Background Wide acceptance of the colony-forming-unit (CFU) assay as a reliable potency test for stem cell products is hindered by poor inter-laboratory reproducibility. The goal of this study was to ascertain current laboratory practices for performing the CFU assay to identify practices that could be standardized to improve overall reproducibility. Materials and Methods A survey to evaluate current laboratory practices for performing CFU assays was designed and internationally distributed. Results A total of 105 individuals initiated the survey, of which 68% perform CFU assays. A majority specified that an automated rather than a manual cell count was performed on pre-diluted aliquots of stem cell products. Viability testing methods employed a variety of stains and when multiple sites used the same viability stain the methods differed. Cell phenotype used to prepare working cell suspensions for inoculating the CFU assay differed among sites. Most respondents scored CFU assays between 14–16 days of incubation, but culture plates were read with a variety of different microscopes. Of 57 respondents, 42% had not performed a validation study or established assay linearity. Only 63% of laboratories had criteria for determining if a plate was overgrown with colonies. Conclusion Survey results revealed inconsistent inter-laboratory practices for performing the CFU assay. Moreover, the relatively low number of centers with validated CFU assays raises concerns about assay accuracy and emphasizes a need for the establishment of central standards. The survey results shed light on a number of steps of the methodology that could be targeted for standardization across laboratories.

Understanding current practice will help in the design of future studies for CTP storage and transportation.

The survey results revealed a variety of tests and inconsistent interlaboratory practices for performing the viability assay. Flow cytometry with a 7-AAD dye was suggested as a first step toward standardization.

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