Methods and systems relating to assessing mitochondrial efficiency of an individual in the presence or absence of a health condition

Abstract

A method for assessing a mitochondrial efficiency of an individual may include: treating cultured cells with a biological sample from the individual; and measuring a physiological characteristic related a mitochondrial efficiency of the treated cultured cells, wherein the physiological characteristic relates to one or more of: mitochondrial stress, adenosine triphosphate (ATP) production rate, reactive oxygen species, and mitochondrial networks.

Description

METHODS AND SYSTEMS RELATING TO ASSESSING MITOCHONDRIAL EFFICIENCY OF AN INDIVIDUAL IN THE PRESENCE OR ABSENCE OF A HEALTH CONDITIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application No. 63 / 515,925 filed July 27, 2023, the full disclosure of which is incorporated by reference in its entirety for all purposes.FIELD OF THE INVENTION

[0002] The present disclosure is directed to methods and systems for assessing mitochondrial efficiency and, optionally, utilizing the assessment for monitoring and diagnosing health conditions based on the mitochondrial efficiency of an individual.BACKGROUND

[0003] Mitochondria generate adenosine triphosphate (ATP) via oxidative phosphorylation using the energy released during the oxidation food. ATP is the primary energy source for most biochemical and physiological processes, such as growth, movement, and homeostasis. Mitochondria are especially important in organs that demand a high amount of energy for proper functioning, such as heart, liver, muscles, and brain. For example, about 40% of each heart muscle cell and about 25% of each liver cell are made up of mitochondria.

[0004] Mitochondria are involved in the pathogenesis and progression of numerous human chronic and non-chronic diseases. As such, the function, dynamics, and structure of the mitochondria (or referred to herein as the “mitochondrial efficiency”) may provide insight into the presence of disease, its progression and severity. Current methods to measure the mitochondrial condition of an individual include taking a biopsy and directly measuring the mitochondrial structure and function using a variety of assays. For these assays, the biopsy sample or the biological sample extracted therefrom require fresh samples that have not been frozen or stored. Further, depending on the number of assays to be performed, the testing can take over 8 hours. As such, these current methods to assess the mitochondrial condition of an individual are not only invasive and have potential for adverse events but also not widely available, pose challenges related to logistics, sample integrity and viability and are costly.

[0005] Alternative, less-invasive methods are being investigated using blood samples and measuring the concentration of individual components in the blood sample that would affectmitochondrial activity. However, the isolation of components from the blood sample is time consuming and requires specific blood draw techniques and special sample handling. Accordingly, while using blood samples in said assays allows for shipping, the blood samples still need to be relatively fresh for accurate results. Therefore, these alternative methods are not widely available, pose challenges related to logistics, sample integrity and viability and are still relatively costly.

[0006] Thus, there exists a need in the art for assessing mitochondrial efficiency in a way that increases availability, increases sample durability, integrity, viability and reduces biological sample handling requirements, and is less costly.SUMMARY

[0007] The present disclosure is directed to methods and systems for assessing mitochondrial efficiency and, optionally, utilizing the assessment for monitoring and diagnosing health conditions based on the mitochondrial efficiency of an individual.

[0008] A nonlimiting method for assessing a mitochondrial efficiency of an individual may comprise: treating cultured cells with a biological sample from the individual; and measuring a physiological characteristic related a mitochondrial efficiency of the treated cultured cells, wherein the physiological characteristic relates to one or more of: mitochondrial stress, adenosine triphosphate (ATP) production rate, reactive oxygen species, and mitochondrial networks.

[0009] A nonlimiting method for assessing a mitochondrial efficiency of an individual may comprise: reconstituting dried serum from the individual in the presence of a reconstitution medium; treating cultured cells with a biological sample comprising about 0.1 vol% to about 10 vol% of the reconstituted serum; and measuring a physiological characteristic related a mitochondrial efficiency of the treated cultured cells, wherein the physiological characteristic relates to one or more of: mitochondrial stress, adenosine triphosphate (ATP) production rate, reactive oxygen species, and mitochondrial networks.

[0010] A nonlimiting method for assessing a mitochondrial efficiency of an individual may comprise: treating cultured cells with a biological sample from the individual; and measuring a plurality of physiological characteristics related a mitochondrial efficiency of the treated cultured cells, wherein the plurality of physiological characteristics relates to two or more of: non-mitochondrial respiration, basal metabolic respiration, mitochondrial proton leak, ATP production-linked oxygen consumption, maximal mitochondrial respiration,mitochondrial respiratory reserve capacity, extracellular acidification rate, ATP production rate by glycolysis, ATP production rate by mitochondria, concentration of reactive oxygen species, cell membrane structure, cell membrane integrity, cell fission, cell fusion, cell apoptosis, mitochondrial network structures, cell membrane structure, cell membrane integrity, cell fission, cell fusion, and cell apoptosis, including kinetic rates of change in said physiological characteristics.

