Patent Description:
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 (<NPL>; <NPL>; <NPL>; <NPL>). Because of these many functions, mitochondrial damage can have detrimental effects (<NPL>). To investigate mitochondrial function and dysfunction, several mitochondrial isolation methods have been described. The earliest published accounts of mitochondrial isolation appear to date to the <NUM> (<NPL>; <NPL>; <NPL>; <NPL>). One attempt demonstrated mitochondrial isolation by grinding liver tissue in a mortar followed by centrifugation in a salt solution at low speed (<NPL>; <NPL>). Other groups expanded upon the original procedure and demonstrated tissue fractionation based on differential centrifugation (<NPL>; <NPL>; <NPL>). These early methods formed the basis of current art-known techniques, which often incorporate homogenization and/or differential centrifugation (<NPL>; <NPL>; <NPL>; <NPL>; <NPL>; <NPL>; <NPL>). The number of homogenization and centrifugation steps varies among protocols. These repetitive steps increase the time for mitochondrial isolation and ultimately reduce viability. In addition, manual homogenization can cause mitochondrial damage and inconsistent results if not properly controlled (<NPL>; <NPL>). Document <CIT> discloses a nucleic acid purification apparatus and method.

The present disclosure is based, at least in part, on the discovery that viable, respiration-competent mitochondria can be isolated with high yield and high purity by differential filtration. In particular, applicants have found that a filter with a pore size of about <NUM> to about <NUM>, e.g., about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or about <NUM>, allows mitochondria to be collected in a filtrate, while cell debris and other organelles are retained by the filter. Accordingly, the present specification provides, e.g., filtration apparatuses, kits, and methods to isolate viable, respiration-competent mitochondria. The invention is disclosed in the appended set of claims.

In one aspect, the present disclosure provides filtration apparatuses that has a tubular body configured to be received in a centrifuge tube and having a lumen and first and second ends, each end having an opening; a first filter disposed and secured within the lumen, wherein the filter has a pore-size of about <NUM> to about <NUM>, e.g., about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or about <NUM>; and a second filter disposed and secured within the lumen adjacent to the first filter and having a pore-size of about <NUM> to about <NUM>, e.g., about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or about <NUM>. In some embodiments, the filtration apparatuses can have a third filter disposed and secured within the lumen adjacent to the first filter and the second filter and having a pore-size of about <NUM> to about <NUM>, e.g., about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or about <NUM>. In some embodiments, the first filter and the third filter have the same pore-size, e.g., <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or about <NUM>. In some embodiments, the second filter and the third filter have the same pore-size, e.g., <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or about <NUM>. In one embodiment, the filtration apparatuses can be sterile. In some embodiments, the first, second, and third filters comprise nylon, mylar, stainless steel, wire mesh, aluminum, synthetic mesh, spectra, Kevlar, plastic, paper, or any combination thereof. In yet another embodiment, the centrifuge tube is a <NUM> centrifuge tube.

The present disclosure also refers to kits, which are not part of the invention, having at least one, e.g., two, three, five, or ten or more, filtration apparatuses described above, e.g., apparatuses that have a tubular body configured to be received in a centrifuge tube and having a lumen and first and second ends, each end having an opening; a first filter disposed and secured within the lumen, wherein the filter has a pore-size of about <NUM> to about <NUM>, e.g., about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or about <NUM>; and a second filter disposed and secured within the lumen adjacent to the first filter and having a pore-size of about <NUM> to about <NUM>, e.g., about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or about <NUM>. In one embodiment, the kit can include a first solution having <NUM> sucrose; <NUM> K+HEPES, pH <NUM>; and <NUM> K+EGTA, pH <NUM>; a second solution having <NUM> Subtilisin A per <NUM> of the first solution; a third solution having <NUM> BSA per <NUM> of the first solution; a fourth solution having <NUM> BSA per <NUM> of the first solution; a <NUM> centrifuge tube; and a <NUM> microcentrifuge tube. In some embodiments, the <NUM> centrifuge tube and the <NUM> microcentrifuge tube are sterile.

