Five questions to answer before you move your assays from 2D to 3D

This paid-for advertorial by Promega appeared in DDW Volume 27 – Issue 3, Summer 2026. The shift from 2D monolayers to 3D cell culture models like spheroids, organoids, and complex tissue constructs, is gaining momentum in the drug discovery space. And for good reason: 3D models better recapitulate in vivo biology, improving the predictive value of early screening data and reducing costly late-stage failures. But moving to 3D isn't as simple as swapping your culture vessel. The assays you rely on in 2D may not perform the same way when transitioned to 3D models. Before you make the switch, these five questions are worth asking: Are your current assays validated for 3D models? An assay that works well in a monolayer doesn't automatically translate to a spheroid or organoid. 3D structures introduce physical barriers that can interfere with reagent access; barriers like dense cellular cores, extracellular matrix components and necrotic centres in larger constructs. Look for assays with published 3D performance data across multiple model types and sizes. Can your reagents access the entire 3D structure? This is the most common failure point in the transition to 3D models. Incomplete lysis or inadequate reagent diffusion into the interior of a 3D model means you're measuring only the outer cell layers, potentially underestimating viability or missing cytotoxic effects entirely. Luminescence assays that use strong lytic reagents have demonstrated more complete ATP recovery from large spheroids compared to colorimetric or weaker formulations. How will you manage batch-to-batch variability? 3D cultures are inherently more variable than monolayers. Spheroid diameter, organoid maturation stage, and matrix lot-to-lot differences all affect readouts. Your assay needs to deliver a wide enough dynamic range and sufficient sensitivity to detect real cellular differences above the noise of model variability. How much data can you get from a single sample? 3D samples take longer to produce and consume more resources than 2D cultures. Sacrificing an entire well for a single endpoint is expensive. Non-lytic assays, particularly real-time viability and cytotoxicity chemistries, allow you to monitor the same well over time, then follow with a terminal readout or nucleic acid purification. Multiplexing from a single 3D sample turns one data point into several. Are you building the right model for your question? The most physiologically relevant 3D model isn't always the right one for your experiment. Your model should be fit for purpose: as simple as possible, as complex as needed. A spheroid may answer your screening question just as well as a fully matured organoid, with less variability and faster turnaround. Moving from 2D to 3D is a meaningful step forward for any drug screening programme. Asking these questions before you start ensures the data you generate is as reliable as the models you're building. From DDW Volume 27 – Issue 3, Summer 2026 – Read the digital issue here