Stop using the misidentified cell line LO2 as a human hepatocyte

Bifidobacterium pseudolongum-generated acetate suppresses non-alcoholic fatty liver disease-associated hepatocellular carcinomaJournal of HepatologyVol. 79Issue 6PreviewRecent studies have highlighted the role of the gut microbiota and their metabolites in non-alcoholic fatty liver disease-associated hepatocellular carcinoma (NAFLD-HCC). We aimed to identify specific beneficial bacterial species that could be used prophylactically to prevent NAFLD-HCC. Full-Text PDF Open AccessReply to: "Stop using the misidentified cell line LO2 as a human hepatocyte"Journal of HepatologyVol. 80Issue 5PreviewWe appreciate the comment by Shao and Chen,1 with reference to our recent paper,2 that LO23 might be a misidentified cell line not representative of normal human hepatocyte. Full-Text PDF Recently, we read the article by Song et al. in the Journal of Hepatology.1Song Q. Zhang X. Liu W. et al.Bifidobacterium pseudolongum-generated acetate suppresses non-alcoholic fatty liver disease-associated hepatocellular carcinoma.J Hepatol. 2023; 79: 1352-1365Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar Using cellular and animal models, the authors concluded that B. pseudolongum protected against non-alcoholic fatty liver disease-associated hepatocellular carcinoma (NAFLD-HCC) by secreting the antitumor metabolite acetate through the gut-liver axis, and thus, suggested that B. pseudolongum is a potential probiotic for NAFLD-HCC prevention. Undoubtedly, the results of this study are very promising regarding a novel probiotic for the potential prevention and treatment of NAFLD-HCC in humans. However, some in vitro data still need to be questioned. In particular, the authors did not clearly describe the cell origin and falsely employed "LO2 cells" in a group of experiments as a "human immortalized hepatocyte cell line". LO2, also written as L02, L0-2, LO-2, or HL-7702, is a cell line established by Chinese researchers. In the 1970s, 10 cases of primary hepatocytes were derived from adult human liver quadrate lobes by Yeh et al. of our institute (formerly named Shanghai Institute of Cell Biology, Chinese Academy of Sciences). Four were able to be cultured continuously for a long period of time (the exact time was not recorded). One of them was cultured continuously for 16 months in vitro and was named Human Liver-7702 (HL-7702).2Yeh H.J. Chu T.H. Shen T.W. Ultrastructure of continuously cultured adult human liver cell.Acta Biologiac Experimentalis Sinica. 1980; 13: 361-369Google Scholar In that study published in 1980, Yeh et al. demonstrated that before the 10th subculture, HL-7702 cells appeared to have characteristics that were similar to those of human normal liver cells in vivo under an electron microscope. From the 11th to 32nd passage, the HL-7702 cells underwent a change in morphology, including the nuclear shape and the number and structure of cytoplasmic organelles, especially mitochondria. After decades, this cell line was gradually misconsidered as a human fetal hepatocyte cell line.3Hu X. Yang T. Li C. et al.Human fetal hepatocyte line, L-02, exhibits good liver function in vitro and in an acute liver failure model.Transplant Proc. 2013; 45: 695-700Crossref PubMed Scopus (43) Google Scholar In 2015, it came to our attention that the LO2 cell line was possibly contaminated with the cervical cancer cell line HeLa,4Ye F. Chen C. Qin J. et al.Genetic profiling reveals an alarming rate of cross-contamination among human cell lines used in China.FASEB J. 2015; 29: 4268-4272Crossref PubMed Scopus (78) Google Scholar as we failed to establish a correct STR profile of the LO2 cells derived from early passage samples preserved in referenced repositories or different laboratories in China. According to the findings by Yeh et al., the LO2 cell line might have been contaminated or misidentified at the time of lineage establishment. There is also the possibility that contamination or misidentification was caused by long-term cultivation. Similar to several problematic hepatocellular carcinoma (HCC) cell lines (BEL7402, SMMC7721, MHCC97L, BEL7404, QGY7701, QGY7703, and QSG7701) described previously,5Rebouissou S. Zucman-Rossi J. Moreau R. et al.Note of caution: contaminations of hepatocellular cell lines.J Hepatol. 2017; 67: 896-897Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar LO2 was clearly shown to be a HeLa derivative and listed with registration ID ICLAC-00575 as a misidentified cell line released by the International Cell Line Authentication Committee (ICLAC). Unfortunately, this misidentified cell line is still frequently used as a hepatocyte derivative in many laboratories, especially in China. A total of 2,195 results were found when the terms "LO2 cells", "L-O2 cells", "L02 cells" or "L-02 cells" were searched for together with the word "liver" on the Web of Science. In the past 5 years, this mistaken cell line has been used in over 150 articles annually (Fig. 1A). Articles originating in China accounted for 93.35% of these, and those from the United States (2.55%), Japan (0.36%), Singapore (0.32%), and South Korea (0.32%) round out the top five countries (Fig. 1B). The top ten research areas of this cell were as follows: biochemistry molecular biology (1,282 hits), gastroenterology hepatology (1,179 hits), pharmacology pharmacy (1,136 hits), cell biology (1,124 hits), genetics heredity (819 hits), oncology (671 hits), toxicology (541 hits), chemistry (384 hits), general internal medicine (368 hits), and pathology (270 hits) (Fig. 1C). To avoid possible academic controversy, cell line authentication remains the best approach to addressing the problem of cross-contamination and misidentification. Although it does not directly prevent contamination, it allows researchers to verify the authenticity of a cell line before performing any experiments on it. Here, we urgently call on scientists and researchers to take necessary action and perform regular quality control for potential contaminants that commonly occur in culture. We highly recommend that researchers use THLE-2 or THLE-3 cell lines as normal human hepatocytes.6Pfeifer A.M. Cole K.E. Smoot D.T. et al.Simian virus 40 large tumor antigen-immortalized normal human liver epithelial cells express hepatocyte characteristics and metabolize chemical carcinogens.Proc Natl Acad Sci USA. 1993; 90: 5123-5127Crossref PubMed Scopus (215) Google Scholar Moreover, we appeal to the Journal of Hepatology to no longer consider accepting experiments using LO2 as a normal hepatocyte cell line. This work was supported by grants from the National Science and Technology Infrastructure, the Biological Resources Program of Chinese Academy of Sciences (BRP-CAS) and the Technical Talents Program of CAS. The authors declare no potential conflicts of interest. Please refer to the accompanying ICMJE disclosure forms for further details. TS conceived the idea, conducted the data analysis and drafted the manuscript. YLC supervised the study, provided financial support and revised the manuscript. All authors have read and approved the manuscript for publication. The data that support the findings of our study are available from the corresponding author upon request. The following are the supplementary data to this article: Download .pdf (.18 MB) Help with pdf files Multimedia component 1