In yet another aspect, the present disclosure provides methods to isolate viable, respiration-competent mitochondria. The methods includes providing a cell homogenate having a viable mitochondrion; providing a filtration apparatus described above, e.g., an apparatus that has a tubular body configured to be received in a centrifuge tube and having a lumen and first and second ends, each end having an opening; a first filter disposed and secured within the lumen, wherein the filter has a pore-size of about <NUM> to about <NUM>, e.g., about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or about <NUM>; and a second filter disposed and secured within the lumen adjacent to the first filter and having a pore-size of about <NUM> to about <NUM>, e.g., about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or about <NUM>; optionally, situating the filtration apparatus in a relatively upright position; introducing the cell homogenate into the opening at the first end such that the cell homogenate contacts and is filtered through the first filter and subsequently the second filter to thereby form a filtrate; and collecting the filtrate, thereby isolating the viable mitochondrion.

In one embodiment, the method includes homogenizing a tissue, e.g., mammalian tissue, e.g., mammalian tissue from a tissue biopsy, in a solution comprising <NUM> sucrose; <NUM> K+HEPES, pH <NUM>; and <NUM> K+EGTA, pH <NUM>, to thereby provide the cell homogenate. In some embodiments, the method can include, prior to introducing the cell homogenate, wetting the second filter with a solution comprising <NUM> BSA in <NUM> of a solution comprising <NUM> sucrose; <NUM> K+HEPES, pH <NUM>; and <NUM> K+EGTA, pH <NUM>. In one embodiment, the method can include centrifuging the apparatus at about <NUM> x g for three minutes prior to collecting the filtrate. In some embodiments, the filtrate is centrifuged at <NUM> rpm at <NUM> for five minutes. In one embodiment, the cell homogenate can be provided by homogenizing tissue in a sterile glass-grinding vessel.

Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting.

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation typically require <NUM> to <NUM> minutes to complete. Described herein are filtration apparatuses, kits, and methods for rapid isolation of mitochondria from tissues. Certain methods described herein employ a tissue dissociator and differential filtration. In this method, manual homogenization can be replaced with the tissue dissociator's standardized homogenization cycle, which allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the cell homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than <NUM> minutes. A typical isolation using the filtration apparatuses, kits, and methods described herein can yield approximately <NUM> x <NUM><NUM> viable and respiration-competent mitochondria from <NUM> ± <NUM> (wet weight) of tissue sample.

Filtration apparatuses described herein can be used to rapidly isolate intact, viable mitochondria in <NUM> minutes or less, e.g., <NUM> minutes, <NUM> minutes, or <NUM> minutes or less. Employing differential filtration in place of standard differential centrifugation in methods of isolating mitochondria significantly reduces procedure time and subjects mitochondria to less mechanical stress than using standard differential centrifugation protocols. For example, protocols incorporating several centrifugation steps can take <NUM> minutes to <NUM> minutes to isolate mitochondria (<NPL>; <NPL>; <NPL>; <NPL>; <NPL>). Another advantage of the present filtration apparatuses, kits, and isolation methods is that tissue homogenization is standardized. A tissue dissociator provides a standardized cycle and yields consistent and reproducible results. This is in contrast to manual homogenization that is subject to user variability and inconsistency. The isolation time frame provided by the present methods is compatible for clinical and surgical therapeutic intervention (<NPL>; <NPL>).

Filtration apparatuses described herein feature a body (e.g., a tubular body) configured to house multiple filters, which are further described below and represented in <FIG>. Body (<NUM>) can be shaped to fit into a centrifuge tube (<NUM>). Body (<NUM>) and centrifuge tube (<NUM>) can be of any size. Body (<NUM>) has first and second ends (<NUM>) and (<NUM>), each end having an opening (<NUM>) and (<NUM>). The body has a lumen (<NUM>), such that a sample can be placed into the body at one end, travel through lumen (<NUM>), and be retrieved at the opposite end, after the sample progresses through at least two filters disposed within the body, e.g., by force of gravity, capillary action, and/or centrifugation. Skilled practitioners will appreciate that one or both ends (<NUM> and/or <NUM>), while each has an opening, can in some embodiments be reversibly capped, e.g., to preserve sterility and/or provide an area to collect filtrate. Typically, the body will have a roughly circular cross-section along the length of the body. However, skilled practitioners will appreciate that the cross-section of the body can be of any shape desired, e.g., oval, square, rectangular, triangular, etc. In some embodiments, the cross-section is roughly circular so that the body can be received within a commercially-available centrifuge tube (<NUM>), e.g., a <NUM> centrifuge tube. Skilled practitioners will appreciate that tubular body (<NUM>) can be constructed from any art-known material, e.g., polypropylene or polystyrene. One end of the body, e.g., end (<NUM>), can have a tapered configuration (shown in <FIG>) to facilitate insertion of the body into a centrifuge tube and/or to aid in retaining the filter(s) within the body.

