RAPID PROTOCOL FOR PREPARING HUMAN ENDOMETRIAL MITOCHONDRIA
FOR TRANSPLANTATION
DIMITAR PARVANOV*, IVAYLO STOYKOV,
GEORGI STAMENOV, TODOR CHAUSHEV
Nadezhda Women’s Health Hospital, 3 “Blaga vest” Street, Sofia, Bulgaria
*Corresponding author: dimparvanov@abv.bg
Keywords: mitochondria, human endometrium, cell culture, transplantation
Abstract: Most of the applied methods for isolation of intact mitochondria are time consuming, required special equipment and are based on expensive commercial kits. Here, we have developed a simple, fast, cost-effective, and labor-efficient protocol for preparing endometrial functionally active mitochondria from primary cell culture. Primary endometrial cells were grown on microplates, detached with 2.9 mM EDTA (pH 6.14) added directly to wells containing cell culture medium (RPMI 1640 and 10% FBS) and harvested mechanically with cell scrapers, centrifuged at 600 g for 10 minutes, homogenized by using syringes with 27G and 30G needles. The mitochondria were isolated by differential two-step centrifugation (1300 g for 5 minutes and 12000 g for 15 minutes). All procedures were done on ice and the centrifugation steps were carried out at 4 C in STE buffer with 10 μl/ml protease inhibitor. The quality of the obtained mitochondria have been analyzed using flow cytometric assessment of mitochondrial membrane potential after staining with dye JC-1 and luminescence analysis of adenosine triphosphate (ATP) measured by ATP determination kit. This isolation protocol requires about 40 minutes and yields approximately 2 x 107 viable mitochondria from 1 x 109 culture cells with good membrane potential and high ATP content. The developed procedure bypasses washing steps, differential filtration, reducing the cell and mitochondrial loss that occurs in standard multistep protocols. The obtained viable mitochondria were intended for respiration studies and following in vivo transplantation as an alternative treatment of women with endometrial pathologies and recurrent implantation failure (RIF).