Isolation of trophoblast cells from first trimester placentas - Optimizing a protocol for future investigation of placenta and platelet alloimmunization

Abstract

Background: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a pregnancy complication caused by an HPA-incompatibility between mother and fetus. In Caucasians, mainly maternal anti-HPA-1a antibodies cause FNAIT. It has been hypothesized that maternal anti-HPA-1a antibodies might affect not only the fetal platelets, but also the placenta, in alloimmunized pregnancies. However, the link between maternal platelet antibodies and placenta is far from fully understood. Investigating the properties of live trophoblast/EVT cells in the context of anti-HPA-1a antibodies would be an important part in unravelling these mechanisms. Therefore, the aim was to optimize the first steps of a protocol for isolation of trophoblast cells from first trimester placentas. If successful, the protocol could be used in future research on how anti-HPA-1a antibodies are related to placenta.

Method: Existing protocols were used as basis to test two different strategies for trophoblast isolation, which mainly differed in the enzymatic processing of the tissue. Strategy 1 utilized trypsin-EDTA and collagenase I, whereas strategy 2 utilized trypsin-EDTA and DNase I.

Results: Of eight conducted experiments, four were considered partly successful, in that cells had adhered to the fibronectin coated plate, in turn indicating that the cells had differentiated overnight.

Conclusion: Strategy 2 seemed to be superior to strategy 1. However, the protocol still requires further testing and adjusting before it is fully optimized. Flow cytometry is warranted to confirm differentiation of trophoblasts to EVT cells.