Agarose overlay selectively improves macrocolony formation and radiosensitivity assessment in primary fibroblasts

Abstract

Purpose: Primary fibroblasts are not suitable for in vitro macrocolony assay due to their inability to form distinct colonies. Here we present a modification of agarose overlay that yielded extensive improvement in their colony formation and assessment of radiosensitivity.

Materials and methods: Macrocolony formation was assessed in primary human fibroblasts VH10 and HDFn with or without overlay using 0.5% agarose in growth medium at 24 h post-seeding. Malignant human cell lines (A549, U87) and transformed non-malignant fibroblasts (AA8 hamster, MRC5 human) were used for comparison.

Results: Agarose overlay caused significant improvement marked by early appearance (one week) of distinct colonies with high cell density and multifold higher plating efficiency than conventional macrocolony assay in VH10 and HDFn human fibroblasts. Compared to conventional assay or feeder cell supplementation, agarose overlay resulted in broader cell morphology due to improved adherence, and yielded more compact colonies. Gamma-radiation dose-response survival curves could be successfully generated for both fibroblast cell lines using this method, which yielded no such effects in the transformed/malignant cell lines tested.

Conclusion: This easy and inexpensive ‘agarose overlay technique’ significantly and selectively improves the fibroblast plating efficiency, thus considerably reducing time and effort to greatly benefit the survival studies on primary fibroblasts.

Keywords::

• Agarose overlay macrocolony assay
• radiosensitivity

Acknowledgements

Authors are thankful to Dr R.P. Tripathi, Director, Institute of Nuclear Medicine & Allied Sciences (INMAS) for his special contribution in facilitating the collaboration between INMAS (Delhi) and Stockholm University (Stockholm). Authors thank Mr Ravi Soni and Ms Anjali for help during irradiation.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.