“…1 A). Our cryopreservation protocol relies on a specific DMSO-based buffer [UW, able to both prolong organ preservation and to prevent cell and organelles swelling (Sakata et al 1997 )] and on a gradual freezing procedure from the on-ice to liquid nitrogen and − 80 °C storage. Fig.…”
Section: Resultsmentioning
confidence: 99%
“…Placental biopsies were cryopreserved as described previously (García-roche et al 2018 ; Giovarelli et al 2021 ). Placental biopsies in ice-cold BIOPS buffer were quickly dried in blotting paper and soaked in cryovials containing 1 mL of ice-cold modified University of Wisconsin solution (UW) (20 mM histidine, 20 mM succinate, 3 mM glutathione, 1 μM leupeptin, 2 mM glutamate, 2 mM malate, 2 mM ATP, 0.5 mM EGTA, 3 mM MgCl2·6H2O, 60 mM MOPS, 20 mM taurine, 10 mM KH2PO4, 20 mM HEPES, 110 mM sucrose, 1 g/L bovine serum albumin (BSA) and 10% (v/v) dimethyl sulfoxide (DMSO) (Sakata et al 1997 )). DMSO was added to the solution immediately before cryopreservation.…”
Background
High-resolution respirometry (HRR) of human biopsies can provide useful metabolic, diagnostic, and mechanistic insights for clinical research and comparative medical studies. Fresh tissues analysis offers the potential best condition, the drawback being the need to use them shortly after dissection for mitochondrial respiratory experiments. The development of effective long-term storage protocols for biopsies that allow the assessment of key Electron Transport System (ETS) parameters at later stages is thus a major need.
Methods
We optimised a cryopreservation protocol that preserves mitochondrial membranes intactness, otherwise affected by direct tissue freezing. The protocol is based on a gradual freezing step from on-ice to liquid nitrogen and − 80 °C storage using a specific DMSO-based buffer.
Results
Placenta is a suitable tissue to design and test the effectiveness of long-term storage protocols being metabolically active foetal tissue with mitochondrial dysfunctions contributing to placental disease and gestational disorders. Here we designed and tested the effectiveness of the cryopreservation protocol using human placenta biopsies; we measured the ETS activity by HRR of placenta specimens comparing fresh, cryopreserved, and snap frozen conditions.
Conclusions
By this protocol, Oxygen Consumption Rate (OCR) measurements of fresh and cryopreserved placental specimens are comparable whereas snap frozen procedure impairs mitochondrial activity.
Graphical Abstract
“…1 A). Our cryopreservation protocol relies on a specific DMSO-based buffer [UW, able to both prolong organ preservation and to prevent cell and organelles swelling (Sakata et al 1997 )] and on a gradual freezing procedure from the on-ice to liquid nitrogen and − 80 °C storage. Fig.…”
Section: Resultsmentioning
confidence: 99%
“…Placental biopsies were cryopreserved as described previously (García-roche et al 2018 ; Giovarelli et al 2021 ). Placental biopsies in ice-cold BIOPS buffer were quickly dried in blotting paper and soaked in cryovials containing 1 mL of ice-cold modified University of Wisconsin solution (UW) (20 mM histidine, 20 mM succinate, 3 mM glutathione, 1 μM leupeptin, 2 mM glutamate, 2 mM malate, 2 mM ATP, 0.5 mM EGTA, 3 mM MgCl2·6H2O, 60 mM MOPS, 20 mM taurine, 10 mM KH2PO4, 20 mM HEPES, 110 mM sucrose, 1 g/L bovine serum albumin (BSA) and 10% (v/v) dimethyl sulfoxide (DMSO) (Sakata et al 1997 )). DMSO was added to the solution immediately before cryopreservation.…”
Background
High-resolution respirometry (HRR) of human biopsies can provide useful metabolic, diagnostic, and mechanistic insights for clinical research and comparative medical studies. Fresh tissues analysis offers the potential best condition, the drawback being the need to use them shortly after dissection for mitochondrial respiratory experiments. The development of effective long-term storage protocols for biopsies that allow the assessment of key Electron Transport System (ETS) parameters at later stages is thus a major need.
Methods
We optimised a cryopreservation protocol that preserves mitochondrial membranes intactness, otherwise affected by direct tissue freezing. The protocol is based on a gradual freezing step from on-ice to liquid nitrogen and − 80 °C storage using a specific DMSO-based buffer.
Results
Placenta is a suitable tissue to design and test the effectiveness of long-term storage protocols being metabolically active foetal tissue with mitochondrial dysfunctions contributing to placental disease and gestational disorders. Here we designed and tested the effectiveness of the cryopreservation protocol using human placenta biopsies; we measured the ETS activity by HRR of placenta specimens comparing fresh, cryopreserved, and snap frozen conditions.
Conclusions
By this protocol, Oxygen Consumption Rate (OCR) measurements of fresh and cryopreserved placental specimens are comparable whereas snap frozen procedure impairs mitochondrial activity.
Graphical 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.
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