In vitro Cytotoxicity Assay: Colony Forming Assay

In vitro Cytotoxicity Assay: Colony Forming Assay

(hamster cell line V79)

In this assay the cytotoxicity of leachable substances released from the test item is assessed through the colony forming ability after treatment with various concentrations of the test item extract compared to the controls.

The dilution that reduces colony formation to 50% (IC₅₀) can be determined by plotting the relative colony forming rates (%) versus the dilutions of the extracts (%).

The mean number of colonies formed in the cultures treated with fresh culture medium represents the absolute colony forming ability of cells, which reflects the survival rate of the cells after passage and seeding in the culture plates (100% control).

Colony Forming Assay

Assessment of Cytotoxicity by Colony Forming Ability

The test item is extracted for a defined period of time at 37 ± 1°C with cell culture medium and V79 cells are incubated with the extract for several days.
After the incubation period, the cultures are observed microscopically and washed carefully with PBS buffer. After fixation with methanol, the colonies are stained with Giemsa‘s staining solution.
The stained colonies are dried at room temperature. The colonies consisting of more than 50 cells are counted manually.

Colony Forming Assay

Test Item Acceptance Criteria:

IC₅₀ acceptance limit according to MHLW: ≥ 90%
Reduction of relative plating efficiency at 100% concentration of the test solution by more than 30% is considered as a cytotoxic effect

Protocol
Cell line	V79 cells (ATCC, CCL-93).
Analysis	The mean number of colonies formed in the cultures represents the absolute colony forming ability of cells, which reflects the survival rate after treating with the test item
Concentrations	6 concentrations of the test extract
Extraction time	24 h at 37 ± 1°C
Incubation time	6 - 7 days at 37 ± 1°C
Quality controls	100% control: Fresh MEM 10% FBS Solvent control: MEM 10% FBS Negative control: High-density polyethylene extracted in MEM 10% FBS Positive controls: ZDEC- and ZDBC- polyurethane film extracted in MEM 10% FBS
Data delivery	Colony Forming Ability
Positive prediction	IC₅₀ acceptance limit according to MHLW: < 90% Reduction of relative plating efficiency at 100% concentration of the test solution by more than 30% is considered as a cytotoxic effect

References

ISO 10993-12: 2012 “Sample preparation and reference materials”
MHLW (Ministry of Health, Labour and Welfare, Japan) Notification by Director of OMDE Yakushokuki-hatsu No. 0301-20, “Basic Principles of Biological Safety Evaluation Required for Application for Approval to Manufacture/Market Medical Devices”, March 1, 2012
MHLW (Ministry of Health, Labour and Welfare, Japan) Ministerial Notification by Director of Health, Labour and Welfare Yasuhisa Shiozaki No. 64, The Japanese Pharmacopoeia, Seventeenth Edition, March 07, 2016, chap.7.03, 4. “Cytotoxictity test”

In vitro Cytotoxicity Assay: Colony Forming Assay