Autologous mitochondrial transplantation in circulating leukocytes Ya Wang1,2, Maryam Shojaei1,2, Antony McLean2, David Booth1, Marek Nalos2, Mandira Chakraborty2, and Benjamin Tang1,2 1Centre for Immunology and Allergy Research, The Westmead Institute for Medical Research, 176 Hawkesbury Rd, Westmead NSW2145, Australia; 2Nepean Genomic Research Group, Department of Intensive Care Medicine, Nepean Hospital , Penrith NSW2751, Australia Corresponding author email: email@example.com Aim: To identify the technical barriers which may hinder the transfer of viable mitochondria obtained from a healthy donor into the peripheral blood mononuclear cells (PBMCs) of the same donor
Methods: Human whole blood was collected from healthy volunteers into tubes containing anticoagulant EDTA. Peripheral blood mononuclear cells (PBMCs) were prepared from whole blood using Ficoll. Mitochondria were isolated from human PBMCs using Mitochondria Isolation Kit (Miltenyi Biotech). Mitochondria isolated from 1x107 PBMCs were co-cultured with 5x105 recipient PBMCs in RPMI+10% FBS at 37°C CO2 incubator for 24hrs. To determine the transplantation efficiency, isolated mitochondria were labeled with FITC labeling check reagent (Miltenyi Biotech) prior to transplantation and transplanted PBMCs were subjected to Flow Cytometry CantoII analyses (Becton Dickinson). Transplanted PBMCs were also subjected to DeltaVision microscopy (GE Healthcare) to visualize the pre-labeled mitochondria within the cells. Results:We were able to achieve 13% of transplantation efficiency in total PBMCs, among which large cells (mostly monocytes) seemed to take up more of the added mitochondria than small cells (mostly lymphocytes). We observed 70-80% of large cells were FITC positive whereas only 0.1-0.2% of small cells were FITC positive. And the transplantation efficiency is dose dependent. Microscopy results confirmed the presence of FITC-labeled particles in PBMCs with mitochondria transplantation which was absent in control non-transplanted PBMCs. Discussion and conclusion: Our results demonstrated that it is possible to transplant mitochondria into leukocytes. It will be important to show if the transplanted mitochondria are functional and if it is possible to restore mitochondria dysfunction via autologous mitochondria transplantation.
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