The apparatuses include a first filter (<NUM>) disposed and secured within the lumen, wherein the filter has a pore-size of about <NUM> to about <NUM>, e.g., about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or about <NUM>. The first filter can be constructed from any art-known filter material, e.g., nylon, mylar, stainless steel, wire mesh, aluminum, synthetic mesh, spectra, Kevlar, plastic, paper, or any combination thereof. The apparatuses also include a second filter (<NUM>) disposed and secured within the lumen adjacent to the first filter and have a pore-size of about <NUM> to about <NUM>, e.g., about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or about <NUM>. The second filter can be constructed from any art-known filter material, e.g., nylon, mylar, stainless steel, wire mesh, aluminum, synthetic mesh, spectra, Kevlar, plastic, paper, or any combination thereof. The first and second filters can be situated within the body such that they contact each other or are spaced apart some distance apart. For example, the first and second filters can be disposed within the body at a distance apart of at least or about <NUM>, e.g., at least or about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or at least or about <NUM>, depending on the intended use and/or the length of the body.

The apparatuses can optionally include a third filter disposed and secured within the lumen adjacent to the first filter and the second filter and have a pore-size of about <NUM> to about <NUM>, e.g., about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or about <NUM>. The third filter can be constructed from any art-known filter material, e.g., nylon, mylar, stainless steel, wire mesh, aluminum, synthetic mesh, spectra, Kevlar, plastic, paper, or any combination thereof. The third filter can be disposed between the first and second filters. In some embodiments, the third filter is disposed between the first filter and first opening, i.e., closer to the first opening than the first filter. In other embodiments, the third filter is disposed between the second filter and the second opening, i.e., closer to the second opening than the second filter. The third filter can be disposed such that it contacts the first filter and/or the second filter, or the three filters can be evenly spaced apart, or unevenly spaced apart. For example, when the third filter is disposed between the first and second filters, the first and third filters can be in contact with each other, or at least or about <NUM> apart, e.g., at least or about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or at least or about <NUM> apart; and the third and second filters can be in contact with each other, or at least or about <NUM> apart, e.g., at least or about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or at least or about <NUM> apart.

Skilled practitioners will appreciate that further filters (e.g., a fourth filter, fifth filter, sixth filter, etc.) may in some instances be added, depending on the intended use. In some embodiments, the apparatus is sterile. Skilled practitioners will appreciate that filters can be secured within the body using any art-known method, e.g., using adhesive, a pressure fit, and/or configuring the lumen walls in a way that causes the filters to be retained in the lumen (e.g., by designing lumen walls to have ridges, grooves, or other retentive elements).

The present disclosure also provides kits featuring the filtration apparatuses described herein to isolate viable mitochondria. Such kits include at least one, e.g., two, three, five, or ten, filtration apparatus described above. The kits can further include one or more solutions useful for performing the mitochondria isolation methods described herein. For example, a kit may include a first solution comprising <NUM> sucrose; <NUM> K+HEPES, pH <NUM>; and <NUM> K+EGTA, pH <NUM>. Alternatively or in addition, the kit may include a second solution comprising <NUM> Subtilisin A per <NUM> of the first solution. Alternatively or in addition, the kit may include a third solution comprising <NUM> BSA per <NUM> of the first solution. Alternatively or in addition, the kit may include solutions comprising inactive human serum albumin or acetylated human serum albumin. Alternatively or in addition, the kit may include a fourth solution comprising <NUM> BSA per <NUM> of the first solution. In some instances, the kit may include a <NUM> centrifuge tube, into which the filtration apparatus can be fitted. Alternatively or in addition, the kit can include a <NUM> microcentrifuge tube. In some embodiments, the <NUM> centrifuge tube and the <NUM> microcentrifuge tube are sterile.

