed to position the spheroids. Vacuum pressure was determined using a 30-gauge needle, and experimental studies were conducted to identify the lower and upper vacuum limits for spheroid positioning.

Results, Conclusions, and Discussions: Results and

Discussion: A relationship was discovered with the polynomial equation where y represents the spheroid diameter and x, the cell number. The experimental setup included a 96-well plate attached to a 3D bioprinter, which was calibrated to apply an optimal suction force of -9 kPa suitable for spheroids with diameters ranging from 400 to 800µm. This suction force enabled the sequential lifting and precise positioning of three spheroids into a triangular formation. Each spheroid was positioned approximately 1 mm apart, determined by the centers of their respective circles. Our analysis observed an average positioning error of 91.095 μm (±62.69 μm) between the targeted and actual spheroid positions. Cell migration within a controlled microenvironment was investigated across samples. Cell culture media was introduced via syringe pump at a flow rate of 500 μl/hr through PEGDA/GelMA chip. Spheroids were observed to gradually migrate within the hydrogel microfluidic chip under active perfusion, indicating the effectiveness of the designed setup in facilitating controlled TME.

Conclusion: We have engineered a vacuum nozzle system for the precise placement of tumor spheroids on a PEGDA/GelMA chip, designed to study tumor dynamics. Directed by G-code, the system facilitates automated and accurate positioning of spheroids, thereby improving the consistency and scalability of tumor models. Future research will focus on exploring tumor behavior within a vascularized system to test anti-vascularization drugs in our model.

Acknowledgements (Optional):

References:
[1] Dogan E. et al. Adv Nanobiomed Res. 2021; 1(11).
[2] Dogan E. et al. Acta Biomaterialia, 2024 (submitted).
[3] Bhusal et al. Biofabrication, 2021.