this may happen when a drug / compound with high solubility in DMSO, but low aqueous solubility, may precipitate out of solution when added to an aqueous cell culture media and crystalise out of solution as the DMSO goes from 100% to less than 1%.

As a control you should test whether it happens in just media.

Thank you for the reply Alwyn Dart. We did a troubleshooting experiment with just the media for a couple of days. But we didn’t find anything.

Hi Prathiksha,

Most “crystals” in these setups are precipitated small molecules or vehicle: pre-warm media, add stocks dropwise with mixing, keep final DMSO ≤0.05–0.1%, and 0.22 µm-filter working solutions; include vehicle-only controls. (I used to work in media development)

Salt precipitation happens with Ca²⁺/phosphate or pH drift: avoid mixing high-phosphate media with Ca²⁺ concentrates, keep CO₂/bicarbonate stable, limit edge evaporation (use humidified chambers or plate seals).

Proteinaceous flakes can come from ECMs (e.g., Matrigel/laminin) after multiple freeze–thaws or overheating; switch to fresh lots and simplify the matrix during the small-molecule exposure window.

If precipitates are disrupting adhesion, a cleaner base layer helps: DendroTEK dPGA | Protease-Resistant Culture Matrix under a laminin overlay maintains attachment and reduces debris.

Best of luck,

Jason Hamlin

Source: I currently work at DendroTEK Biosciences.

www.dendrotek.ca