Products and methods to isolate mitochondria
Filtration apparatuses, kits, and methods for rapid isolation of intact, viable mitochondria from tissues are described with mitochondria isolated by differential filtration through nylon mesh filters. Mitochondria can be isolated in less than 30 minutes using the filtration apparatuses, kits, and methods described.
1. A method for isolating a viable and respiration-competent mitochondrion, the method comprising:
providing a mammalian cell homogenate comprising a viable mitochondrion;
passing the cell homogenate through a first filter having a pore-size of 30 μm to 50 μm, and
subsequently passing the cell homogenate through a filter having a pore-size of 5 μm to 20 μm, to thereby form a filtrate; and
collecting the filtrate, centrifuging the filtrate, and removing supernatant, thereby isolating the viable and respiration-competent mitochondrion,
wherein the method of isolating a viable and respiration-competent mitochondrion from the cell homogenate does not comprise repetitive centrifugation steps,
wherein the method takes less than 30 minutes to isolate the mitochondrion.
2. The method of claim 1 , wherein the method further comprises, after passing the cell homogenate through the filter having a pore size of 30 μm to 50 μm and prior to passing the cell homogenate through the filter having a pore-size of 5 μm to 20 μm, passing the cell homogenate through a filter having a pore-size of 15 μm to 50 μm.
3. The method of claim 1 , wherein the method comprises homogenizing a tissue in a solution comprising 300 mM sucrose, 10 mM K + HEPES, and 1 mM K + EGTA, to thereby provide the cell homogenate.
4. The method of claim 1 , wherein the method comprises, prior to passing the cell homogenate through the filter having a pore-size of 5 μm to 20 μm, wetting the filter having a pore-size of 5 μm to 20 μm with a solution comprising 1 mg BSA in 1 mL of a solution comprising 300 mM sucrose, 10 mM K + HEPES, and 1 mM K + EGTA.
5. The method of claim 1 , wherein the filter having a pore-size of 30 μm to 50 μm and the filter having a pore-size of 5 μm to 20 μm are disposed in an apparatus and the apparatus is centrifuged at 1×g prior to collecting the filtrate.
6. The method of claim 1 , wherein the filtrate is centrifuged at 9000×g.
7. The method of claim 1 , wherein the cell homogenate is provided by homogenizing tissue in a sterile vessel.
8. The method of claim 2 , wherein the pore-size for the filter having a pore-size of 30 μm to 50 μm is 40 μm, the pore-size for the filter having a pore-size of 5 μm to 20 μm is 10 μm, and the pore-size for the filter having a pore-size of 15 μm to 50 μm is 40 μm.
9. The method of claim 2 , wherein the method further comprises passing the filtrate through an additional filter following passing through the filter having a pore-size of 5 μm to 20 μm.
10. The method of claim 9 , wherein the additional filter has a pore-size of 5 μm to 20 μm.
11. The method of claim 5 , wherein the apparatus is centrifuged at 1×g for three minutes prior to collecting the filtrate.
12. The method of claim 7 , wherein the cell homogenate is provided by homogenizing tissue in a sterile vessel at 4° C.
13. The method of claim 1 , wherein the cell homogenate is passed through each filter without centrifugation.
14. The method of claim 1 , wherein the filtrate is centrifuged at 4° C.
15. The method of claim 2 , wherein the cell homogenate is passed through each filter without centrifugation.
16. The method of claim 9 , wherein the cell homogenate is passed through each filter without centrifugation.
17. The method of claim 1 , wherein the cell homogenate is from a cellular source.
18. The method of claim 17 , wherein the cellular source is a human cellular source.
19. The method of claim 1 , wherein the cell homogenate is from a tissue sample.
20. The method of claim 19 , wherein the tissue sample is a tissue biopsy.