Respiratory analysis of coupled mitochondria in cryopreserved liver biopsies

Abstract

The aim of this work was to develop a cryopreservation method of small liver biopsies for in situ mitochondrial function assessment. Herein we describe a detailed protocol for tissue collection, cryopreservation, high-resolution respirometry using complex I and II substrates, calculation and interpretation of respiratory parameters. Liver biopsies from cow and rat were sequentially frozen in a medium containing dimethylsulfoxide as cryoprotectant and stored for up to 3 months at −80 °C. Oxygen consumption rate studies of fresh and cryopreserved samples revealed that most respiratory parameters remained unchanged. Additionally, outer mitochondrial membrane integrity was assessed adding cytochrome c, proving that our cryopreservation method does not harm mitochondrial structure. In sum, we present a reliable way to cryopreserve small liver biopsies without affecting mitochondrial function. Our protocol will enable the transport and storage of samples, extending and facilitating mitochondrial function analysis of liver biopsies.

Impaired hepatic mitochondrial function during early lactation in dairy cows: Association with protein lysine acetylation

“…Mitochondrial function was studied measuring oxygen consumption rate in a high-resolution respirometer OROBOROS Oxygraph—2k at 37 °C as described previously [32,34]. Electrodes were calibrated in modified MIR05 respiration medium (0.5 mM EGTA, 3mM MgCl 2 •6H 2 O, 60 mM MOPS, 20 mM taurine, 10 mM KH 2 PO 4 , 20 mM HEPES, 110 mM sucrose, 1 g.L -1 BSA, pH 7.1) with a calculated saturated oxygen concentration of 191 μM at 100 kPa barometric pressure at 37 °C [34].…”

“…Two dates representative of early and late lactation (35 and 250 DPP, respectively) were taken into consideration for further molecular studies. Biopsies for oxygen consumption rate measurements were cryopreserved as described previously [ 32 ]. Biopsies for western blot analysis and enzyme activity assays were immediately frozen in liquid nitrogen.…”

“…All respiratory parameters and indices were obtained as described in [ 27 , 32 ]. Briefly, the non-mitochondrial oxygen consumption rate was determined after adding antimycin A or rotenone and subtracted from all other values before calculating the respiratory parameters.…”

The contralateral kidney presents with impaired mitochondrial functions and disrupted redox homeostasis after 14 days of unilateral ureteral obstruction in mice

“…The results demonstrate profound functional mitochondrial alterations in the CL kidney of mice when the UUO kidney has tissue alterations similar to those found in CKD [13]. The considerable recent advance comes from taking organ biopsies for mitochondrial oximetric studies in a preserved cell environment [41]. However, we have used here isolated mitochondria: (i) to investigate mitochondrial ROS formation without interference from the cytosolic environment, and (ii) for the assessment of mitochondrial swelling in vitro with raising Ca 2+ concentrations.…”

Restoring Mitochondrial Function While Avoiding Redox Stress: The Key to Preventing Ischemia/Reperfusion Injury in Machine Perfused Liver Grafts?

“…A well-established method for assessing mitochondrial function is high-resolution respirometry (HRR), which has already been used to analyse rat [59], pig [60] and human liver biopsy samples [61][62][63]. With adequate HRR protocols, the state of the mitochondrial OXPHOS machinery can be analysed, which reflects the damage to mitochondria during IRI [64].…”