CellTrypase Cell Dissociation Protocol, Animal-Free Trypsin / TrypLE™ Alternative

A simple, animal-free protocol for dissociating adherent cells with CellTrypase, a defined recombinant trypsin-like enzyme that is dosed at the same volume per unit growth area as your existing trypsinization workflow, with no inhibitor step. Dosing translates directly; detachment kinetics do not — verify detachment time and viability in your own cell model before switching.

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)

1. Aspirate the spent medium and rinse the cell monolayer once with PBS.
2. Add CellTrypase to fully cover the cells, approximately 1 mL of 1× solution per 25 cm² of surface area.
3. 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.
4. Quench by adding fresh medium, no inhibitor needed.
5. Optional: for complete enzyme removal, centrifuge and resuspend the cells in fresh medium.

Release time varies widely by cell line. On the manufacturer's own data, MDCK takes roughly ten times longer than CHO-K1 or HEK 293 (see the table below). Determine the endpoint visually on your own line rather than fixing a single incubation time across a facility.

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 efficient release while maintaining high viability.

Tips & troubleshooting

Validated performance

The manufacturer reports the following on four adherent lines, the only ones with a performance table in the product information sheet. Release time, viability and yield are the single values published there; the doubling times are means across five passages (P0–P4) from a Kerry (formerly c-LEcta) scientific poster, given as industry-standard control → CellTrypase. Figures are reproduced as published, including the MDCK result:

Source: CellTrypase Product Information Sheet v4.0 (Kerry, 18 August 2026); doubling times from Kerry scientific poster #11462, “Advancing cell dissociation in bioprocessing with CellTrypase” (February 2026), n = 3 technical replicates, mean ± SD. Yield is reported relative to a reference condition defined by the manufacturer; the product information sheet does not name a comparator product. Values are the manufacturer's; BioHippo has not independently repeated these experiments.

Kerry has since published five-passage data (P0–P4) for CHO-K1, HEK 293, MDCK and Vero, covering viability, relative yield and population doubling time. Doubling times were equal to or shorter than the industry-standard control on all four lines, so proliferation is maintained over serial passaging rather than merely assumed.

Human iPSC data is now published. In 2D colonies, 1× CellTrypase reached a single-cell suspension in 5 minutes against 7 minutes for the industry-standard control, with 94% viability for both enzymes, 99% relative yield, residual aggregates below 5%, and pluripotency markers (Oct3/4, SSEA4, TRA-1-60, CD30, Nanog, Sox2, SSEA3) above the 75% acceptance threshold by flow cytometry. In 3D iPSC aggregates both enzymes took about 10 minutes; CellTrypase gave 87% viability against 85%, 104% relative yield and fewer residual aggregates (12% against 16%). Source: Kerry scientific poster #11462, “Advancing cell dissociation in bioprocessing with CellTrypase” (February 2026); n = 3 technical replicates, mean ± SD.

Published comparative data covers CHO-K1, HEK 293, MDCK, Vero and human iPSC in 2D and 3D. Other cell types carry no published figures — including hESC, primary cells and organoids, which the manufacturer names as intended applications, and everything else besides — so qualify the enzyme on your own cells before adopting it in a routine or regulated workflow.

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.

Trademark notice: TrypLE™ is a trademark of Thermo Fisher Scientific Inc. Any reference is nominative and for identification or comparison only; no affiliation with, sponsorship by, or endorsement from the trademark owner is implied. BioHippo is not affiliated with, endorsed by, or sponsored by Thermo Fisher Scientific.
Research use statement: CellTrypase R&D grade is supplied For Research Use Only (RUO) and is not for use in diagnostic or therapeutic procedures. GMP-grade material is supplied as a processing aid for biopharmaceutical manufacturing and is not for direct administration to humans or animals.
Change control: Substituting a raw material in a validated, licensed or clinical-phase process is a change governed by your own quality system. Nothing in this protocol determines the scope of qualification or revalidation work required on your side.
Data source: The four-line release time, viability and yield figures are taken from the CellTrypase Product Information Sheet v4.0 (Kerry, 18 August 2026), while the doubling times and the five-passage and iPSC figures are taken from Kerry scientific poster #11462, “Advancing cell dissociation in bioprocessing with CellTrypase” (February 2026).