A simple method to isolate term trophoblasts and maintain them in extended culture

Area of Science:

• Cell Biology
• Reproductive Biology
• Biotechnology

Background:

• Primary trophoblast cultures are crucial for studying placental development and function.
• Existing methods for culturing term trophoblasts have limitations, including short lifespan (approx. 7 days) due to overgrowth by contaminating cells.
• Term trophoblasts, initially mononuclear, spontaneously fuse to form multinucleated syncytia in vitro.

Purpose of the Study:

• To develop an optimized method for the prolonged isolation and culture of primary human term trophoblasts.
• To overcome the limitations of short culture lifespan and cellular contamination in existing trophoblast culture techniques.
• To enable extended in vitro experimentation for a better understanding of trophoblast behavior.

Main Methods:

• Trophoblasts were isolated from term placentae using either trypsin/DNase or dispase/DNase digestion.
• Trophoblast purity was assessed via flow cytometry and immunocytochemistry.
• Cell viability and fusion were monitored using propidium iodide and PKH67 staining, respectively.
• Cultures were maintained in Advanced DMEM/F12 supplemented with 2% fetal bovine serum.

Main Results:

• Dispase/DNase digestion yielded significantly more trophoblasts compared to trypsin/DNase.
• The chosen culture medium (Advanced DMEM/F12 with 2% FBS) supported trophoblast viability and minimized contamination for up to 30 days.
• Over 50% of cultured trophoblasts remained mononuclear even after prolonged culture.
• Trophoblasts exhibited syncytialization, forming multinucleated clusters as expected.

Conclusions:

• A simple and effective method for prolonged trophoblast culture (up to 30 days) from term placentae has been established.
• This method reduces contamination and extends the utility of trophoblast cultures for long-term in vitro studies.
• The extended culture period facilitates further investigation into trophoblast biology and function.