Creation of a synthetic amniotic fluid for use in fetal therapy

Amnioinfusion fluid during fetal interventions (saline or Ringer’s) is more acidic and nutrient poor than amniotic fluid. We created a synthetic amniotic fluid for use in fetal therapy and evaluated it's impact upon amniotic epithelium in vitro. Placentas from unlabored cesareans were collected and amniotic epithelium was isolated/cultured per protocol. A synthetic fluid was created with equivalent pH, electrolyte, albumin, and glucose concentrations, to human amniotic fluid, termed “Amnio-well” (AL). Cultured amniotic epithelium was exposed to normal saline (NS), Lactated Ringer’s (LR) or AL. Culture media was a control. To simulate the normal turnover of amniotic fluid, at 6, 12, 18, and 24 hours, 25%, 50%, 75%, and 100% (respectively) of the fluid was replaced with culture media. Cells were evaluated for rates of early and late apoptosis, and necrosis. To examine if cells could be "rescued" after amnioinfusion or if damage was progressive, experimentation was repeated and cells were grown in culture for an additional 48 hours. Experimentation was repeated on full thickness amnion explants. Flow cytometry revealed high fidelity of culture, with > 95% of cells expressing PanCK and EPCAM, < 1% expression of hematopoetic/immune markers, and < 5% expression of mesenchyme markers. After exposure to NS or LR, only 44% and 52% of cells were alive at 24 hours, compared with 85% for control and 89% in AL (Figure 1, p < 0.001). After exposure and 48 hour “rescue,” flow cytometry revealed that only only 21% and 44% of cells exposed to NS and LR were living, compared to 88% and 94% for control and AL (Figure 1, p < 0.001). Tissue staining after amnioinfusion on full thickness amnion tissue revealed that 32% and 20% of cells exposed to NS and LR were apoptotic within 24 hours of exposure, vs only 7% in AL and 4% in control (p< 0.001). The current fluids used for amnioinfusion in fetal cases are toxic to human amnion in vitro. It is possible this impacts the rate of membrane rupture post-surgery and consideration should be given to use of a fluid similar to amniotic fluid.View Large Image Figure ViewerDownload Hi-res image Download (PPT)