Modeling Trophoblast Differentiation using Equine Chorionic Girdle Vesicles

A method for isolating and culturing placental cells from failed early equine pregnancies

“…Whilst the culture of placental cells isolated from healthy, terminated equine pregnancies has previously been described [9,10,15], to the best of the authors' knowledge, this is the first demonstration of a method to isolate and culture placental cells from failed early pregnancies in domestic species. This material will allow genetic characterisation and detection of further aetiologies associated with EPL in the horse and may be applied to a range of species to increase our understanding of this common and economically important condition.…”

“…However, proliferative factors that may be included in this medium may be detrimental to cell functionality and so EqTM medium may be better suited for studies that assess gene expression or cell function, providing a known, controlled environment [14]. Further, EqTM has been used successfully to culture and study the functionality of chorionic girdle trophoblast cells isolated from clinically normal early equine conceptuses [9,10].…”

“…Dependent on the developmental stage, 2 -9 cm 2 allantochorion and chorion were dissected free of surrounding tissue. Tissue was finely chopped in a petri dish with 3 ml of equine trophoblast medium (EqTM) [9].The material was split evenly between three tubes and centrifuged at 250 RCF at 10°C for 5 minutes. All media were removed and the remaining cell pellets resuspended at a concentration of 0.2 cm 2 tissue/ml medium in either (i) AmnioChrome™ Plus (Amnio) (Lonza, Slough, Berkshire, UK) supplemented with 2.5 µg/ml amphotericin B, (ii) Chang D ® (Chang) (Irvine Scientific, Newton, Co. Wicklow, Ireland) supplemented with 2.5 µg/ml amphotericin B and 100 µg/ml kanamycin sulphate or (iii)…”

“…EqTM containing Dulbecco's Modified Eagle's Medium (Gibco), 10% HyClone Fetal Bovine Serum, 1% l-glutamine 100x concentrate (Gibco), 10 units/ml Penicillin-Streptomycin and 100 µg/ml streptomycin [9]. AmnioChrome™ Plus (Amnio) and Chang D ® (Chang) are commercial media developed for the primary culture of amniotic fluid and chorionic villi cells for the purpose of karyotyping.…”

“…Coverslips were removed when cells reached confluency and fixed in ice cold acetone for 10 minutes and stored at -20° C. Immunohistochemistry was performed as previously described [9] using an equine anti-trophoblast (102.1) [11] monoclonal antibody that has been shown to label all equine trophoblast tested [11], an anti-equine endoderm/mesoderm (102.5) monoclonal antibody as a isotype control [11] and a goat anti mouse Ig peroxidase conjugate (Invitrogen, Paisley, UK ) as a secondary antibody. Four randomly selected images were collected for each labelling reaction using a Leica DM4000B microscope and Leica DC500 camera (Leica, Milton Cell number and viability were measured from 1 well of each medium using trypan blue exclusion after 10 days of culture.…”