TITLE: Primary Prostate Cell Culture from prostatectomies
AUTHORS: Mira Jung

[DESCRIPTION]
Based on the tissue collection protocol under the Georgetown University’s Institutional Review Board approval, fresh clinical specimens were transferred from the operating room to surgical pathology, processed and examined by the pathologist for areas of tumor or normal (non-tumor) regions for biopsies. The specimens were placed on ice and transferred to the laboratory for processing, outgrowth and expansion. To generate primary cell cultures, the minced tissues were cultured in collagen-coated cell culture dishes with keratinocyte serum-free medium (K-SFM) supplemented with human recombinant epidermal growth factor (rEGF) and bovine pituitary extract (BPE) at the time of use. Tissue explants were grown to confluence, and aliquots of the primary cultures were frozen and stored in liquid nitrogen. To further expand primary cells, irradiated 3T3 J-2 cells derived from mouse embryonic fibroblast cells were used as feeder cells for growing epithelial cells. J2 cells were cultured in complete DMEM, 10% heat inactivated FBS and 1 % Pen Strep. The complete DMEM and Ham’s F-12 nutrient mix (25%) supplemented with hydrocortisone, epidermal growth factor, bovine insulin, cholera toxin, amphotericin B, gentamicin, and Rock Inhibitor (Y-27632) was used to grow primary epithelial cells with irradiated J2 cells with 30 Gy of gamma radiation. 

[STEPS]
SECTION: Isolation of primary cells from human prostate tissue
1. Working under a biosafety cabinet, transfer the fresh tissue into the plate.
SECTION: Isolation of primary cells from human prostate tissue
2. With a sterile blade, make a fine mince of the tissues.
SECTION: Isolation of primary cells from human prostate tissue
4. Add 5 ml of collection media and place dishes into a 37°C 5% CO 2 incubator. 







