Benign human prostate specimens

Dear author,

Thanks for your interest on our protocol. Below is a more comprehensive description of our protocol for benign prostate epithelial cell lines establishment from our eLife publication together with some tips:

1.    Dissociation and preparation of single cell suspension: Human prostate tissue specimens was cut into small pieces with scalpels, washed with PBS with 4 mg/ml Gentamicin (Gibco), and then minced with scissors. Human prostate tissues were then incubated in DMEM/F12 (Gibco) supplemented with 5% FBS, 1:10 dilution of collagenase/hyaluronidase (STEMCELL Technologies) at 37°C for 3 hr. Dissociated tissues were spun at 350 g for 5 min, and resuspended in ice-cold 0.25% trypsin-EDTA (STEM-CELL Technologies), followed by incubation at 4°C for 1 hr. Trypsinization was stopped by addition of Modified Hank’s Balanced Salt Solution (HBSS) (STEMCELL Technologies) supplemented with 2% FBS. After centrifugation at 350 g, pelleted cells were resuspended with pre-warmed 5 U/ml dispase (STEMCELL Technologies) supplemented with 1:10 dilution of 1 mg/ml DNase I (STEMCELL Technologies), triturated vigorously for 1 to 2 min, and diluted by addition of HBSS/2% FBS. Finally, the cell suspension was passed through a 40 µm cell strainer (Falcon).

      Additional notes: The specimens should be subjected for dissociation within 1 hour of surgery to ensure higher cell viability. In addition, all dissociation reagents should be freshly prepared to obtain optimal results. Moreover, consistent and vigorous trituration step is crucial for dissociating tissues or cell clusters into single cells.    

2.    Isolation of human prostate epithelial cells: For flow sorting of human prostate epithelial cells, cell suspensions were stained on ice for 25 min with fluorescent-tagged EpCAM (BioLegend #324208, specific for human) and E-cadherin (eBioscience #46-3249-82) antibodies. The stained cells were spun, and cell pellets washed with HBSS/2% FBS, followed by resuspension in HBSS/2% FBS with 10 mM Y-27632 (ROCK inhibitor; STEMCELL Technologies) and a 1:1000 dilution of 0.5 mg/ml DAPI to exclude dead cells. Both side-scatter pulse width (SSC-W) vs. area (SSC-A) and forward side-scatter pulse area (FSC-A) vs. heights (FSC-H) were used to isolate single dissociated cells. Human prostate epithelial cells were isolated based on EpCAM and/or E-cadherin expression.

Additional notes: DAPI exclusion step is crucial to remove all dead cells, which could affect the success of prostate epithelial cell line establishment. In addition, all EpCAM or E-cadherin single positive cells as well as EpCAM and E-cadherin double positive cells are sorted out for culture.        

3.    Establishment of prostate epithelial cell lines: To derive benign prostate epithelial adherent cell lines, the isolated prostate epithelial cells were seeded at 100,000 per well on a 6-well Primaria plate (Corning)and cultured with the identical prostate organoid medium (Chua et al., 2014, Nature Cell Biology). The medium consisted of hepatocyte medium supplemented with 10 ng/ml epidermal growth factor (EGF) (Corning), 10 mM Y-27632 (STEMCELL Technologies), 1x glutamax (Gibco), 5% Matrigel (Corning), 5% charcoal-stripped FBS (Gibco) heat-inactivated at 55°C for 1 hr, and supplemented with 1 nM DHT (Sigma). Passaging of adherent cultures was performed by removal of accumulated Matrigel on surface of the cells by gentle washing with cold PBS, treated with 0.25% trypsin for 5 min at 37°C, and mechanically dissociated. Medium was changed every 4 days. Adherent cells were frozen in media consisting of 80% FBS, 10% complete medium, and 10% DMSO.

Additional notes: Cells grow much better on a Primaria plate compared to other ordinary culture plates. In addition, once the medium is prepared, it should be good for three weeks. However, Rock inhibitor, Y-27632, Matrigel and DHT should only be added into the medium prior to applying to the culture. In particular, Matrigel should only be added into cold medium and then the mixture should be warmed for 15-30 minutes prior to use.

For more detailed information about tissue dissociation and the medium preparation, please kindly refer to our previous publication in Nature Cell Biology (Chua et al., 2014) as well as the book chapter that we wrote and published recently in Methods in Molecular Biology (Yu and Chua, 2019). Please don’t hesitate to contact me if you have more specific questions.

Regards,

Chee Wai

Chee Wai Chua, PhD

Principal Investigator 

Laboratory of Prostate Tumor Biology and Modeling

Renji-Med X Clinical Stem Cell Research Center;

State Key Laboratory of Oncogenes and Related Genes; and

Department of Urology

Renji Hospital, School of Medicine

Shanghai Jiao Tong University

Shanghai 200127, China

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