Automated microfluidic system for analysis of lifespan and health span in nematodes

Abstract

To provide an automated microfluidic system for the analysis of life and health span of organisms, for example, nematodes.SOLUTION: The present invention includes a system and method for analyzing an animal including a media reservoir and a media pump in fluid communication with the media reservoir, a food reservoir and a food pump in fluid communication with the food reservoir, an input port in fluid communication with the media pump and the food pump, a microfluidic device in fluid communication with the input port, the microfluidic device including a micropillar arena or a plurality of micropillar chambers, an outlet port in fluid communication with the microfluidic device, a light source positioned outside the micropillar arena to illuminate an interior of the micropillar arena, an imager positioned outside the micropillar arena to image the interior of the micropillar arena, and a controller connected to the media pump, the food pump, the microfluidic device, the light source, and the imager.SELECTED DRAWING: Figure 2A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS Not applicable.

[0002] The present invention relates generally to the field of automated microfluidic systems for the analysis of lifespan and healthspan of organisms, such as nematodes.

[0003] Statement of Federally Funded Research This invention was made with a grant from the National Institutes of Health awarded by A This invention was made with government support under grants G050503 and NNX15AL16G awarded by NASA. The government has certain rights in this invention. [Background technology]

[0004] Without limiting the scope of the present invention, its background will be described in the context of imaging devices.

[0005] Aging is a significant risk factor for a wide range of diseases, including neurodegenerative diseases, diabetes, and cancer (Harman, D. The aging process: Major risk factor for disease and death. Proceedings of the National Academy of Science 88, 5360-5363 (1991); Niccoli, T. & Partridge, L. Ageing as a Risk Factor for Disease. Current Biology 22, R741-R752, doi:10.1016 / j.cub.2012.07.024 (2012); Farooqui, T. & Farooqui, AA Aging: An important factor for the pathogenesis of neurodegenerative diseases. Mechanisms of aging and development 130, 203-215 (2009); North, BJ & Sinclair, DA The intersection between aging and cardiovascular disease. Circulation research 110, 1097-1108 (2012), White, MC et al. Age and cancer risk: a potentially modifiable relationship. American journal of preventive medicine 46, S7-15 (2014)). people As the population ages, the socioeconomic burden caused by age-related diseases is staggering, making it essential to develop therapies that promote healthy aging. C. elegans has a short lifespan (3–5 weeks) and a remarkable genetic similarity to humans (approximately 38% orthologs (Shaye, 2014)). D. & Greenwald, I. OrthoList: A Compendium of C. elegans Genes with Human Orthologs. Plos One 6, doi:10.1371 / journal.pone.0020085 (2011))) signaling pathways It is highly conserved (Kenyon, CJ The genetics of aging. Nature 464, 504-512 (2010)) and is a powerful model organism for investigating aging. In addition, the genome has been completely mapped (Consortium, C. e. S. & Consortium, C. e. S. Genome sequence of the nematode C. elegans: A platform for investigating biology. Science 282,2012-2018, doi:10.1093 / glycob / cwi075 (1998)) and genetic plasticity is impressive (Kenyon, C. The plasticity of aging: Insights from long-lived mutants. Cell 120, 449-460, doi:10.1016 / j.cell.2005.02.002|10.1016 / j.cell.2006.02.002 (2005); Fielenbach, N. & Antebi, A. C. elegans dauer formation and the molecular basis of plasticity. Genes & Development 22, 2149-2165, doi:10.1101 / gad.1701508 (2008)), making C. elegans an attractive tool for aging research.Advances in fluorescence microscopy (Chalfie, M., Tu, Y., Euskirchen, G., Ward, WW & Prasher, DC. Green fluorescent protein as a marker for gene expression. Science 263, 802-805 (1994)) and genomic technologies (RNAi, CRISPR) (Ran, F. et al. Genome engineering using the CRISPR-Cas9 system. Nature Protocols 8, 2281-2308, doi:10.1038 / nprot.2013.143 (2013); Fire, A. et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811, doi:10.1038 / 35888 (1998)) have further expanded the number of possible ways in which healthy aging can be studied using C. elegans.

[0006] Lifespan analysis has become a classic method for assessing the effects of a wide variety of genes, proteins, and pharmaceutical compounds on aging and age-related diseases. However, traditional lifespan analysis is generally low-throughput and lacks the ability to analyze non-invasive health indicators. Aging assays generally involve the use of nematode growth media (NGM) containing spores on which animals crawl. This method is performed with C. elegans reared on agar plates containing 100% methylated fertilized eggs. During the breeding season, adults must be manually transferred to new plates to separate the progeny from the original sample. To reduce the need for manual transfers, many laboratories utilize a potent progeny blocker (2'-deoxy-5-fluorouridine, FUdR) to maintain adult-only populations (Mitchell, D.H., Stiles, J.W., Santelli, J. & Sanadi, D.R. Synchronous growth and aging of Caenorhabditis elegans in the presence of Fluorodeoxyuridine. The Journal of gerontology 34, 28-36 (1979), Gandhi, S., Santelli, J., Mitchell, D. H., Stiles, JW & Sanadi, DR. A simple method for maintaining large, aging populations of Caenorhabditis elegans. Mechanisms of aging and development 12, 137-150 (1980); Bishop, N. & Guarente, L. Two neurons mediated diet-restriction-induced longevity in C. elegans. Nature 447, 545-+ (2007)). An alternative to this approach is to use sterile mutants (Vanvoorhies, W. Production of sperm reduces nematode life-span. Nature 360, 456-458, doi:10.1038 / 360456a0 (1992)); Zhang, WB et al. Extended twilight among isogenic C. elegans causes a disproportionate scaling between lifespan and health. Cell Systems 3, 333-345(2016), Pittman, W., Sinha, D., Zhang, W., Kinser, H. & Pincus, Z. A simple culture system for long-term imaging of individual C. elegans. Lab on a Chip 17, 3909-3920, doi:10.1039 / c7lc00916j (2017)).

