LICORbio’s ULA plate is compatible with a wide variety of human- and animal-derived cell lines or primary cells. The plate’s surface is engineered to be highly non-adhesive, enabling normally adherent cells to interact primarily with each other rather than attach to the plate surface. This creates an ultra-low-attachment environment that supports spheroid formation in a broad variety of cells.

In general, cells that readily attach and grow in culture tend to have a higher likelihood of forming spheroids. However, not all cell types naturally form spheroids. Their ability to aggregate depends on factors such as the original tissue’s structure, function, and morphology, as well as how the cells were isolated.

To optimize spheroid formation, experimental conditions may need adjustment. This can include changing cell seeding density, modifying media composition, or adding supportive components such as ECM supplements or matrix-producing cells. When working with a new cell type, it is best to test a range of conditions to determine the most effective approach.

LICORbio’s ULA plates are optically clear and suitable for many applications, including imaging directly in the plate, fluorescent image analysis including live/dead cell assay of spheroids, fluorescent staining of cell structures, and antigen detection, as well as brightfield image analysis, such as quantitative evaluation of spheroid size and morphology.

Irregular spheroids or the formation of multiple aggregates within a well can result from several factors, including:

• Excessively high seeding densities
• Poor single-cell suspension quality
• Cell clumping prior to seeding
• Inconsistent pipetting
• Use of flat-bottom plates instead of round-bottom ULA plates
• Cell lines with inherently weak cell–cell adhesion

To improve spheroid uniformity:

• Filter cells before seeding
• Optimize seeding density
• Use round-bottom ULA plates
• Minimize vibrations or plate disturbance during early aggregation
• Monitor spheroid formation within the first 24–48 hours

Optimal spheroid size depends on the application and cell type. In general, smaller spheroids may provide better imaging clarity and nutrient penetration, while larger spheroids may better model physiologic gradients such as hypoxia or necrosis.

For many imaging and screening workflows, spheroids between approximately 200–500 µm in diameter provide a good balance between reproducibility, viability, and biological relevance.

Users should optimize seeding density and culture duration to achieve the desired spheroid size at the assay endpoint.

Because spheroids in ULA-coated microplates are not attached to the surface, removing all the media can disturb or displace them. Instead, only a portion of the used media should be removed and replaced with fresh media. To minimize disruption, perform the media exchange along the side of each well rather than directly over the spheroid. Using a slower pipetting speed, especially with electronic pipettors, can further help keep spheroids intact. A common and simple approach is to replace 50% of the media. For example, if each well contains 200 µL, gently remove 100 µL from the side and add 100 µL of fresh media. If a complete media change or wash is required, leave at least 20 µL of liquid in the well to prevent disturbing the spheroids. When using automated liquid handling systems, position the pipette tip about 2–3 mm above the bottom of the well and slightly off-center, and use low aspiration/dispense speeds for best results.

Round-bottom microplates with ULA coatings, such as LICORbio’s ULA plate, are designed to consistently produce a single, uniform spheroid in each well. The curved well shape helps guide cells toward the center, promoting even aggregation. In contrast, flat-bottom ULA wells often lead to the formation of multiple irregular cell clusters. These clusters can vary in size and distribution within a well, resulting in greater variability between wells. Due to this inconsistency, flat-bottom formats are generally less suitable for applications that require reproducibility or high-throughput screening.

If your cells are prone to sticking together or forming aggregates, passing the suspension through a 30–70 µm cell strainer can help to remove the clumps and produce a more uniform single-cell suspension.

The following cell lines have been used in-house and observed to form spherical cultures: PANC-1, BxPC-3, Hep G2, T-47D, BT-474, and LNCaP. MDA-MB-231 and SW480 were also grown in these plates with the addition of ECM.

For imaging using the LICORbio Atlas Imager, the following specifications should be entered into the Acquire ribbon, in Image Studio Software:

• Under Channels select 630 trans channel
• Under Focus > Select Focus choose
  • 3.1 mm focus offset with focus refinement off.
  • 2.0 mm air gap.
• Under Scan Controls select 20 µm resolution.

Note: Depending on your spheroid size, you may have to adjust the focus in 0.1 mm increments.