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In vitro Cytotoxicity Assay: Colony Forming Assay

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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

  1. ISO 10993-12: 2012 “Sample preparation and reference materials”
  2. 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
  3. 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”