Establishment of Day 13 Rat Junctional Zone Primary Cell Culture

  

1.            PROTOCOL (Based on a procedure developed by Lu and Soares, 1994)

 Junctional zones of day 13 rat chorioallantoic placentas are dissected under sterile conditions (see Soares, 1987).  Tissues are cut into small pieces with iris scissors and dissociated with Dispase (4.8 mg/ml; Sigma) and DNase I (Sigma) for one hour at 37 C with continuous shaking.  At the end of the digestion, the suspension of cells and tissue fragments are mixed several times with the aid of a pasteur pipet, and centrifuged.  The harvested cells are then resuspended in DMEM culture medium (Sigma) supplemented with 10% fetal bovine serum (JRH Biosciences) and filtered through a nylon mesh (74 microns).  The cell suspension is then centrifuged through a 60% cushion of Percoll (Sigma) for 15 minutes at room temperature.  Cells at the interface are collected, washed with DMEM supplemented with 10% fetal bovine serum, and cultured in the same medium.  The cells can be maintained in 1-10% fetal bovine serum containing DMEM medium for 7-10 days.

 

2.            COMMENTS

 a.         The isolation of the rat day 13 junctional zone is facilitated by the use of a dissecting microscope (see Soares, 1987).  Junctional zone tissue from the mouse and hamster are not as easy to isolate.

 b.         Dissection of the junctional zone from tissue obtained earlier than day 13 of gestation is more time consuming and yields a nominal amount of starting tissue for the dissociation.  Junctional zones from later in gestation are easy to dissect but do not make for a good starting cell population.  In a limited series of experiments, we have found that the establishment of junctional zone cultures from placentas obtained later during gestation is difficult and the cells show poor viability.    

 c.         The rat day 13 junctional zone cell cultures show no evidence of proliferation of any cell type.  These cultures also show modest contamination of vimentin-positive cells (2-3%).

 d.         The rat day 13 junctional zone cells in primary culture spontaneously differentiate as demonstrated by expression of members of the placental prolactin gene family (see Soares et al., 1991 for a reference about the family).    

  

3.            REFERENCES

 

Lu X-J, Deb S, Soares MJ 1994 Spontaneous differentiation of trophoblast cells along the spongiotrophoblast cell pathway:  expression of members of the placental prolactin gene family and modulation by retinoic acid.  Dev Biol 163, 86-97