Breast milk and mastopexy excised tissue are suitable sources of material for normal mammary ductal epithelium, and purer epithelial cells can be obtained from breast milk. Preliminary digestion and isolation with collagenase is preferred. Culturing cells on the feeder layer of fetal small intestine inhibits stromal cell contamination of normal and cancerous tissues, and the use of optimal culture medium allows for serial passaging and clonal culture of epithelial cells. The collagen gel culture method facilitates the construction of three-dimensional structures similar to those of donor tissues. Source: Animal Cell Culture: A Guide to Basic Techniques, Fifth Edition

Operation method

Mammary epithelial cell culture

Principle

In the presence of endogenous macrophages, breast milk epithelial cells obtained by centrifugation in early lactation were cultured in nutrient-rich culture medium and could be passaged with a mixed protease chelating solution.

Materials and Instruments

RPMI 1640 Fetal bovine serum Human serum Insulin Hydrocortisone Cholera toxin Trypsin solution
Culture flasks Centrifuge tubes

Move

makings

non-sterile

1. Growth medium: RPMI 1640

2. fetal bovine serum (FBS)

3. human serum: blood bank expired serum, Australian antigen negative)

4. stockpile of spare fluids:
(a) 1 mg/ml insulin (Sigma): prepared with 6 mmol/L hydrochloric acid;
(b ) 0.5 mg/ml hydrocortisone: prepared in saline;
(c) 50ug/ml cholera toxin: prepared in saline.

Precautions

Serum and storage reserves of insulin, hydrocortisone, cholera toxin, pancreatic enzymes and trypsin should be kept at -20°C.

5. TEGPED: Trypsin solution, containing the following components
(a) 13 mmol/L EGTA (ethylene glycol-bis-(β-aminoethylether) -N,N,N',N'-tetraacetic acid, Sigma): 10 ml, prepared in D-PBSA;
(b) 7 mmol/L EDTA (diaminoethane tetraacetic acid, Sigma): 4 ml, prepared in D-PBSA;
(c) 0.2% trypsin (Difco, B-I) Bioscience): 4 ml, prepared in HBSS;
(d) 1% Trypsin (Difco, B-D Bioscience): 2 ml in HBSS.

6. Growth medium: RPMI 1640 with 15% FBS, 10% human serum, 50 ng/ml cholera toxin.

7. 0.5ug/ml cortisone oxide and 1ug/ml insulin

8. 50 mm Nunc plastic petri dish or 25 ^cm2 culture bottle

9. Plain container or 20-50 ml centrifuge tube

Procedure

Collect the milk, preferably in a hospital room, 2 to 7 days after delivery. The breasts are scrubbed with sterile water and the milk is then squeezed by hand into a sterile container. Generally, 5-20 ml of milk is collected from each patient. The collected milk is mixed and diluted with RPMI 1640 (1:1) to facilitate centrifugation.

Primary culture

1. Centrifuge the diluted milk (600-1000 g, 20 min) and gently aspirate the supernatant. A small amount of liquid should be left in order to avoid affecting the precipitated cells.

2. Wash the cells 2 to 4 times with RPMI 1640 containing 5% FCS until the supernatant is not cloudy.

3. Suspend the cells with growth medium by adding 5ul of cell suspension to a 50 mm Petri dish (Nunc). Then, add 6 ml of growth medium and incubate the cells at 37°C and 5 % _CO2 .

4. The culture medium was changed after 3-5d and then twice a week. 6-8d clones appeared, and the growth of clonal cells crowded out macrophages. At the beginning, macrophages acted as feeder cells.

Passage culture

5. Incubate the cells with TEGPED (1.5 ml per 50 mm dish) for 5-15 min at 37°C. The incubation time depends on how long the cells have been incubated. Then, suspend the cells.

6. After centrifugation (100 g, 5 min ), suspend the cells with 6 ml of culture medium.

7. Dispense the cell suspension into 50 mm dishes and 25 ^cm2 flasks at 2 ml per 3 dishes or per flask.

8. Dilute the cell suspension 3-fold by adding 4 ml of fresh culture medium.

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