In an exemplary method, intact, viable mitochondria are isolated from tissue, e.g., mammalian tissue, e.g., mammalian tissue from a tissue biopsy. For example, tissue from a mammal can be minced, e.g., with a scalpel, and homogenized in a sterile glass-grinding vessel (Thomas, Philadelphia, PA) with a motor-driven pestle for <NUM> to <NUM> seconds at <NUM> in a first solution containing <NUM> sucrose; <NUM> K+HEPES, pH <NUM>; and <NUM> K+EGTA, pH <NUM>. A solution containing <NUM> Subtilisin A per <NUM> of the first solution is then added to the homogenate and incubated on ice for <NUM> minutes.

After incubation on ice, the cell homogenate is introduced to a sterile filtration apparatus that is positioned relatively upright, as described herein. In some embodiments, a volume of the cell homogenate, e.g., about <NUM>µL, <NUM>µL, <NUM>µL, <NUM>µL, <NUM>µL, <NUM>µL, <NUM>µL, <NUM>µL, <NUM>µL, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or about <NUM>, is introduced into the opening at the first end such that the cell homogenate contacts the first filter prior to contacting the second filter, and a filtrate is collected after passing through both filters, e.g., by gravity or by centrifugation, in a tube, e.g., a centrifuge tube, a vial, a microcentrifuge tube, or a test tube, to isolate the intact, viable, respiration-competent mitochondria. Alternatively or in addition, the filtration apparatus can have a cap on the second end that is able to collect the filtrate, and the cap can be uncapped or unscrewed to collect the filtrate after the filtrate has flowed through the first filter, second filter, and, if present, third filter, by gravity or centrifugation. In some embodiments, a volume of the cell homogenate can be passed through a filter with a pore-size of about <NUM> to about <NUM>, e.g., about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or about <NUM>, and optionally, the filtrate passed through another filter with a pore-size of about <NUM> to about <NUM>, e.g., about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or about <NUM>, before being passed through a filter with a pore-size of about <NUM> to about <NUM>, e.g., about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or about <NUM>.

Prior to introducing the cell homogenate to the filtration apparatus, the filter with a pore-size of about <NUM> to about <NUM>, e.g., about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or about <NUM>, can be wetted with a solution comprising <NUM> BSA in <NUM> of a solution comprising <NUM> sucrose; <NUM> K+HEPES, pH <NUM>; and <NUM> K+EGTA, pH <NUM>. While not always required in the methods described herein, skilled practitioners will appreciate that filtrate collection can be facilitated by centrifuging the apparatus, e.g., at <NUM> x g for three minutes. Skilled practitioners will appreciate that mitochondria can be concentrated by centrifuging the filtrate at <NUM> rpm at <NUM> for five minutes.

Several general protocols are described below, which may be used in any of the methods described herein and do not limit the scope of the invention described in the claims.

The following solutions were prepared to isolate intact, viable, respiration-competent mitochondria. To successfully isolate mitochondria using the present methods, all solutions and tissue samples should be kept on ice to preserve mitochondrial viability. Even when maintained on ice, isolated mitochondria will exhibit a decrease in functional activity over time (<NPL>). All solutions should be pre-prepared if possible.

