Optimization of mitochondrial isolation techniques for intraspinal transplantation procedures

Abstract

Background Proper mitochondrial function is essential to maintain normal cellular bioenergetics and ionic homeostasis. In instances of severe tissue damage, such as traumatic brain and spinal cord injury, mitochondria become damaged and unregulated leading to cell death. The relatively unexplored field of mitochondrial transplantation following neurotrauma is based on the theory that replacing damaged mitochondria with exogenous respiratory-competent mitochondria can restore overall tissue bioenergetics. New Method We optimized techniques in vitro to prepare suspensions of isolated mitochondria for transplantation in vivo. Mitochondria isolated from cell culture were genetically labeled with turbo-green fluorescent protein (tGFP) for imaging and tracking purposes in vitro and in vivo. Results We used time-lapse confocal imaging to reveal the incorporation of exogenous fluorescently-tagged mitochondria into PC-12 cells after brief co-incubation. Further, we show that mitochondria can be injected into the spinal cord with immunohistochemical evidence of host cellular uptake within 24 hours. Comparison to Existing Methods Our methods utilize transgenic fluorescent labeling of mitochondria for a nontoxic and photostable alternative to other labeling methods. Substrate addition to isolated mitochondria helped to restore state III respiration at room temperature prior to transplantation. These experiments delineate refined methods to use transgenic cell lines for the purpose of isolating well coupled mitochondria that have a permanent fluorescent label that allows real time tracking of transplanted mitochondria in vitro, as well as imaging in situ. Conclusions These techniques lay the foundation for testing the potential therapeutic effects of mitochondrial transplantation following spinal cord injury and other animal models of neurotrauma.

Effects of Mitochondrial Transplantation on Bioenergetics, Cellular Incorporation, and Functional Recovery after Spinal Cord Injury

“…PC12 cells were transgenically labeled with a mitochondriatargeting tGFP, as previously described. 46 Briefly, PC-12Adh cells were transfected with a plasmid vector containing tGFP and a mitochondrial targeting sequence for the cytochrome c oxidase complex subunit VIII. Following transfection, cells were kept under constant selection using media containing 3 lg/mL puromycin in complete media (F-12K Medium (ATCC cat # 30-2004 Manassas, VA) with 2.5% fetal bovine serum (Atlanta Biologicals # S1111OH, Atlanta, GA), 15% horse serum (Gibco # 26050-070), and 1.1% penicillin streptomycin (Corning # 30-002-CI, Tewksbury, MA).…”

“…Purified mitochondria were isolated from transfected tGFP-PC12 cells as previously described. 46 Briefly, when cells were 95% confluent they were removed manually from culture plates using a cell scraper. Mitochondria were isolated using multiple centrifugation and resuspension steps to yield Ficoll purified mitochondria.…”

“…45 Additional premise for the current studies is based on our recent report that exogenous transgenically labeled mitochondria can be incorporated into cells both in vitro and in rat spinal cords at 24 and 48 h post-transplantation. 46 Herein, we investigated the therapeutic effects of mitochondrial transplantation immediately after SCI. We hypothesized that supplementation of the injured spinal cord with a pool of healthy, wellcoupled mitochondria would provide a new source of increased energy production, decreased oxidation, and dampened effects of glutamate excitotoxicity, resulting in maintenance of tissue bioenergetics.…”

Stem cell-derived mitochondria transplantation: a novel strategy and the challenges for the treatment of tissue injury

“…Recently, there was inspirational discovery of a new tool called MitoCeption to track the transplanted mitochondria, providing a facility for research or application [ 83 ]. In addition, using transgenic mitochondrial labeling such as tGFP may be a viable option for tracking transplanted mitochondria both in vitro and in vivo, since the inner mitochondrial membrane can be labeled with high transfection efficiency and expression stability [ 84 ]. Finally, the consequence and security of mitochondrial transfer from different kinds of stem cells need to be further explored and evaluated.…”

Challenges in Promoting Mitochondrial Transplantation Therapy

“…Mt isolated from PC-12 cells (allogeneic) and soleus muscle of rat (allogeneic) by Gollihue's method [4] Injection into the mediolateral gray matter Maintenance of OCR Gollihue et al, 2018 [14] ALT, alanine aminotransferase; LDL-C, low density lipoprotein cholesterol; Mt, mitochondria; OCR, oxygen consumption rate; RI, regional ischemia; ROS, reactive oxygen species; SCI, spinal cord injury; TC, content of cholesterol; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. MSCs, mesenchymal stem cells; Mt, mitochondria; OCR, oxygen consumption rate; PTECs, proximal tubular epithelial cells; RI, regional ischemia; ROS, reactive oxygen species; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.…”