Placental and trophoblastic in vitro models to study preventive and therapeutic agents for preeclampsia.

In the field of preeclampsia, enormous efforts are ongoing to identify biomarkers predicting the syndrome already in the first trimester of pregnancy. At the same time, there is the need for in vitro models to test such biomarkers prior to their use in clinical trials. In addition, in vitro models may accelerate the development and evaluation of the benefit of any putative therapeutics. Therefore, in vitro systems have been established to evaluate the release of biomarkers and measure the effect of putative therapeutics using placental villous explants as well as the choriocarcinoma cell line BeWo. For explants, a cryogenic method to freeze, transport and thaw villous explants was developed to use such tissues for a multi-site tissue culture evaluation. Here we focus on three out of many in vitro models that have been established for human placental trophoblast. (1) Choriocarcinoma cell lines such as BeWo, Jeg-3 and Jar cells (2) isolated primary trophoblast cells, and (2) villous explants from normal placentas delivered at term. Cell lines were used to assess the effect of differentiation and fusion on the expression and release of a preeclampsia marker (placental protein 13; PP13) and beta-hCG. Moreover, cell lines were used to study the effect of putative preeclampsia therapeutics such as vitamins C and E, heparin and aspirin on marker release and viability. Cryopreservation of villous explants enabled shipment to a remote laboratory and testing of parameters in different countries using explants from one and the same placenta. Recently published data make it tempting to speculate that the choriocarcinoma cell line BeWo as well as fresh and cryogenically stored placental villous explants may well serve as in vitro models to study preventive and therapeutic agents in the field of preeclampsia.

Cryogenic and low temperature preservation of human placental villous explants - a new way to explore drugs in pregnancy disorders.

BACKGROUND: A major handicap in cell culture studies using human tissues is the insufficient availability of fresh material on site. A method was developed for cryogenic storage and low temperature preservation of human placental villous explants, facilitating multi-site distribution for functional studies. METHODS: Explants from term placentas were incubated with cryoprotectant agents (dimethyl-sulfoxide (DMSO), ethylene glycol, propanediol or Aedesta), frozen in liquid nitrogen, thawed and then cultured in-vitro. Viability was assessed by comparing frozen and thawed explants with non-frozen controls for morphological changes, lactate dehydrogenase (LDH) release, placenta protein 13 (PP13) secretion, and PCNA Western blotting. Functional studies determined the effect of oxygen and magnesium on explant viability. RESULTS: Cryoprotection by 3 M DMSO best maintained explants' viability, morphological integrity and PP13 release after freezing and thawing from liquid nitrogen. The effect of oxygen and magnesium was used to test the functional viability of cultured explants, after freezing in liquid nitrogen and transfer to dry ice for 1-5 days on site or for shipment to a remote lab. The tested parameters were similar between controls and cryogenically treated explants in the remote lab and the lab of origin, demonstrating the possibility of cryostoring explants for functional studies. CONCLUSION: Cryogenically stored placental villous explants shipped frozen can serve as a useful tool for comparative functional studies of placental villous tissues. The results of this pilot study also open the way for multi-site studies associated with drug tailoring for pregnancy disorders.