From early post-implantation stages to approximately 18 weeks' gestation, cytotrophoblast at the tips of anchoring villi at the placental periphery proliferate to form columns. From the distal edges of these, single cells break away and infiltrate the adjacent maternal decidual stroma, progressing as far as the inner one third of the myometrium. These cells invade maternal spiral arteries and transform their walls with loss of smooth muscle and the associated elastic and collagenous extracellular matrix which is replaced by a fibrinoid substance.
This method enables the researcher to explant terminal placental villi onto a three dimensional matrix of either collagen or Matrigel and monitor the formation of anchoring cytotrophoblast columns. The resulting cells can be stimulated to assume a migratory phenotype by growth factors. TGFb1 inhibits outgrowth.
Explant culture of first trimester placental tissue for de novo generation of extravillous trophoblast
The protocols described include explant on either collagen I or Matrigel. The results are similar, but trophoblast outgrowth on collagen occurs radially at the surface while on Matrigel it occurs predominantly in a downward direction into the gel. The former experiment is therefore more conveniently monitored by whole mount microscopy (Figure 6). Under control conditions of 20% oxygen and serum free medium, cell proliferation occurs during the first 24 h after which it ceases and further outgrowth on collagen, which continues for up to about 5 days, represents the reorganisation of an existing pool of cells from a thick column at the villous tip to a sheet only a few cells thick at the collagen gel surface.
Explant culture method
Equipment and reagents
6, 12 or 24 well culture plates (Costar)
Pastettes
9 cm petri dishes
5 cm petri dishes
Rat tail collagen type I (Becton Dickenson, distributed by Stratech)
Matrigel (Becton Dickenson, distributed by Stratech)
10 x DMEM (Sigma)7.5% sodium bicarbonate (Gibco/BRL)
Culture medium: 1:1 mixture of DMEM and Ham's F12
25 gauge needles
1 ml syringes
10 ml stripettes
Antibiotic, antimycotic mix AAM (Sigma)
Fetal calf serum (Gibco/BRL)
Growth factors
Humid box
70% ethanol
50 mg collagenase (Sigma type IA)