[0011] These and other aspects of the present invention may be realized as shown and described in the following figures and related description.BRIEF DESCRIPTION OF THE FIGURES

[0012] FIG. 1 illustrates a nonlimiting example method of the present disclosure for assessing a mitochondrial efficiency of an individual.

[0013] FIG. 2 illustrates the oxygen consumption rate (OCR) per 1,000 cells as a function of time.

[0014] FIG. 3 is a bar graph of ATP production rate with distinction between glycolysis ATP production rate per 1,000 cells and mitochondrial ATP production rate per 1,000 cells (values are normalized per 1,000 cells).

[0015] FIG. 4 is an energy phenotype map graph of oxidation phosphorylation percent as a function of glycolysis percent.

[0016] It will be appreciated that the drawings are illustrative and not limiting of the scope of the invention, which is defined by the appended claims. The embodiments shown accomplish various aspects and objects of the invention. It is appreciated that it is not possible to clearly show each element and aspect of the invention in a single figure, and as such, multiple figures are presented to separately illustrate the various details of the invention in greater clarity. Similarly, not every embodiment need accomplish all advantages of the present invention. Elements and acts in the figures are illustrated for simplicity and have not necessarily been rendered according to any particular sequence or embodiment.DETAILED DESCRIPTION OF THE INVENTION

[0017] The present disclosure is directed to methods and systems for assessing mitochondrial efficiency and, optionally, utilizing the assessment for monitoring and diagnosing health conditions based on the mitochondrial efficiency of an individual, Generally, the methods and systems of the present disclosure transfer the metabolic state ofthe mitochondria (or the mitochondrial efficiency) from a biological sample of an individual to cultured cells by treating the cultured cells with said biological sample. Then, conventional in vitro assays may be performed to assess the mitochondrial efficiency of the cultured cells having been treated with the biological sample from the individual.

[0018] Advantageously, the transfer the mitochondrial condition may be performed using a variety of different biological samples and using a variety of different cultured cell lines. The ability to use different biological samples, including dried blood and dried serum as described in more detail below, may allow for a wider availability to the methods and systems of the present disclosure. Further, the ability to use a variety of different cultured cell lines may allow for a more comprehensive assessment or holistic approach by examining the mitochondrial efficiency over a plurality of organs and systems. For example, using a biological sample of serum, a variety of cultured cell lines, for example, relating to the lung, liver, kidneys, and heart, may be treated. Then, the changes (increase, reduce, or not affect at all) to the mitochondrial condition of the cultured cells in response to the individual’s serum allow inferences to the general health state of the individual. For example, if the individual’s serum reduces metabolism of the mitochondria in a muscle cell line, then the presence or absence of one or more components in the individual’s serum may be reducing the functioning of the individual’s muscles. If the person were to make life-style changes and / or start (or change) a course of treatment, monitoring the metabolism of the mitochondria in a muscle cell line over time (e.g., in 2 week to 3 month, 6 month or other intervals) may provide insight into their effect on the health of the muscle cells of the individual.

[0019] Further, advantageously, in vitro assays for various aspects of the mitochondrial efficiency can be performed in multi-well plates, sometimes with more than one assay per well, with fast throughput. Accordingly, several indicators of the function, dynamics, and structure of the mitochondria can be performed with a reasonable turnaround, which reduces cost and further opens up the availability to more persons.

[0020] FIG. 1 illustrates a nonlimiting example method 100 of the present disclosure. The method includes obtaining 102 a biological sample of an individual, then treating 104 cultured cells with the biological sample, and measuring 108 a physiological characteristic related a mitochondrial efficiency of the treated cultured cells. Optionally, after treating 104, the method 100 may include washing 106 the treated cultured cells before measuring 108 the physiological characteristic of the treated cultured cells.

[0021] The biological sample is or contains a biological specimen or an extract thereof from an individual. Examples of biological specimen may include, but are not limited to, muscle tissue, whole blood, serum, plasma, fibroblasts, buccal cells, urine, saliva, the like, and any combination thereof. Preferred biological specimen to increase availability to persons and reduce cost may include, but are not limited to, serum, plasma, buccal cells, saliva, urine, the like, and any combination thereof.

[0022] The biological sample may be a solution, semi-solid, solid, or suspension comprising the biological specimen or the extract thereof. For example, the biological sample may be serum from the individual dispersed in cell culture medium.

