prostate epithelium

Summary

The prostate was removed and the specimen processed (30 min), and the prostate tissue was digested with collagenase (1 h) to collect individual cells and small cell clusters (1 h). Cells were inoculated and cultured until a monolayer was formed (7 d).

Operation method

Program 23.11 Prostate Epithelium

Principle

The prostate was removed and the specimen processed (30 min), and the prostate tissue was digested with collagenase (1 h) to collect individual cells and small cell clusters (1 h). Cells were inoculated and cultured until a monolayer was formed (7 d).

Materials and Instruments

Fetal bovine serum or equine serum Penicillin Kanamycin Cholera toxoid Dexamethasone EGF Goat prolactin or rat prolactin Insulin Partially refined prostate conditioner or refined prostate conditioner Type I collagenase Rat
Growth medium Saline culture medium
Petri Petri dishes Surgical scissors Syringes and 14-G cannulas 25 ml Erlenmeyer flasks Metal wire filters 50 ml conical plastic centrifuge tubes Nylon filters 24-well plastic petri plates Shaking water baths Centrifuges Hematocrit plates or Coulter counters

Move

1. Under sterile conditions, remove the desired prostate lobe and place it in a sterilized 60 mm Petri dish (glass or plastic, 60 mm diameter).

2. Trim the fat from the prostate lobe and weigh it.

3. Add 2 ml of collagenase (675 U/ml, prepared with MSS containing 100 U/ml penicillin and 100 μg/ml kanamycin).

4. Using surgical scissors, cut the prostate lobe into pieces of tissue approximately 1 mm in size. The block should be small enough to pass through the 14-G cannula.

5. Using a syringe and 14-G cannula, transfer the tissue block to a sterilized 25 ml Erlenmeyer flask and add collagenase, 1 ml per 0.1 g of wet tissue.

6. Incubate for 1 h at 37°C in a shaking water bath.

7. The digested cell suspension is aspirated in 3 batches through a 14-G cannula and then filtered through a 1 mm pore size wire screen (1 mm pore size for 50 ml conical centrifuge tubes) into a 50 ml conical plastic centrifuge tube to remove debris and undigested tissue. The cell suspension is washed with an equal amount of MSS containing 5 % fetal bovine serum or horse serum.

8. Centrifuge at 4°C (100 g for 5 min) and collect the cells.

9. Suspend the cells with 5 ml of MSS containing 5% serum and filter the cell suspension through nylon filters (with 50 ml conical centrifuge tubes) with pore sizes of 250 μm, 150 μm, 100 μm, and 40 μm, rinsing the filters with 5 ml of MSS containing 5% serum each time.

10. Collect the cells by centrifugation and suspend the cells with 5 ml of WAJC404 culture medium.

11. Count the cells and adjust the cell concentration to 4 x ¹⁰⁶ cells/ml.

12. Inoculate the cells into a 24-well plate with 50 μl of cell suspension (containing 2 x ¹⁰⁵ cells) per well (area of approximately 2 ^cm2 ). Each well contains 1 ml of WAJC404 culture medium, 10 ng/ml cholera toxin, 1 μmol/L dexamethasone, 10 ng/ml EGF, 1 μg/ml prolactin, 5 μg/ml insulin, 10 ng/ml prostate conditioner, 100 U/m penicillin and 100 μg/ml streptomycin. Partially refined prostaglandin can be substituted as described by McKeehan et al. [1984] and Chaproniere and McKeehan [1986].

13. Incubate in a humidified environment of 95 % air and 5 % _CO2 at 37°C. Change the culture on days 3 and 5. Change fluids on days 3 and 5. Cells may be near monolayer by day 7.

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