Abstract
BackgroundMollicutes contamination is recognized to be a critical issue for the cultivation of continuous cell lines. In this work we characterized the effect of Mycoplasma hyorhinis contamination on CD133 expression in human colon cancer cell lines.MethodsMycoAlert® and mycoplasma agar culture were used to detect mycoplasma contamination on GEO, SW480 and HT-29 cell lines. Restriction fragment length polymorphism assay was used to determine mycoplasma species. All cellular models were decontaminated by the use of a specific antibiotic panel (Enrofloxacin, Ciprofloxacin, BM Cyclin 1 and 2, Mycoplasma Removal Agent and MycoZap®). The percentage of CD133 positive cells was analyzed by flow cytometry on GEO, SW480 and HT-29 cell lines, before and after Mycoplasma hyorhinis eradication.ResultsMycoplasma hyorhinis infected colon cancer cell lines showed an increased percentage of CD133+ cells as compared to the same cell lines rendered mycoplasma-free by effective exposure to antibiotic treatment. The percentage of CD133 positive cells increased again when mycoplasma negative cells were re-infected by Mycoplasma hyorhinis.ConclusionsMycoplasma hyorhinis infection has an important role on the quality of cultured human colon cancer cell lines giving a false positive increase of cancer stem cells fraction characterized by CD133 expression. Possible explanations are (i) the direct involvement of Mycoplasma on CD133 expression or (ii) the selective pressure on a subpopulation of cells characterized by constitutive CD133 expression.In keeping with United Kingdom Coordinating Committee on Cancer Research (UKCCCR) guidelines, the present data indicate the mandatory prerequisite, for investigators involved in human colon cancer research area, of employing mycoplasma-free cell lines in order to avoid the production of non-reproducible or even false data.
Highlights
Mollicutes contamination is recognized to be a critical issue for the cultivation of continuous cell lines
Mycoplasma hyorhinis infection has an important role on the quality of cultured human colon cancer cell lines giving a false positive increase of cancer stem cells fraction characterized by CD133 expression
The aim of this study is to show how and to what extent the expression of CD133 antigen can be altered by Mycoplasma hyorhinis contamination
Summary
Mollicutes contamination is recognized to be a critical issue for the cultivation of continuous cell lines. The aim of this study is to show how and to what extent the expression of CD133 antigen can be altered by Mycoplasma hyorhinis contamination To address this issue, we employed the well-established GEO, SW480 and HT-29 human colon cell lines [4,5,6] and multidimensional flow cytometry. In spite of a bright positivity for CD133 antigen during Mycoplasma hyorhinis contamination, after antibiotic decontamination it was possible to evidence a decrease in the percentage of CD133 positive cellular population. On these bases we decided to demonstrate that Mollicutes agents can have remarkable effects on the correct identification and characterization of CD133 positive CSCs subpopulation
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