[0023] A biological specimen of an individual may be collected by any suitable procedure. For example, whole blood samples may be collected by blood draw or finger prick. Serum and plasma samples may be collected from separated whole blood samples. Buccal cell samples may be collected from a cheek swab. Whole blood, serum, plasma, and buccal cell samples and other sample forms may be dried. For example, a blood spot card may be used to collect a whole blood sample where an individual, for example, pricks their finger and places one or more drops of blood in a designated location on the card. In another collection example, a serum card may be used where an individual, for example, pricks their finger and places one or more drops of blood in a designated location. The serum card includes a paper or other material strip, synthetic or natural, single or multiple layer, bonded or unbonded, of any shape or size, that separates the components of the serum from the blood cells.

[0024] The biological specimen or the extract thereof may be dried, refrigerated (e.g., at about 10 °C or less), frozen (e.g., at about -5 °C or less), otherwise treated, or any combination thereof to preserve the biological specimen or the extract thereof for transport and / or storage between collection and evaluation. If so, then, obtaining the biological sample of the individual may include reconstituting (e.g., rehydrating and / or warming) the biological specimen or the extract thereof. For example, the serum portion of whole blood may dry when using a serum card. As such, the methods of the present disclosure may include reconstituting the dried serum on the serum card and producing a biological sample of the individual from the reconstituted serum. For example, a portion of paper of the serum card that contains serum and few to no blood cells may be cut or through other technique removed from the serum card. Then, the cut or removed portion may be hydrated in a reconstitution medium (e.g., natural, chemical or synthetic liquids or solvents alone or in combination suchas water of various natures and / or methanol containing a serum albumin protein). After a period of time (e.g., about 10 minutes to about 24 hours) in the reconstitution medium, the reconstitution medium having the reconstituted serum therein may be separated from the cut portion of the serum card, optionally purified (e.g., vortexed, stirred, filtered), and used in make the biological sample of multiple concentrations.

[0025] The biological specimen or the extract thereof may be dried, refrigerated, frozen, otherwise treated, or any combination thereof for about 1 hour to about 24 months or more (e.g., about 1 hour to about 24 hours, or about 12 hours to about 72 hours, or about 2 days to about 20 days, or about 15 days to about 60 days, or about 1 month to about 24 months or more) before reconstitution and use in producing the biological sample used in the methods and systems of the present disclosure. Advantageously, the methods and systems of the present disclosure may use dried, refrigerated, frozen, or otherwise treated biological specimens or extracts thereof when producing the biological samples being evaluated, which reduces the complexity in collecting the biological specimen from the individual and may, consequently, reduce cost and increase availability.

[0026] The biological sample used to treat the cultured cells may comprise the biological specimen or the extract thereof at a concentration of about 0.1 wt% to about 100 wt% (or about 0.1 wt% to about 3 wt%, or about 1 wt% to about 5 wt%, or about 3 wt% to about 10 wt% or up to 100wt%).

[0027] The biological sample may include further components, which may include, but are not limited to, cell medium, cell medium additives, water, methanol, dimethyl sulfoxide, ethanol, pH buffer, carbon, nitrogen, serum of varying nature or source, minerals and vitamins, antibiotics, saline, the like, and any combination thereof. Examples of cell medium additives may include, but are not limited to, a serum albumin protein (e.g., bovine serum albumin), glucose, pyruvate, glutamine, buffers, the like, and any combination thereof. The choice of components in the biological sample that are included with the biological specimen, or the extract thereof should be chosen to not degrade the biological specimen or the extract thereof and to be compatible with the cultured cells to be treated.

[0028] Examples of cell types that may be used as the cultured cells may include cells from the 11 organ systems of the body, but are not limited to, blood cells (e.g., erythrocytes, leukocytes, and thrombocytes), muscle cells (e.g., skeletal muscle cells, cardiac muscle cells, and smooth muscle cells), fat cells, nerve cells, cartilage cells, skin cells, endothelial cells,epithelial cells, bone cells, stem cells, endocrine cells, liver cells the like, and any combination thereof.

[0029] The cultured cells may be from any organ including, but not limited to, brain, nerves, heart, lung, skin, bone, muscle, stomach, intestine, kidney, liver, bladder, uterus, cervix, ovaries, breasts, prostate, testes, rectum, lymph node, thyroid, and the like.

[0030] The cultured cells may be from any suitable species including, but not limited to, murine, rabbit, bovine, porcine, canine, monkey, human, and the like. The cultured cells may be from the individual or a matched or unmatched donor to the individual undergoing the evaluation.

[0031] Treating the cultured cells with the biological sample may include exposing the cultured cells to the biological sample. For example, the cultured cells may be in a multi-well plate with cell medium, and the biological sample may be added to the cell medium.

