A simple, animal-free protocol for dissociating adherent cells with CellTrypase — a defined recombinant trypsin-like enzyme that drops into your existing trypsinization workflow with no inhibitor and no protocol changes.
Before you start
Bring CellTrypase and culture medium to room temperature or 37 °C (warmer temperatures accelerate detachment). Have PBS and fresh medium ready, and work under aseptic conditions. No trypsin inhibitor is required — the enzyme is inactivated by dilution.
Dissociation procedure (1× ready-to-use)
- Aspirate the spent medium and rinse the cell monolayer once with PBS.
- Add CellTrypase to fully cover the cells — approximately 1 mL of 1× solution per 25 cm² of surface area.
- Incubate at room temperature or 37 °C until cells detach. Monitor visually and gently tap the vessel to aid release. Optional: for sensitive or tightly adherent cells, gently pipette up and down to complete dissociation.
- Quench by adding fresh medium — no inhibitor needed.
- Optional: for complete enzyme removal, centrifuge and resuspend the cells in fresh medium.
Using the 10× concentrate
The 10× stock can be used undiluted for fast detachment of strongly adherent cells, or diluted to 1× in PBS containing 1 mM EDTA under sterile conditions. For most applications a 1× working concentration gives gentle, efficient release while maintaining high viability.
Tips & troubleshooting
| Issue | Likely cause | What to do |
|---|---|---|
| Incomplete detachment | Under-incubated or strongly adherent line | Extend incubation, warm to 37 °C, gently pipette, or use the 10× stock undiluted |
| Cell clumping | Enzyme didn’t fully cover the monolayer; harsh pipetting | Ensure complete coverage; pipette gently; resuspend fully in medium |
| Lower-than-expected viability | Over-digestion | Minimize incubation time and quench promptly with fresh medium |
Validated performance
In c-LEcta’s validation, CellTrypase (1×) matched the current industry-standard trypsin-like enzyme on dissociation, with high viability and equal-or-higher yield:
| Cell line | Release time | Mean viability | Yield vs standard |
|---|---|---|---|
| CHO-K1 | 2:25 | 98% | 106% |
| HEK 293 | 2:27 | 95% | 100% |
| MDCK | 23:32 | 99% | 102% |
| Vero | 4:07 | 99% | 103% |
Yield as a percentage of the current industry-standard trypsin-like enzyme (control). Source: CellTrypase PIS v2.0 (c-LEcta).
Get CellTrypase
CellTrypase is available in an R&D grade (in-house, ISO 9001-compliant manufacturing) and a GMP grade (in-house GMP production under a QMS compliant with the EXCiPACT® certification standard), in 100 mL and 500 mL. BioHippo offers a free 100 mL sample — one per lab, while supplies last. Request a free sample or view the CellTrypase product page.
Have a question about dissociating a tricky or sensitive cell line? Ask a scientist — our team will help you adapt the protocol to your cells.
