Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Summary

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is <30 min compared to 60 - 100 min using alternative methods.

Abstract

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator’s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 10¹⁰ viable and respiration competent mitochondria from 0.18 ± 0.04 g (wet weight) tissue sample.

Introduction

Mitochondria exist in every cell in the body except red blood cells and are involved in a large number of important cellular and metabolic processes^1-4. Because of these many functions, mitochondrial damage can have detrimental effects³. In order to investigate mitochondrial function and dysfunction several mitochondrial isolation methods have been described. The earliest published accounts of mitochondrial isolation date to the 1940s^5-8. The first documented attempt demonstrated mitochondrial isolation by grinding liver tissue in a mortar followed by centrifugation in a salt solution at low speed^5,8. Later, other groups exp....

Protocol

Preparation

1. Prepare 1 M K-HEPES Stock Solution (adjust pH to 7.2 with KOH).
2. Prepare 0.5 M K-EGTA Stock Solution (adjust pH to 8.0 with KOH).
3. Prepare 1 M KH₂PO₄ Stock Solution.
4. Prepare 1 M MgCl₂ Stock Solution.
5. Prepare Homogenizing Buffer (pH 7.2) 300 mM sucrose, 10 mM K-HEPES, and 1 mM K-EGTA. Store at 4 °C.
6. Prepare Respiration Buffer 250 mM sucrose, 2 mM KH₂PO_4, 10 mM MgCl_2, 20 mM K-HEPES Buffer (pH 7.2) and 0.5 mM K-EGTA (pH 8.0). Store at 4 °C.
7. Prepare 10x PBS Stock Solution by dissolving 80 g of NaCl, 2 g of KCl, 14.4 ....

Results

A figure outlining the procedural steps in the isolation of mitochondria using tissue dissociation and differential filtration is shown in Figure 1. Total procedural time is less than 30 min.

Tissue samples were obtained using a 6 mm biopsy punch. Tissue weight was 0.18 ± 0.04 g (wet weight). The number of mitochondria isolated as determined by particle size counting was 2.4 x 10¹⁰ ± 0.1 x 10¹⁰ mitochondria for skeletal muscle and 2.75 x 10¹⁰ ±.......

Discussion

To successfully isolate mitochondria using this protocol it is essential to keep all solutions and tissue samples on ice to preserve mitochondrial viability. Even when maintained on ice, isolated mitochondria will exhibit a decrease in functional activity over time¹⁹. We recommend that all solutions and additions be pre-prepared. We pre-weigh and store Subtilisin A in 4 mg aliquots in 1.5 ml microfuge tubes and store them at -20 °C. Similarly BSA is pre-weighed and stored in 20 mg aliquots in 1.5 ml micro.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sucrose	Sigma Aldrich	84100
HEPES	Sigma Aldrich	H4034
EGTA	Sigma Aldrich	E4378
Substilsin A	Sigma Aldrich	P5380
BSA	Sigma Aldrich	A7906
KH2PO4	Sigma Aldrich	P5379
MgCl2	Sigma Aldrich	M8266
NaCl	Sigma Aldrich	S6191
KCl	Fisher Scientific	P2173
Na2HPO4	Fisher Scientific	S374
ATPlite Luminescence Assay System, 1,000 Assay Kit	Perkin Elmer	6016941
50 ml Conical Tubes	BD	352098
40 μm Nylon Filters	BD	352340
GentleMACS C tube	Miltenyl Biotech	120-005-331
1.5 ml Eppendorf tube	Fisher Scientific	05-408-129
6 mm biopsy punch	Miltex	33-36
10 μm Pluristrainer	Pluriselect	43-500-10-03
Eppendorf Centrifuge 5415C	Marshall Scientific	EP-5415C
GentleMACS Dissociator	Miltenyl Biotech	130-093-235
96-well plates, tissue culture treated	VWR	82050-732
Rotomax 120 orbital shaker	Heidolph	544-41200-00
Synergy H4 Hybrid Multi-Mode  Microplate Reader	BioTek
Multisizer 4 Coulter Counter	Beckman Coulter	A63076
Oxytherm System	Hansatech Instruments
Hemacytometer	Fisher Scientific	267110