SECTION: Isolation of primary cells from human prostate tissue
3. Add 10 ml of collection medium, mix well, and transfer into collagen-coated dishes.
SECTION: Isolation of primary cells from human prostate tissue
4. Add
SECTION: Isolation of primary cells from human prostate tissue
5. ml of collection media and place dishes into a 37°C 5% CO 2 incubator.
SECTION: Isolation of primary cells from human prostate tissue
6. Within a week the minced tissue should be completely attached to the bottom surface of the culture dishes.
SECTION: Isolation of primary cells from human prostate tissue
7. Do not change the medium before 1 week or the cells may not have a chance to adhere and grow.
SECTION: Isolation of primary cells from human prostate tissue
8. After
SECTION: Isolation of primary cells from human prostate tissue
9. days of culture, aseptically remove the collection medium and add 10 ml of keratinocyte growth medium (KGM) (CC-4152, Lonza) with no 5%FBS into a petri dish.
SECTION: Isolation of primary cells from human prostate tissue
10. Incubate the cells at 37° C in humidified air of 5% CO 2 until reaching semiconfluency (about 70–80%), changing the medium twice weekly.
SECTION: Isolation of primary cells from human prostate tissue
11. Freeze aliquots of primary cells using 10% DMSO in KGM.
SECTION: Isolation of primary cells from human prostate tissue
5. Within a week the minced tissue should be completely attached to the bottom surface of the culture dishes.
SECTION: Isolation of primary cells from human prostate tissue
6. Do not change the medium before 1 week or the cells may not have a chance to adhere and grow.
SECTION: Isolation of primary cells from human prostate tissue
7. After 7 days of culture, aseptically remove the collection medium and add 10 ml of keratinocyte growth medium (KGM) (CC-4152, Lonza) with no 5%FBS into a petri dish.
SECTION: Isolation of primary cells from human prostate tissue
8. Incubate the cells at 37° C in humidified air of 5% CO 2 until reaching semiconfluency (about 70–80%), changing the medium twice weekly.
SECTION: Isolation of primary cells from human prostate tissue
9. Freeze aliquots of primary cells using 10% DMSO in KGM.
SECTION: Expansion of primary cells under conditional reprograming cell culture (CRC)
10. Steps:
SECTION: Expansion of primary cells under conditional reprograming cell culture (CRC)
11. Grow J2 feeder cells with 25 mL Complete DMEM to 75-80% confluency (1-1.5 x 107 cells) in T-175 flask.
SECTION: Expansion of primary cells under conditional reprograming cell culture (CRC)
12. Remove media and trypsinize cells using 10 mL of 0.25% Trypsin for minimum of 2 min.
SECTION: Expansion of primary cells under conditional reprograming cell culture (CRC)
13. Make sure cells are floating as single cells and not in clumps or clusters by pipetting several times or vortexing cell suspension and verify by microscope.
SECTION: Expansion of primary cells under conditional reprograming cell culture (CRC)
14. Neutralize Trypsin with 10 mL of Complete DMEM.
SECTION: Expansion of primary cells under conditional reprograming cell culture (CRC)
15. Transfer cell suspension into a conical tube and spin down cells at 3000 rpm for 5 min.
SECTION: Expansion of primary cells under conditional reprograming cell culture (CRC)
16. Aspirate out the supernatant.
SECTION: Expansion of primary cells under conditional reprograming cell culture (CRC)
17. Resuspend cells with 10 mL Complete DMEM.
SECTION: Expansion of primary cells under conditional reprograming cell culture (CRC)
18. Irradiate cell suspension with a Cesium Irradiator at 30 Grey.
SECTION: Expansion of primary cells under conditional reprograming cell culture (CRC)
19. Spin down cells and aspirate out the supernatant.
SECTION: Expansion of primary cells under conditional reprograming cell culture (CRC)
20. Resuspend cells with fresh 10 mL F-Medium (less than 2 weeks old or refresh older F-Medium by adding fresh Rock Inhibitor (10 M Y-27632).
SECTION: Expansion of primary cells under conditional reprograming cell culture (CRC)
21. Plate irradiated J2 cells in a T25 flask (1 × 104 cells/cm2) together with epithelial cells in complete F medium.
SECTION: Expansion of primary cells under conditional reprograming cell culture (CRC)
22. Maintain cocultures at 37 °C, 95% humidity and 5% CO2 until confluent.
SECTION: Expansion of primary cells under conditional reprograming cell culture (CRC)
23. Passage cocultured cells using differential trypsin treatment.
SECTION: Expansion of primary cells under conditional reprograming cell culture (CRC)
24. Rinse coculture with PBS and incubate with 1 ml of 0.05% trypsin/EDTA solution (for a T25 flask) at room temperature for 20 s.
SECTION: Expansion of primary cells under conditional reprograming cell culture (CRC)
25. Monitor closely using a phase-contrast microscope. When the feeder cells become round and begin to detach from the substrate, tap the flask gently and remove the detached cells by aspiration.
SECTION: Expansion of primary cells under conditional reprograming cell culture (CRC)
26. Colonies of epithelial cells should remain tightly adherent. Do not extend trypsin/EDTA incubation time longer than 1 min.
SECTION: Expansion of primary cells under conditional reprograming cell culture (CRC)
27. Rinse the epithelial cells with PBS and re-treat them with 1 ml of 0.05% trypsin/EDTA at 37 °C for 5 min.
SECTION: Expansion of primary cells under conditional reprograming cell culture (CRC)
28. Stop the trypsin/EDTA incubation by adding four volumes of complete DMEM to the cells and transfer the mixture to a 15-ml centrifuge tube.
SECTION: Expansion of primary cells under conditional reprograming cell culture (CRC)
29. Centrifuge the cells at 300g for5  min at 4 °C.
SECTION: Expansion of primary cells under conditional reprograming cell culture (CRC)
30. After centrifugation, resuspend the cell pellet in complete F medium.