[0032] Any suitable vessel can be used for treating the cultured cells with the biological sample and assaying the effect of the biological sample on the cultured cells. In some embodiments, cells are treated and / or assayed at a concentration of between about 35,000 and about 115,000 cells / ml, between about 45,000 and about 105,000 cells / ml, between about 55,000 and about 95,000 cells / ml, between about 65,000 and about 85,000 cells / ml, or about 75,000 cells / ml. In some embodiments, treatments and / or assays are performed using 96-well plates containing between about 5,000 and about 25,000 cells / well, between about 7,500 and about 22,500 cells / well, between about 10,000 and about 20,000 cells / well, between about 12,500 and about 17,500 cells / well, or about 15,000 cells / well. Treatments and / or assays can be performed using any suitable volume for the vessel being used and the effects being measured. For example, wells of a 96-well plate may contain about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, or about 250 pl volume / well. In some embodiments, the well volume for an assay is between about 10 and about 50 pl / well, between about 15 and about 45 pl / well, between about 15 and 40 pl / well, between about 20 and 35 pl / well, or between about 25 and 30 pl / well.

[0033] Treating the cultured cells (e.g., exposure of the cultured cells to the biological sample) may occur for about 10 minutes to about 8 hours or longer (or about 10 minutes to about 180 minutes, or about 2 hours to about 8 hours).

[0034] Optionally, after treating the cultured cells with the biological sample, the treated cultured cells may be washed in preparation for measuring the physiological characteristic. In some instances, treating the cultured cells may include one or more steps relating to the measuring of the physiological characteristic. For example, a reagent of an assay used in measuring the physiological characteristic may be present during at least a portion of the time the cultured cells are treated with the biological sample.

[0035] A variety of assays may be used to measure a physiological characteristic that is related to the mitochondrial efficiency of the treated cultured cells. The physiological characteristic may relate to one or more of: mitochondrial stress, ATP production rate, reactive oxygen species, mitochondrial networks, the like, and any combination thereof. The physiological characteristic for each of these may be measured for when the cells are normal, healthy, diseased, stressed, or recovering from stress.

[0036] Physiological characteristics that relate to mitochondrial stress may include, but are not limited to, non-mitochondrial respiration, basal metabolic respiration, mitochondrial proton leak, ATP production-linked oxygen consumption, maximal mitochondrial respiration, mitochondrial respiratory reserve capacity, extracellular acidification rate, the like, and any combination thereof including kinetic rates of change in said physiological characteristics.

[0037] Physiological characteristics that relate to ATP production rate may include, but are not limited to, ATP production rate by glycolysis, ATP production rate by mitochondria, the like, and any combination thereof.

[0038] Physiological characteristics that relate to reactive oxygen species may include, but are not limited to, concentration of reactive oxygen species, cell membrane structure, cell membrane integrity, cell fission, cell fusion, cell apoptosis, the like, and any combination thereof.

[0039] Physiological characteristics that relate to mitochondrial biology may include, but are not limited to, mitochondrial network structures, cell membrane structure, cell membrane integrity, cell fission, cell fusion, cell apoptosis, the like, and any combination thereof.

[0040] One or more assays may be performed to measure or characterize desired physiological characteristic relating to mitochondrial efficiency. When multiple assays are used (e.g., to measure or characterize a plurality of desired physiological characteristic), two or more the assays may be performed simultaneously optionally with additional assays performed separately. For example, an imaging assay (e.g., mitochondrial network assay) may be combined with another functional assay (e.g. basal respiration, or basal respirationand glycolysis assay) to assess physical characteristics and functionality to establish efficiency of the mitochondria of the cell.

[0041] Once the physiological characteristics are measured, the results may be analyzed to assess the individual’s metabolic efficiency. Again, when discussing metabolic efficiency, the data may be for a single cultured cell line or for many cultured cell lines, or actual cells of an individual, covering a plurality of organs. The metabolic condition may then be used to infer mitochondrial efficiency in the particular organ system, health condition disease state, stage of disease, effect of therapeutic or other interventions (e.g., supplement, pharmaceutical, biologic, lifestyle, diet or other alone or in combination) in that organ system.

[0042] The results (e.g., the data and quantitative measures of each function, dynamic and structural aspect alone or in combination) of the mitochondrial efficiency measurements may be interpreted in a manner to assess mitochondrial efficiency, health status, diagnose and aid in the treatment or management of an individual. For example, in a specific health conditions a pattern of normal energetics (e.g. basal respiration, mitochondrial efficiency, mitochondrial potential and mitochondrial index) presents in the results while mitochondrial stress, structure and dynamic measures (e.g., Reactive oxygen species and mitochondrial networks stressed and un-stressed) present as out of the range of normal. Such changes efficiency, in some instances may precede signs and symptoms of the condition in an individual, thus providing valuable insight into validating a condition during phases of the condition that routine medical examinations, blood chemistry or genetic testing may not detect.

[0043] The methods of the present disclosure may be repeated periodically to monitor the individual’s metabolic efficiency and assess changes in the metabolic efficiency over time. The methods described herein may be performed every 2 weeks to annually (or monthly, or once every 2-3 months, or quarterly, or annually) or any other suitable periodicity depending on the individual’s needs and desires. A dashboard, portal, physical report, digital report or other communication of the result or results may be used for reporting metabolic efficiency and displaying changes in metabolic condition over time.

