Non-Adhesive Well Plates For Spheroid Generation

Spheroid are defined as 3-dimentional cell aggregates that are formed via spontaneous self-assembly of cells when cultivated on a cell repellant, non-adhesive surface. Spheroids are by far one of the most popular scaffold-free 3D cell culture methods, and are low tech and low cost compared to other conventional 3D culture platforms.

Creating Non-adhesive surfaces

Non-adhesive surfaces facilitates the formation of spheroids by preventing protein adsorption and cell attachments thus allowing cell-cell aggregation (1).

Agar and agarose are popular biomaterials used to modify surfaces to become non-adhesive. Both agar and agarose are relatively low cost, are soluble at temperatures equal or higher than 650C and are compatible with autoclave-based sterilization. However, despite these advantages, agar and agarose-coated plates can be used for cell cultivation for a limited period of time (2-4).

An alternative to agar and agarose is poly 2-hydroxyethyl methacrylate (poly-HEMA). This coating material is more expensive, but offers the advantage of extended storage time, and longer usage period over several months. However, surface coating with poly-HEMA requires 5-7 hours, as well as additional time to evaporate the ethanol used as a solvent. Although it is more stable over long periods than agar or agarose, the process is costly and time consuming and thus affects reproducibility (2,5).

In the past, non-adhesive surfaces were made using petri dishes or cell culture flasks. However. spheroids on flat, and wider surface tends to be variable in size (2). In recent times, round-bottomed 96-well plates are used to form spheroids. In these plates, the cells aggregate at the bottom of the wells via gravity. When compared with other conventional spheroid culture methods such as the hanging drop method, these cultures are easier to handle and require little to no external manipulation. One of the biggest advantages is the ability to control spheroid size simply by changing the density of seeded cells per well (6).

Commercialized non-adhesive well plates

Currently, there are many commercialized coated plates available that are highly effective across a wide range of cell lines. These include microplates with treated non-adhesive surfaces and U bottom. Among them,  BiofloatTM 96- and 384-well plates are coated with a special polymeric solution, that renders the surface cell repellant. BiofloatTM Flex solution can also be used to modify plastic and glas surfaces into non-adhesive surfaces. The commercialized products carry the advantage of improving end-user convenience in terms of reducing spheroid formation time, thus contributing to increased uniformity and reproducibility. All in all, microplates with modified non-adhesive surface is by far the simplest method to generate uniform spheroids in a time and cost effective manner (7,8).


  1. Kim, S.J., Kim, E.M., Yamamoto, M., Park, H., Shin, H.: Engineering Multi-Cellular Spheroids for Tissue Engineering and Regenerative Medicine. Adv. Healthc. Mater. 9, e2000608 (2020).
  2. Costa, E.C., de Melo-Diogo, D., Moreira, A.F., Carvalho, M.P., Correia, I.J.: Spheroids formation on non-adhesive surfaces by liquid overlay technique: considerations and practical approaches. Biotechnol. J. 13, 1700417 (2018).
  3. Dalen, H., Burki, H.: Some observations on the three-dimensional growth of L5178Y cell colonies in soft agar culture. Exp. Cell Res. 65, 433–438 (1971).
  4. Su, G., Zhao, Y., Wei, J., Han, J., Chen, L., Xiao, Z., Chen, B., Dai, J.: The effect of forced growth of cells into 3D spheres using low attachment surfaces on the acquisition of stemness properties. Biomaterials 34, 3215–3222 (2013).
  5. Kuroda, Y., Wakao, S., Kitada, M., Murakami, T., Nojima, M., Dezawa, M.: Isolation, culture and evaluation of multilineage differentiating stress-enduring (Muse) cells. Nat. Protoc. 8, 1391–1415 (2013).
  6. Pereira, P.M.R., Berisha, N., Bhupathiraju, N., Fernandes, R., Tome, J.P.C., Drain, C.M.: Cancer cell spheroids are a better screen for the photodynamic efficiency of glycosylated photosensitizers. PLoS ONE 12, e0177737 (2017).
  7. Park, S.Y., Hong, H.J. & Lee, H.J. Fabrication of Cell Spheroids for 3D Cell Culture and Biomedical Applications. BioChip J17, 24–43 (2023).