Large-scale production of functional and miniaturized spheroids for drug screening

App Notes


Large-scale production of functional and miniaturized spheroids for drug screening

Heshuang Qu 2, Salomé Thüler 2, Yacine Bounab 1, Stéphanie van Loo 1, Quentin Graillet 1, Brinton Seashore-Ludlow 2 and Piia Mikkonen 3
1 LiveDrop SA , Belgium, 2 Karolinska Institute and SciLifeLab, Sweden, 3 UPM Biomedicals, Finland


Spheroids have emerged as indispensable tools research and industry, offering improved physiological relevance, accelerated drug screening, and enhanced toxicity assessment compared to traditional 2D cultures. However, current methods like hanging drop micro-plates suffer from labor intensiveness, high costs, and poor reproducibility, limiting their scalability for large-scale screening studies. Moreover, traditional large spheroids pose significant challenges for reproducible analysis using microscopy imaging techniques.


To overcome these challenges, we have developed a streamlined workflow combining the high-throughput (HTP) miniaturized spheroid production capabilities of the OneFlowTM or ModaFlowTM LiveDrop’s microfluidics instruments with the benefits of GrowDex® hydrogel from UPM Biomedicals.

Materials and Methods

Using the OneFlowTM instrument, we successfully produced compact and homogeneous spheroids suitable for high-throughput drug screening assays. Approximately 75 HepG2cells were encapsulated in 3 nL droplets at a rate of 100μl/min. Following an overnight incubation, we recovered and resuspended the spheroids in 0.2% GrowDex®-medium dilution. The overall recovery rate was about 90%, and the spheroids remained compact and maintained their integrity even when exposed to physical manipulation, such as pipetting during the recovery process.

After 48-hour incubation in GrowDex® hydrogel, the average diameter of spheroids was 70 μm (A)and the average roundness was 0.72 out of 1 (B). By introducing the drugs to 384-well spheroidculture for 48 hours, we were able to distinguish the negative (DMSO) and positive (Benzoylchloride) controls (C) by measuring the cell viability by TMRM (orange, staining for live cells)-SyTox(green, staining for dead cells) staining. The average cell viability of native control wells is 94% (D)with example TMRM-SyTox staining of negative (E) and positive (F) control-treated spheroids (scalebars = 200 μm).


This collaborative effort introduces an innovative approach to efficiently produce miniaturized spheroids, yielding 20,000spheroids from 0.8 million cells suspended in 100 μL medium in just 3 minutes, with each spheroid measuring approximately 70 μm in diameter. These spheroids exhibit robustness, homogeneity, and functionality.

The incubation of cells in nanoliter-scale droplets promotes cell contact, facilitating cell aggregation and accelerating spheroid formation. Additionally, the use of GrowDex®hydrogel provides steric hindrance to spheroids, preventing clustering during drug testing and cell staining.

The workflow is characterized by its robustness and simplicity and holds potential for further development to accommodate various cell types, thereby exploring new therapeutic avenues and personalized medicine applications.

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