[0044] If the individual has undergone a lifestyle change (e.g., a change in diet, exercise, and the like) or a medical treatment change (e.g., a change in medication, supplements, and the like), the changes in the individual’s metabolic efficiency may be assessed in view of the lifestyle and / or medical treatment changes, which may inform additional changes to the individual’s lifestyle and / or medical treatment.

[0045] The assessed metabolic efficiency (at a single point or over time) if an individual may be integrated into their medical records along with the individual’s medical history (e.g., disease state like diabetes, heart conditions, liver sclerosis, and the like), demographics (e.g., race, ethnicity, sex, and the like), and, optionally, lifestyle and / or medication changes. Then, the metabolic efficiency may be considered as one of many inputs when making medical decisions.

[0046] As data related to mitochondrial condition grows for multiple individuals integrated with lifestyle and / or medication change data and, optionally, additional medical data and / or demographic data, algorithms may be developed to model mitochondrial condition changes in response to lifestyle and / or therapeutic supplement or medication changes, which may be used for predictive modeling when recommending a lifestyle and / or supplement or medication change, or when being used for assessment and diagnosis of health condition(s), disease or other diagnosable state.

[0047] At least a portion of the methods and systems of the present disclosure may be performed using computer-program products tangibly embodied in non-transitory machine- readable storage media, including instructions configured to cause one or more data processors to perform specified actions. The computer-program products may relate to reporting metabolic condition, displaying changes in metabolic condition over time, modeling changes in metabolic condition in response to lifestyle and / or medication changes, alone or in combination with other measurable components (e.g., blood chemistry, hormones, enzymes, metabolite, proteins, cytokines, stem cells, exosomes and other) implementing a predictive model for estimating changes to the metabolic condition in in response to lifestyle and / or medication changes, or as a diagnostic to determine health states or presence or absence of disease or health conditions and the like.

[0048] Nonlimiting Example Embodiments

[0049] Embodiment 1. A method for assessing a mitochondrial efficiency of an individual, the method comprising: treating cultured cells with a biological sample from the individual; and measuring a physiological characteristic related a mitochondrial efficiency of the treated cultured cells, wherein the physiological characteristic relates to one or more of: mitochondrial stress, adenosine triphosphate (ATP) production rate, reactive oxygen species, and mitochondrial networks.

[0050] Embodiment 2. The method of Embodiment 1 further comprising: separating serum from whole blood of the individual; and producing the biological sample from the serum.

[0051] Embodiment 3. The method of Embodiment 2, wherein the separating of the serum from the whole blood occurs on a serum card.

[0052] Embodiment 4. The method of Embodiment 1 further comprising: reconstituting dried serum present on a serum card; and producing the biological sample from the reconstituted serum.

[0053] Embodiment 5. The method of Embodiment 4, wherein the dried serum has been dry for at least 24 hours.

[0054] Embodiment 6. The method of Embodiment 4, wherein the dried serum experienced temperatures of about 10°C or less.

[0055] Embodiment 7. The method of any preceding Embodiment further comprising: further treating the cultured cells with a reagent.

[0056] Embodiment 8. The method of any preceding Embodiment, wherein the treating of the cultured cells is for about 10 minutes to about 8 hours.

[0057] Embodiment 9. The method of any preceding Embodiment, wherein cultured cells are from a species selected from a group consisting of murine, rabbit, bovine, porcine, canine, monkey, and human.

[0058] Embodiment 10. The method of any preceding Embodiment, wherein cultured cells are selected from the group consisting of: blood cells, muscle cells, fat cells, nerve cells, cartilage cells, skin cells, endothelial cells, epithelial cells, bone cells, and stem cells.

[0059] Embodiment 11. The method of any preceding Embodiment, wherein cultured cells represent one selected from the group consisting of: brain, nerves, heart, lung, skin, bone, muscle, stomach, intestine, kidney, liver, bladder, uterus, cervix, ovaries, breasts, prostate, testes, rectum, lymph node, and thyroid.

[0060] Embodiment 12. The method of any preceding Embodiment, wherein cultured cells are from the individual.

[0061] Embodiment 13. The method of any preceding Embodiment further comprising: repeating the treating of the cultured cells with fresh biological samples of the individual and the measuring of the physiological characteristic.

[0062] Embodiment 14. The method of Embodiment 13, wherein the repeating occurs two weeks to one year after a previous occurrence.