A figure outlining the procedural steps in the isolation of mitochondria using tissue dissociation and differential filtration is shown in <FIG>. Two, <NUM> biopsy sample punches were transferred to <NUM> of Homogenizing Buffer in a dissociation C tube and the samples were homogenized using the tissue dissociator's <NUM> minute homogenization program (A). Subtilisin A stock solution (<NUM>µL) was added to the homogenate in the dissociation C tube and incubated on ice for <NUM> minutes (B). The homogenate was filtered through a pre-wetted <NUM> mesh filter in a <NUM> conical centrifuge tube on ice and then <NUM>µL of BSA stock solution was added to the filtrate (C). The filtrate was re-filtered through a new pre-wetted <NUM> mesh filter in a <NUM> conical centrifuge on ice (D). The filtrate was re-filtered through a new pre wetted <NUM> mesh filter in a <NUM> conical centrifuge tube on ice (E). The filtrate was transferred to <NUM> microfuge tubes and centrifuged at <NUM> x g for <NUM> minutes at <NUM> (F). The supernatant was removed, and pellets containing mitochondria were re-suspended, and combined in <NUM> of Respiration Buffer (G).

Immediately prior to isolation, Subtilisin A was dissolved in <NUM> of Homogenizing Buffer. Immediately prior to isolation, BSA was dissolved in <NUM> of Homogenizing Buffer. Two fresh tissue samples were collected using a <NUM> biopsy sample punch and stored in 1X PBS in a <NUM> conical centrifuge tube on ice. The two <NUM> punches of tissue were transferred to a dissociation C tube containing <NUM> of ice cold Homogenizing Buffer. The tissue was homogenized by fitting the dissociation C tube on the tissue dissociator and selecting the pre-set mitochondrial isolation cycle (<NUM> second homogenization).

The dissociation C tube was removed to an ice-bucket. Subtilisin A Stock Solution (<NUM>µL) was added to the homogenate, mixed by inversion, and the homogenate was incubated on ice for ten minutes. A <NUM> mesh filter was placed onto a <NUM> conical centrifuge tube on ice and the filter was pre-wet with Homogenizing Buffer, and the homogenate was filtered into the <NUM> conical centrifuge tube on ice.

Freshly prepared BSA Stock Solution (<NUM>µL) was added to the filtrate and mixed by inversion. (This step was omitted if mitochondrial protein determination was required. ) A <NUM> mesh filter was placed onto a <NUM> conical centrifuge tube on ice and the filter was pre-wet with Homogenizing Buffer, and the homogenate was filtered into the <NUM> conical centrifuge tube on ice. A <NUM> filter was placed onto the <NUM> conical centrifuge tube on ice, and the filter was pre-wetted with Homogenizing Buffer, and the homogenate was filtered into the <NUM> conical centrifuge tube on ice. The filtrate was transferred to two pre-chilled <NUM> microfuge tubes and centrifuge at <NUM> x g for <NUM> minutes at <NUM>. The supernatant was removed, and the pellets were re-suspended and combined in <NUM> of ice-cold Respiration Buffer.

To determine the metabolic activity of isolated mitochondria, an ATP luminescence assay was performed using an ATP assay kit. The protocol, reagents and standards were supplied in the assay kit. A summary of the procedure is described below.

Kit reagents were equilibrated to room temperature. <NUM> ATP Stock Solution was prepared by dissolving lyophilized ATP pellet in <NUM>,<NUM>µL of double distilled water. ATP standard Stock Solution and prepared mitochondrial samples were stored on ice.

Substrate Buffer solution (<NUM>) was added to a vial of lyophilized substrate solution, mixed gently, and placed in the dark. Respiration Buffer (<NUM>µL) was added to all wells of a black, opaque bottom, <NUM> well plate. Mitochondria from the prepared samples (<NUM>µL) were added to each well of the <NUM> well plate. Samples were plated in triplicate, and a row for standards and three wells for the negative control (Respiration Buffer) were included. Mammalian cell lysis solution (<NUM>µL) was added to all wells, including standards and controls. The <NUM> well plate was incubated at <NUM> for <NUM> minutes on an orbital shaker at <NUM> rpm. During the incubation, ATP standards were prepared in concentrations of <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> ATP from the <NUM> ATP Stock Solution and stored on ice. Following the incubation, <NUM>µL of ATP standards were add to corresponding wells as indicated on the plate map (<FIG>). This plate map illustrates how to set up standards (A1-A12), mitochondria samples (B1-C6), and negative controls (C7-C9) for the ATP assay. During the assay, <NUM>µL of Respiration Buffer, <NUM>µL of mammalian cell lysis solution, and <NUM>µL of reconstituted substrate solution are added to all wells (A1-C9). The reconstituted substrate solution (<NUM>µL) was added to each well, and the <NUM> well plate was incubated at <NUM> on the orbital shaker for <NUM> minutes at <NUM> rpm.

