(668b) High-Throughput Fabrication of Large Breast Cancer Spheroids: Facile Encapsulation of MCF7 Cells in Gelatin Methacryloyl (GelMA) Using a Simple Microfluidic Platform

Cancer is the first or second leading cause of mortality in more than 90 countries worldwide. However, fundamental cancer research and anti-cancer drug development are limited by a lack of accessible and reliable in vitro cancer models. A vast body of evidence is now demonstrating that 3D cultures of cancer cell aggregates can recapitulate the response of tumors to anticancer drugs more faithfully than is achieved with conventional 2D monolayer cultures. Cancer spheroids are arguably the simplest and most commonly used 3D model for conducting cancer research and can be fabricated by diverse methods. However, most fabrication methods are based on cumbersome, artisanal protocols that yield heterogeneously shaped and sized spheroid populations. Moreover, these methods result in the formation spheroids that are significantly smaller (50–400 µm diameter) than the actual sizes of clinically relevant avascular tumors.

We have developed a flexible platform for fabricating breast cancer spheroids as cell-laden gelatin methacryloyl (GelMA) structures using a polymethyl methacrylate (PMMA) microfluidic chip connected to a pneumatic pump. The PMMA chip design is a simple T-junction (two inlets and one outlet) that is easily lab-fabricated using a conventional laser cutter. A 5% GelMA solution containing two photo-initiators (0.067% Lap Cellink^® and 0.067% Lap Allevi^®) and MCF7 cells is fed though one inlet, while mineral oil containing 2% (v/v) Span^® 80 is injected through the other. Flow activation then generates spherical drops of cell-laden GelMA in water. Photocrosslinking is then initiated with a high-power blue-violet laser (wavelength of 405 nm) placed in contact with the upper layer of the device at the chip outlet. Upon recovery, the spheroids are exposed again to UV light using an Ominicure® S2000 system (365 nm wavelength).

This simple platform enables the fabrication of micro-spheroids of different sizes. We can produce homogeneous GelMA micro-spheres with diameters between 300 and 1100 µm and low variation coefficients among batch and days (≤15%) simply by varying the diameter of the chip inlet channel and the flow velocity of the GelMA and mineral oil. We have fabricated MCF7-laden GelMA spheres using different initial cell concentrations (from 1 to 3×10⁶ cells/mL) and monitored their shape and architecture evolution for 30 days by optical and scanning electron microscopy. We have also evaluated cell viability within the constructs at different timepoints using Live/Dead assays. In general, progressive degradations of the GelMA matrix and cell proliferation were observed within the cell-laden spheres. As expected, denser and more compact cellular aggregates were formed in less time with high-density cell spheres than with spheres initially seeded with the low cell concentrations.

Here, we present an easy, reproducible, fast, and cost-effective method for high-throughput production of homogenous cancer spheroids encapsulated in GelMA. This method may facilitate the production of relevant cancer models for medium- to high-throughput anti-cancer drug development or fundamental cancer research. We envision that the strategies presented here may easily translate to the fabrication of other types of cancer spheroids or to the fabrication of multi-cellular spheroids for cancer modeling or other tissue engineering applications.