[0063] Embodiment 15. A method for assessing a mitochondrial efficiency of an individual, the method comprising: reconstituting dried serum from the individual in the presence of a reconstitution medium; treating cultured cells with a biological sample comprising about 0.1 vol% to about 10 vol% of the reconstituted serum; and measuring a physiological characteristic related a mitochondrial efficiency of the treated cultured cells, wherein the physiological characteristic relates to one or more of: mitochondrial stress, adenosine triphosphate (ATP) production rate, reactive oxygen species, and mitochondrial networks.

[0064] Embodiment 16. The method of Embodiment 15, wherein the reconstitution medium comprises one or more of: a serum albumin protein, water, and methanol.

[0065] Embodiment 17. The method of Embodiment 15 or 16, wherein the biological sample further comprises one or more of: medium for the cultured cells, glucose, pyruvate, glutamine.

[0066] Embodiment 18. The method of any one of Embodiments 15-17, wherein the treating of the cultured cells is for about 10 minutes to about 8 hours.

[0067] Embodiment 19. The method of any one of Embodiments 15-18, wherein the treated cultured cells are washed before the measuring of the physiological characteristic.

[0068] Embodiment 20. A method for assessing a mitochondrial efficiency of an individual, the method comprising: treating cultured cells with a biological sample from the individual; and measuring a plurality of physiological characteristics related a mitochondrial efficiency of the treated cultured cells, wherein the plurality of physiological characteristics relates to two or more of: non-mitochondrial respiration, basal metabolic respiration, mitochondrial proton leak, ATP production-linked oxygen consumption, maximal mitochondrial respiration, mitochondrial respiratory reserve capacity, extracellular acidification rate, ATP production rate by glycolysis, ATP production rate by mitochondria, concentration of reactive oxygen species, cell membrane structure, cell membrane integrity, cell fission, cell fusion, cell apoptosis, mitochondrial network structures, cell membrane structure, cell membrane integrity, cell fission, cell fusion, and cell apoptosis, including kinetic rates of change in said physiological characteristics.

[0069] Examples

[0070] Dried Serum Reconstitution '. An individual applied about 4 drops of blood to a dry blood or serum card (e.g., by finger prick collection, whole blood draw where a few drops are taken from the sample and paced on the serum card that included a Whatman or equivalentpaper section for separating the serum from the blood cells. A square are measuring less than, equal to or greater than a 12 mm by 12 mm square, circle or other shape with minimal or without or with minimal red blood cells was cut from the Whatman paper. In a well of a 24 up to a 96-well plate, the serum square or other shaped section was soaked in about 350 pL a 0.1% bovine serum albumin (BSA) solution or other solution of varying concentrations. The plate was shaken in an orbital shaker for about 30 minutes at room temperature to reconstitute the serum. The BSA solution or other solution of varying concentrations with reconstituted serum therein was extracted from the well. A portion of the BSA solution or other solution of varying concentrations with reconstituted serum was stored at -80 °C for future use, and a portion of the BSA solution or other solution of varying concentrations with reconstituted serum was placed on ice for immediate use.

[0071] Mitochondrial Stress'. C2C12 mouse skeletal muscle cells were cultured to a cell density of about 15,000 cells per well in 96-well plates (regular or glass bottom dark plates, depending on the assay) using Dulbecco's modified eagle medium (DMEM) culture media supplemented with 10 wt% fetal bovine serum (FBS) and 1 wt% penicillin / streptomycin.

[0072] Mitochondrial stress was measured by applying a reagent at varying time points to the treated cells and measuring the extracellular acidification rate using an Agilent Seahorse XF Pro and a Cytation5 unit. The reagents used to stress the cells were up to 25 mM or more oligomycin (in-well concentration around 1.5 pm), up to 100 mM carbonyl cyanide 4- (trifluoromethoxy) phenylhydrazone (FCCP) (in-well concentration 2 pm), up to 100 mM or more of rotenone (in-well concentration 0.5 pm or greater), and up to 25 mM or more antimycin A (in-well concentration 0.5 pm).

[0073] The cells were treated with the BSA and other solutions alone or in combination with solutions with reconstituted serum and degassed (to remove ambient CO2 that may interfere with the assay) in the Agilent Seahorse for approximately 1 hour. After treatment and degassing, the media was removed and replaced with fresh media not having reconstituted serum therein. Then, the reagents were used to stress the cells were added to the wells at varying time points. After 1.5 hours of exposure to said reagents, the number of cells is counted by the Cytation5, and the oxygen consumption rate is measured by the Agilent Seahorse. The data generates values and / or curves relating to the following physiological characteristics of the mitochondria: non-mitochondrial respiration, basal respiration, proton leak, ATP production-linked oxygen consumption, maximal respiration, and spare capacity.

[0074] Advantageously, the use of a 96-well plate allows for simultaneously assaying control samples and 20 different biological samples in four technical replicates, or more biologic samples utilizing fewer replicates.

[0075] The control cells (C2C12 cells without serum treatment) are considered as 100%.

