M Y Choy1, G St Whitley, I T Manyonda. 1. Department of Immunology, St. George's Hospital Medical School Cranmer Terrace London SW17 ORE.
Abstract
OBJECTIVE: Human trophoblast cells in primary culture are difficult to use for the rigorous study of trophoblast function because of contamination with other cell types, paucity of numbers, poor viability and inter-experiment variation engendered by the need to prepare fresh cells for each experiment. DNA-transfection to produce immortalized cells, or cells with extended life-span, has been the obvious approach to solve this problem. Although there have been a few reports in the literature describing trophoblast cell lines generated in this way, it is clear that to date the methods are difficult and few lines have been generated. The basic problem is that transfection efficiencies of different methods are cell type-specific. The objectives of this study were therefore to compare the transfection efficiencies of three commonly used techniques, to use the best technique to generate trophoblast cell lines, and to conduct preliminary characterization studies. METHODS: We have compared calcium phosphate co-precipitation, DEAE-dextran and poly-L-ornithine (PLO) DNA transfection protocols. We then used the most efficient to transfect human extravillous trophoblast with pSV3neo. RESULTS: Our modification of the PLO method has a transfection efficiency greater than 30 times that of the next best method. Several cell lines were established which had an extended life span and displayed an invasive phenotype, including the expression of MHC Class I framework antigens, human placental lactogen and human chorionic gonadotrophin, and thus have characteristics of extravillous trophoblast. In addition these cells express the integrin subunits b1, a1, and a3, all of which are known to be expressed in human trophoblast, and respond to IL-1alpha by increased secretion of GM-CSF. CONCLUSIONS: PLO is a highly efficient, rapid, reliable, simple and low-cost technique for the procurement of human trophoblast cell lines which retain most, if not all, the phenotype of the parental cell. These lines are potentially powerful tools in the rigorous study of trophoblast function.
OBJECTIVE:Human trophoblast cells in primary culture are difficult to use for the rigorous study of trophoblast function because of contamination with other cell types, paucity of numbers, poor viability and inter-experiment variation engendered by the need to prepare fresh cells for each experiment. DNA-transfection to produce immortalized cells, or cells with extended life-span, has been the obvious approach to solve this problem. Although there have been a few reports in the literature describing trophoblast cell lines generated in this way, it is clear that to date the methods are difficult and few lines have been generated. The basic problem is that transfection efficiencies of different methods are cell type-specific. The objectives of this study were therefore to compare the transfection efficiencies of three commonly used techniques, to use the best technique to generate trophoblast cell lines, and to conduct preliminary characterization studies. METHODS: We have compared calcium phosphate co-precipitation, DEAE-dextran and poly-L-ornithine (PLO) DNA transfection protocols. We then used the most efficient to transfect human extravillous trophoblast with pSV3neo. RESULTS: Our modification of the PLO method has a transfection efficiency greater than 30 times that of the next best method. Several cell lines were established which had an extended life span and displayed an invasive phenotype, including the expression of MHC Class I framework antigens, human placental lactogen and human chorionic gonadotrophin, and thus have characteristics of extravillous trophoblast. In addition these cells express the integrin subunits b1, a1, and a3, all of which are known to be expressed in human trophoblast, and respond to IL-1alpha by increased secretion of GM-CSF. CONCLUSIONS: PLO is a highly efficient, rapid, reliable, simple and low-cost technique for the procurement of human trophoblast cell lines which retain most, if not all, the phenotype of the parental cell. These lines are potentially powerful tools in the rigorous study of trophoblast function.
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