Júlia Vallverdú Ginés, PhD
Lead Scientist3D models such as spheroids and organoids are gaining ground as human-relevant alternatives to 2D cultures and animal testing, a shift actively encouraged by regulatory frameworks like the FDA Modernization Act 2.0 and the proposed 3.0. But most 3D workflows still rely on manual handling, which introduces morphological variability and fragments the process across multiple instruments. If 3D models are going to fulfill their potential as New Approach Methodologies (NAMs), they need to be generated in a standardized, reproducible, and scalable way.
This is exactly the gap the MO:BOT, our benchtop automated platform designed to standardize all operator-dependent steps of complex 3D cell-based workflows, is designed to close. We have recently validated its potential in a proof-of-concept study to generate HepG2 liver spheroids and perform acetaminophen (APAP) drug-response testing, published in Scientific Reports (Nature Portfolio).
Key takeaways
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The MO:BOT: One Platform for the Full Liver Spheroid Workflow
A typical 3D cell culture experiment runs through four distinct phases: culture generation and maintenance, iterative quality control, compound treatment, and endpoint analysis. Traditionally, each of these phases requires its own dedicated instrument with samples transferred between them at every step. Each transfer adds handling time, sterility risk, and operator-dependent variability.
The MO:BOT consolidates all four phases into a single, modular benchtop platform. On-deck components, including a heater, cooler, tilter, shaker, and our integrated imaging module, MO:CROSCOPE, connect without cables, with a plug-and-play design, and are recognized automatically by the system, so users can configure the setup around the specific needs of each protocol. The MO:BOT software provides an intuitive, integrated protocol library covering multiple human 3D in vitro models and enables straightforward implementation of new, customized workflows.
(A) Typical 3D cell culture workflow phases. (B) Cell culture workflow integration in the MO:BOT. (C) Separate instruments in traditional workflows (e.g. biosafety cabinet, liquid handler, shaker, plate reader, microscope etc.), replaced by the MO:BOT.
MO:RE Uniform, MO:RE Viable: Automation Improves Spheroid Generation
Compared with manual handling, MO:BOT-generated spheroids showed substantially lower variability in size across independent batches (8.3% CV vs 15.3% in manual workflows). Medium exchange was sufficiently gentle and preserved spheroid size and overall structure, while manual handling caused a measurable size drop. Shape metrics (ellipticity, roundness) stayed consistent across both methods.
The MO:BOT achieves a significantly lower coefficient of variation in spheroid area across independent experimental runs compared to manual pipetting on day 4 (p < 0.001). n = 96 per batch. (B) Liver spheroids on day 5 have a higher viability than those generated manually. n = 36 per handling method. (C) Liver spheroids generated with both handling conditions present equivalent albumin secretion. n = 24 per handling method.
Automation also improved biological performance: area-normalized viability was 1.4-fold higher in MO:BOT spheroids, likely due to gentler, more consistent liquid handling. Albumin secretion, a marker of liver function, remained equivalent between both methods, confirming that automation preserves cellular function while improving structural consistency.
From Uniform Spheroids to a Sharper Toxicity Signal
The real test came with the APAP dose-response assay. A seven-concentration titration produced a sigmoidal toxicity curve with an EC₅₀ of 40.93 mM. At this concentration, viability and cytotoxicity were comparable between manual and MO:BOT spheroids, confirming that automation preserves assay sensitivity.
The key difference emerged in ALT activity, a specific marker of hepatocellular injury: MO:BOT spheroids showed a significantly stronger ALT increase upon APAP exposure, with lower variability between replicates. The clearer toxicity signal may be attributed to an increase in uniformity.
MO:BOT-generated spheroids exhibit significantly greater ALT release upon APAP treatment compared to manually handled spheroids (p<0.01), indicating enhanced sensitivity for detecting hepatocellular injury. n=18 per handling method.
This study is a first step: proof that the MO:BOT can automate a complete 3D culture workflow while maintaining, and in some respects improving, biological performance. The next step is extending this validation to more complex models, moving 3D models closer to routine use as robust, reproducible tools in preclinical research.
Figures adapted from Hellmold D, et al. Automation of 3D liver spheroid generation and acetaminophen dose–response on the MO:BOT enhances assay robustness and precision. Sci Rep (2026). https://doi.org/10.1038/s41598-026-58939-4. Licensed under CC BY 4.0.