[0076] FIG. 2 illustrates the oxygen consumption rate (OCR) per 1,000 cells as a function of time. This demonstrates the ability to detect OCR at various time points similarly to nondry processed serum.

[0077] Table 1 provides various physiological characteristics related mitochondrial efficiency for the control and test samples. This demonstrates the ability to detect the measures described at various time points similarly to non-dry processed serum.Table 1

[0078] ATP Production Rate'. C2C12 mouse skeletal muscle cells were cultured as described in the Mitochondrial Stress assessment. The C2C12 cells were exposed to the reconstituted serum sample for 1 hour while degassing the Agilent Seahorse. After treatment and degassing, the media was removed and replaced with fresh media not having reconstituted serum therein. Then, the reagents (same as Mitochondrial Stress assessment) were used to stress the cells were added to the wells except an additional nuclear stain of Hoechst (60 pM final concentration) was also included with the rotenone and antimycin A. The cells are counted in a Cytation5, and the oxygen consumption rate (OCR), extracellularacidification rate (ECAR), and proton efflux rate (PER) values of the Seahorse assay are normalized per 1,000 cells.

[0079] The control cells (C2C12 cells without serum treatment) are considered as 100%.

[0080] FIG. 3 is a bar graph of ATP production rate with distinction between glycolysis ATP production rate per 1,000 cells and mitochondrial ATP production rate per 1,000 cells (values are normalized per 1,000 cells). This demonstrates the ability to detect the ATP production rate similarly to non-dry processed serum.

[0081] FIG. 4 is an energy phenotype map graph of oxidation phosphorylation percent as a function of glycolysis percent. The graph is broken into four quadrants to express quiescent state (low mitochondrial and low glycolytic function), glycolytic (high glycolytic and low mitochondrial function), aerobic (low glycolysis and high mitochondrial function), and energetic (high glycolysis and high mitochondrial function). This demonstrates the ability to detect a cells energetic state or ratio of different means by which the mitochondria produce energy similarly to non-dry processed serum.

[0082] Reactive Oxygen Species'.

[0083] C2C12 mouse skeletal muscle cells were cultured as described in theMitochondrial Stress assessment but in a 96-well glass bottom black plate. The plate was incubated in normal conditions for 24 hours prior to the assay. A commercial kit CELLROX® Green Reagent (Invitrogen, Thermo Fisher Scientific) to assess the effects of the reconstituted serum in the C2C12 cells ROS generation. This assay is based on the principle that when a cell-permeable dye in reduced condition is oxidized by ROS, it emits fluorescence in the range of 485 / 520nm (green fluorescence). By using this assay in live cells, real-time the effects of an acute treatment (Ih) with the reconstituted serum can be observed.

[0084] After 1 hour of treatment with the reconstituted serum (no additional reagents were used to induce stress), the cell media was replaced with fresh media as in the previous assays. Then, the treated C2C12 cells were exposed to the CELLROX® reagent for 30 minutes at 37 °C, as suggested by the manufacture. To protect from photobleaching, this was performed in the dark, and the 96-well plate was covered in foil. After the 30 minutes, the cells were washed three times with fresh lx PBS and treated with Hoescht 3332 dye (60 pM final concentration) to stain the nucleus of each cell. Using the Cytation5 unit, the treated and stained cells were observed with a 60X magnification and green fluorescence intensity was calculated.

[0085] In the foregoing specification, the present invention has been described with reference to specific exemplary embodiments. Various modifications and changes may be made, however, without departing from the spirit and scope of the present invention as set forth in the claims, including combinations of elements of the various illustrated embodiments. The specification and figures are illustrative, not restrictive, and modifications are intended to be included within the scope of the present invention. Accordingly, the scope of the present invention should be determined by the claims and their legal equivalents rather than by merely the examples described.

[0086] For example, the steps recited in any method or process claims may be executed in any order and are not limited to the specific order presented in the claims. Additionally, the components and / or elements recited in any apparatus claims may be assembled or otherwise operationally configured in a variety of permutations and are accordingly not limited to the specific configuration recited in the claims.

[0087] Benefits, other advantages, and solutions to problems have been described above with regard to particular embodiments. Any benefit, advantage, solution to problem, or any element that may cause any particular benefit, advantage, or solution to occur or to become more pronounced are not to be construed as critical, required, or essential features or components of any or all the claims.

[0088] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties (e.g., molecular weight), reaction conditions, and the like used in the present disclosure and associated claims are to be understood as being modified in all instances by the term “about.” At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claim, the term “about” relative to each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0089] As used herein, the meaning of “a,” “an,” or “the” includes singular and plural references unless the context clearly dictates otherwise.

[0090] The phrase “consisting essentially of’ as used herein is intended to cover additional elements or functions that do not materially affect the basic and novel characteristics of the claimed invention. Thus, “consisting essentially of’ is intended to encompass not only those components specifically listed, but also separate or additional components that do not materially alter the specifically recited functions or elements.