The plate was read with a spectrophotometer controlled by Open Gen5 <NUM> software. Higher values correlate with increased ATP levels and higher metabolic activity.

Tissue samples were obtained using a <NUM> biopsy punch. Tissue weight was <NUM> ± <NUM> (wet weight). The number of mitochondria isolated as determined by particle size counting was <NUM> x <NUM><NUM> ± <NUM> x <NUM><NUM> mitochondria for skeletal muscle and <NUM> x <NUM><NUM> ± <NUM> x <NUM><NUM> mitochondria for liver preparations (<FIG>). To allow for comparison, mitochondrial number was also determined by hemocytometer. Mitochondrial numbers were underestimated as determined by hemocytometer as <NUM> x <NUM><NUM> ± <NUM> x <NUM><NUM> mitochondria for skeletal muscle and <NUM> x <NUM><NUM> ± <NUM> x <NUM><NUM> mitochondria for liver preparations (<FIG>). Mitochondrial diameter as determined by size based particle counter is shown in <FIG>. The representative tracing shows the isolated mitochondria are localized under one peak with mean diameter of <NUM> ± <NUM> in agreement with previous reports (<NPL>).

Mitochondrial protein/g (wet weight) starting tissue, as determined by Bicinchoninic Acid (BCA) assay, was <NUM> ± <NUM>/g (wet weight) and <NUM> ± <NUM>/g (wet weight) for skeletal muscle and liver samples respectively (<FIG>).

Mitochondrial purity was determined by transmission electron microscopy and is shown in <FIG>. Mitochondria are shown to be electron dense with less than <NUM>% being fractured or damaged. Contamination by non-mitochondrial particles is less than <NUM>%.

Mitochondrial viability was determined by MitoTracker Red as previously described (<NPL>; <NPL>). The present methods produce isolated mitochondria that maintain membrane potential (<FIG>These images indicate that mitochondria maintained membrane potential. Arrows indicate mitochondria lacking membrane potential or debris.

ATP was determined using a luminescent assay kit. A plate map for the ATP assay is shown in <FIG>. ATP standards were plated in duplicate. Mitochondrial samples and negative controls were plated in triplicate. ATP content was <NUM>± <NUM> nmol/mg mitochondrial protein and <NUM> ± <NUM> nmol/mg mitochondrial protein for skeletal muscle and liver samples, respectively (<FIG>).

Mitochondrial respiration was assessed using a Clark type electrode as previously described (<NPL>; <NPL>). Mitochondrial oxygen consumption rate was <NUM> ± <NUM> O<NUM>/min/mg mitochondrial protein for skeletal muscle and <NUM> ± <NUM> O<NUM>/min/mg mitochondrial protein for liver preparations. Respiratory control index (RCI) values were <NUM> ± <NUM> and <NUM> ± <NUM> for skeletal muscle and liver sample preparations, respectively (<FIG>). These results are similar to those reported in previous studies using manual homogenization and differential centrifugation to isolate mitochondria (<NPL>; <NPL>).

Claim 1:
A method for isolating a viable, respiration-competent mitochondrion, the method comprising: providing a cell homogenate comprising a viable mitochondrion; providing a filtration apparatus comprising:
a tubular body (<NUM>) configured to be received in a centrifuge tube (<NUM>) and comprising a lumen (<NUM>) and first and second ends, each end comprising an opening;
a first filter (<NUM>) disposed and secured within the lumen, wherein the filter has a pore-size between <NUM> and <NUM>; and
a second filter disposed and secured within the lumen adjacent to the first filter and having a pore-size between <NUM> and <NUM>; introducing the cell homogenate into the opening at the first end such that the cell homogenate contacts and is filtered through the first filter and subsequently the second filter to thereby form a filtrate; and
collecting the viable, respiration-competent mitochondrion in the filtrate, thereby isolating the viable, respiration-competent mitochondrion.