HTB-22, lot number 59388743, passage 147, shipped at different time points in October and November 2011) and cells from one vial were grown using identical protocols in two labs, Brown University (BU) and Johns Hopkins University (JHU), while the other vial was never thawed. as shown by morphology, estrogenic growth dose-response, whole genome gene expression and untargeted mass-spectroscopy metabolomics for MCF-7 cells. Using Comparative Genomic Hybridization (CGH), differences were traced back to genetic heterogeneity already in the cells from the original frozen vials from the same ATCC lot, however, STR markers did not differ from ATCC reference for any sample. These findings underscore the need for additional quality assurance in Good Cell Culture Practice and cell characterization, especially using other methods such as CGH to reveal possible genomic heterogeneity and genetic drifts within cell lines. Recently, there has been a call for increased attention to cell line authentication, annotation and quality control, which, if not carefully documented and described, can seriously affect reproducibility and scientific quality1,2,3. Since much of what we Rabbit polyclonal to LIMK1-2.There are approximately 40 known eukaryotic LIM proteins, so named for the LIM domains they contain.LIM domains are highly conserved cysteine-rich structures containing 2 zinc fingers. know about the molecular mechanisms of cancer is derived from these cell lines, and they are broadly used for drug development and regulatory testing, this represents a key concern for putting such investigations on a sound footing. The human breast adenocarcinoma cell line MCF-7 (Michigan Cancer Foundation-7) has served for over 40 years as a standard model for cancer research as well AZ505 as estrogen and progesterone receptor science4,5 and is one of the key malignancy cell lines used as a model for investigation of processes that impact patient care6. Almost 23,000 articles using MCF-7 can be retrieved in PubMed; it is used for both basic and applied sciences such as oncologic mechanisms, characterization of drug effects, as well as endocrine disruption hazard assessment of chemicals. However, it is AZ505 not clear whether all studies of MCF-7 cells actually use the same entity. As early as 1987, Resnicoff identified subpopulations in MCF-7 by Percoll gradient centrifugation that showed differences in growth rate, DNA synthesis and expression of estrogen receptors and pointed out the heterogeneous AZ505 character of MCF-77. Later, these findings were confirmed by others8,9,10 and it is now acknowledged that MCF-7 is usually heterogeneous with respect to both the expression of hormone receptors and to the utilization of the signaling pathways linked to these receptors, differences that result in phenotypic heterogeneity11. Sub-clones vary in estrogen and progesterone receptor expression, as well as epidermal growth factor. However, genotyping analysis shows that all sub-clones are related to the AZ505 parental MCF-7 cell line12. Nonetheless, even though questions have been raised about the reproducibility of results with MCF-7 cells13, many laboratories assume that by using cells obtained from a cell lender, standardizing protocols, limiting the number of passages, and employing SNP or STR cell authentication techniques would ensure that their sub-clone will behave with sufficient stability and reproducibility. Our experience is usually that this may not be necessarily sufficient. Based on data from our Human Toxome Project14,15, we demonstrate by various techniques that there can be marked cellular and phenotypic heterogeneity in a single batch of cells from a cell lender that are invisible with the usual STR cell authentication protocols, and that this heterogeneity has serious consequences for reproducibility and primary outcomes of experiments. Results As part of our Mapping the Human Toxome project, two laboratories (Brown University [BU] and Johns Hopkins University [JHU]) used MCF-7 cells from the same ATCC lot (lot number 59388743, passage 147) combined with rigid adherence to standards for validation (standard operations protocols, formal training, and transfer) for cell culture and analytic methods14 including the recommendations for Good Cell Culture Practice16. In a first step, this work included expansion of the cells from the original ATCC AZ505 vials using three passages for BU and eight passages for JHU, respectively to create vials for use in experiments, each of which were then passaged up to 10 occasions after which another vial was thawed for continuing experiments. Recommended genomic typing of short tandem repeat markers (STR) showed that all MCF-7 cell markers were the same lengths as provided by the reference ATCC genotyping panel for all those 9 typed markers (Table 1). Nonetheless, significant differences were observed between the two laboratories in terms of phenotype, gene expression patterns, metabolomics, and (most crucially) sensitivity to estradiol-driven proliferation. To exclude any possible inter-laboratory and/or inter-operator effects, the JHU cells were shipped to BU to verify the results. The results of the morphological, phenotypical and gene expression differences that have been performed in one laboratory (BU) by one individual are given in Fig. 1: Morphologic assessment of the MCF-7 cells showed that BU cells grow in large aggregations while JHU cells grow flat, with cobblestone morphology (Fig. 1A). Following 72?hours of exposure to estradiol (E2), BU MCF-7 cells displayed significant increases in proliferation (cell count) at concentrations of.