[0091] The terms “comprise,” “comprises,” “comprising,” “having,” “including,” “includes” or any variations of such terms, are intended to reference a non-exclusive inclusion, such that a process, method, article, composition, or apparatus that comprises a list of elements does not include only those elements recited, but may also include other elements not expressly listed or inherent to such process, method, article, composition or apparatus. Other combinations and / or modifications of the above-described structures, arrangements, applications, proportions, elements, materials, or components used in the practice of the present invention, in addition to those not specifically recited, may be varied, or otherwise particularly adapted to specific environments, manufacturing specifications, design parameters, or other operating requirements without departing from the general principles of the same.

Claims

WHAT IS CLAIMED IS:

1. A method for assessing a mitochondrial efficiency of an individual, the method comprising: treating cultured cells with a biological sample from the individual; and measuring a physiological characteristic related a mitochondrial efficiency of the treated cultured cells, wherein the physiological characteristic relates to one or more of: mitochondrial stress, adenosine triphosphate (ATP) production rate, reactive oxygen species, and mitochondrial networks.

2. The method of claim 1 further comprising: separating serum from whole blood of the individual; and producing the biological sample from the serum derived from a dry whole blood sample.

3. The method of claim 2, wherein the separating of the serum from the whole blood occurs on a serum card.

4. The method of claim 1 further comprising: reconstituting dried serum present on a serum card; and producing the biological sample from the reconstituted serum.

5. The method of claim 4, wherein the dried serum has been dry for at least 24 hours.

6. The method of claim 4, wherein the dried serum experienced temperatures of about 10°C or less.

7. The method of any preceding claim further comprising: further treating the cultured cells with a reagent.

8. The method of any preceding claim, wherein the treating of the cultured cells is for about 10 minutes to about 8 hours.

9. The method of any preceding claim, wherein cultured cells are from a species selected from a group consisting of murine, rabbit, bovine, porcine, canine, monkey, and human.

10. The method of any preceding claim, wherein cultured cells are selected from the group consisting of: blood cells, muscle cells, fat cells, nerve cells, cartilage cells, skin cells, endothelial cells, epithelial cells, bone cells, stem cells, and cells from any of the 11 body systems.

11. The method of any preceding claim, wherein cultured cells represent one selected from the group consisting of: brain, nerves, heart, lung, skin, bone, muscle, stomach, intestine, kidney, liver, bladder, uterus, cervix, ovaries, breasts, prostate, testes, rectum, lymph node, thyroid, and cells from any of the 11 body systems.

12. The method of any preceding claim, wherein cultured cells are from the individual or a matched donor.

13. The method of any preceding claim further comprising: repeating the treating of the cultured cells with fresh biological samples of the individual and the measuring of the physiological characteristic.

14. The method of claim 13, wherein the repeating occurs two weeks to one year after a previous occurrence.

15. A method for assessing a mitochondrial efficiency of an individual, the method comprising: reconstituting dried serum from the individual in the presence of a reconstitution medium; treating cultured cells with a biological sample comprising about 0.1 vol% to about 10 vol% of the reconstituted serum; and measuring a physiological characteristic related a mitochondrial efficiency of the treated cultured cells, wherein the physiological characteristic relates to one or more of: mitochondrial stress, adenosine triphosphate (ATP) production rate, reactive oxygen species, and mitochondrial networks.

16. The method of claim 15, wherein the reconstitution medium comprises one or more of: a serum albumin protein, water, and methanol.

17. The method of claim 15 or 16, wherein the biological sample further comprises one or more of: medium for the cultured cells, glucose, pyruvate, glutamine.

18. The method of any one of claims 15-17, wherein the treating of the cultured cells is for about 10 minutes to about 8 hours.

19. The method of any one of claims 15-18, wherein the treated cultured cells are washed before the measuring of the physiological characteristic.

20. A method for assessing a mitochondrial efficiency of an individual, the method comprising: treating cultured cells with a biological sample from the individual; and measuring a plurality of physiological characteristics related a mitochondrial efficiency of the treated cultured cells, wherein the plurality of physiological characteristics relates to two or more of: non-mitochondrial respiration, basal metabolic respiration, mitochondrial proton leak, ATP production-linked oxygen consumption, maximal mitochondrial respiration, mitochondrial respiratory reserve capacity, extracellular acidification rate, ATP production rate by glycolysis, ATP production rate by mitochondria, concentration of reactive oxygen species, cell membrane structure, cell membrane integrity, cell fission, cell fusion, cell apoptosis, mitochondrial network structures, cell membrane structure, cell membrane integrity, cell fission, cell fusion, and cell apoptosis, including kinetic rates of change in said physiological characteristics.

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