Modified Procedure for the Establishment of Primary Decidual Cell Cultures

1.         On day 4 of pseudopregnancy or pregnancy, induce the decidual reaction via intraluminal injection of 50-100 μl of sesame oil.

2.         On day 7 of pseudopregnancy or pregnancy, remove uteri, place into sterile Hank's buffered salt solution, make an antimesometrial midline incision opening the uteri and dissect decidual tissue by scraping.

3.         Cut decidual tissue into small segments (1-2 cm²) with irredectomy scissors, wash several times with Hank's buffer.

4.         Incubate tissue segments with Dispase solution (2.4 mg/ml, BMB), containing DNase I (40-80 units/ml) with vigorous shaking (setting of 8-10) at 37 C in a CO₂ incubator.  The ratio of tissue to enzyme solution should be approximately 3-decidua/10 ml of enzyme solution.  Agitate for 1 hour.  Remove dispersed cells and reincubate nondispersed tissue fragments in the enzyme solution for an additional hour.

5.         Pool dispersed cells, recover by centrifugation, and resuspend in DMEM containing 10% fetal bovine serum.

6.         Filter through 74 micron nylon mesh

7.         Plate the equivalent of 1-uterine horn of decidual cells per 35 mm dish.  In order to obtain a more even distribution of cells within the plate, initially plate the cells on a rocker with the setting at 1-2 in a CO₂ incubator for approximately 1-3 hours.

8.         After attachment is complete, remove the culture medium (with the nonattached cells), add 3-ml/well of fresh DMEM supplemented with 1% fetal bovine serum and place in standard CO₂ incubator without shaking.