Establishment and characterization of an immortalized epithelial cell line from human gallbladder

Ziyi Wang; Shijia Wang; Ziheng Jia; Yuhao Zhao; Mao Yang; Weikang Yan; Tao Chen; Dongxi Xiang; Rong Shao; Yingbin Liu

doi:10.3389/fonc.2022.994087

Establishment and characterization of an immortalized epithelial cell line from human gallbladder

Front Oncol. 2022 Oct 28:12:994087. doi: 10.3389/fonc.2022.994087. eCollection 2022.

Authors

Ziyi Wang^{1

2

3

4}, Shijia Wang^{1

2

3

4}, Ziheng Jia^{1

2

3

4}, Yuhao Zhao^{1

2

3

4}, Mao Yang^{1

2

3

4}, Weikang Yan^{1

2

3

4}, Tao Chen^{1

2

3

4}, Dongxi Xiang^{2

3

4}, Rong Shao^{1

2

3

4

5}, Yingbin Liu^{1

2

3

4}

Affiliations

¹ Department of Biliary-Pancreatic Surgery, Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China.
² Shanghai Cancer Institute, State Key Laboratory of Oncogenes and Related Genes, Shanghai, China.
³ Shanghai Key Laboratory of Biliary Tract Disease, Renji Hospital, Shanghai, China.
⁴ Shanghai Research Center of Biliary Tract Disease, Renji Hospital, Shanghai, China.
⁵ Department of Pharmacology and Biochemistry, Shanghai Jiao Tong University School of Medicine, Shanghai, China.

Abstract

Background: Although a plethora of studies have employed multiple gallbladder cancer (GBC) cell lines, it is surprisingly noted that there is still lack of a normal gallbladder epithelial cell line as a normal counterpart, thus impeding substantially the progress of mechanistic studies on the transformation of normal epithelial cells to cancer. Here, we created a normal gallbladder epithelial cell line named L-2F7 from human gallbladder tissue.

Methods: Gallbladder tissues from a diagnosed cholecystitis female patient were collected, and epithelial cells were enriched by magnetic cell sorting. Then, the cells were immortalized by co-introduction of human telomerase reverse transcriptase (hTERT) and Simian virus 40 large T antigen (LT-SV40) via a lentivirus infection system. After clonal selection and isolation, L-2F7 cells were tested for epithelial markers CK7, CK19, CK20, and CD326, genomic feature, cell proliferation, and migration using Western blot, immunofluorescence, whole genome sequencing, karyotyping, and RNA sequencing. L-2F7 cells were also transplanted to Nude (nu/nu) mice to determine tumorigenicity.

Results: We successfully identified one single-cell clone named L-2F7 which highly expressed epithelial markers CD326, CK7, CK19, and CK20. This cell line proliferated with a doubling time of 23 h and the epithelial morphology sustained over 30 passages following immortalization. Transient gene transduction of L-2F7 cells led to expression of exogenous GFP and FLAG protein. L-2F7 cells exhibited both distinct non-synonymous mutations from those of gallbladder cancer tissues and differential non-cancerous gene expression patterns similar to normal tissue. Although they displayed unexpected mobility, L-2F7 cells still lacked the ability to develop tumors.

Conclusion: We developed a non-cancerous gallbladder epithelial cell line, offering a valuable system for the study of gallbladder cancer and other gallbladder-related disorders.

Keywords: cell lines; epithelium cell; gallbladder; gallbladder cancer; immortalization.