[0007] The simplicity of using FUdR or germ-free mutants has led to a new technique for large-scale lifespan analysis in crawling Caenorhabditis elegans. A technique known as the Lifespan Machine (LSM) allows the analysis of populations of thousands of animals grown on agar supplemented with FUdR, automatically capturing serial images to score animal death and determine lifespan (Stroustrup, N. et al. The Caenorhabditis elegans Lifespan Machine. Nature Methods 10, 665-+ (2013)). The LSM technique provides insight into the temporal scaling of aging dynamics (Stroustrup, N. et al. The Caenorhabditis elegans Lifespan Machine. Nature Methods 10, 665-+ (2013), Stroustrup, N. et al. The temporal Scaling of Caenorhabditis elegans aging. Nature 530, 103-+, doi:10.1038 / nature16550 (2016)) has helped identify compounds with robust longevity effects (Lucanic, M., Plummer, WT, Lithgow, GJ, Driscoll, M. &Phillips, PC Impact of genetic background and experimental reproducibility on identifying chemical compounds with robust longevity effects. Nature communications 8, 14256 (2017)). Similarly, WorMotel technology allows for longitudinal analysis of individuals in agar-filled microfabricated well plates. This makes it easier to do so (Churgin, MA et al. Longitudinal imaging of Caenorhabditis elegans in a microfabricated device reveals variation in behavioral decline during aging. eLife 10 (2017)). Despite the large-scale capabilities of such technologies, FUdR activates stress response pathways (Anderson, E. et al. C. elegans lifespan extension by osmotic stress requires FUdR, base excision repair, FOXO, and sirtuins. Mechanisms of Ageing and Development 154, 30-42 (2016); Angeli, S. et al. A DNA synthesis inhibitor is protective against proteotoxic stressors via modulation of fertility pathways in Caenorhabditis elegans. Aging-Us 5,759-769, doi:10.18632 / aging.100605 (2013)), increasing fat accumulation (Aitlhadj, L. & Sturzenbaum, S. The use of FUdR can cause prolonged longevity inmutant nematodes. Mechanisms of Ageing and Development 131, 364-365 (2010)) alters lifespan in some genotypes (Anderson, E. et al. C-elegans lifespan extension by osmotic stress requires FUdR, base ex cision repair, FOXO, and sirtuins. Mechanisms of Ageing and Development 154,30-42 (2016), Aitlhadj, L. & Sturzenbaum, S. The use of FUdR can cause prolonged longevity in mutant nematodes. Mechanisms of Ageing and Development 131, 364-365 (2010), Van Raamsdonk, J. & Hekimi, S. FUdR causes a twofold increase in the lifespan of themitochondrial mutant gas-1. Mechanisms of Ageing and Development 132, 519-521(2011)), so the use of FUdR in LSM and WorMotel techniques is confusing.

[0008] Additionally, current techniques such as LSM and WorMotel lack the ability to study the effects of temporary environmental manipulations on lifespan. Such manipulations over user-defined time intervals have been shown to significantly impact cognitive aging (Kauffman, AL, Ashraf, JM, Corces-Zimmerman, MR, Landis, JN, & Murphy, CT. Insulin signaling and dietary restriction differentially influence the decline of learning and memory with age. PLos Biology). 8, e1000372 (2010)). Conventional lifespan techniques lack the ability to rapidly and reversibly manipulate environmental conditions, limiting their usefulness for survival analyses involving animals exposed to unique environments.

[0009] In recent years, microfluidic approaches have begun to address the limitations of agar-based lifespan assays (Hulme, S. et al. Lifespan-on-a-chip: microfluidic chambers for performing lifelong observation of C. elegans. Lab on a Chip 10, 589-597, doi:10.1039 / b919265d(2010); Wen, H., Shi, W. & Qin, J. Multiparameter evaluation of the longevity in C. elegans under stress using an integrated microfluidic device. Biomedical Microdevices 14, 721-728 (2012); Wen, H., Yu, Y., Zhu, G., Jiang, L. & Qin, J. A droplet microchip with substance exchange capability for the developmental study of C. elegans. Lab on a Chip 15, 1905-1911) (2015), Xian, B.et al. WormFarm: a quantitative control and measurement device toward automatedCaenorhabditis elegans aging analysis. Aging Cell 12, 398-409 (2013), Dong, L., Cornaglia, M., Lehnert, T. & Gijs, M. On-chipmicrofluidic biocommunication assay for studying male-induced demise in C.elegans hermaphrodites. Lab on a Chip 16, 4534-4545, doi:10.1039 / c6lc01005a(2016)).Some key advantages of using PDMS-based microfluidic technology are: (i) excellent permeability to oxygen and carbon dioxide, which allows animals to experience natural atmospheric conditions (Halldorsson, S., Lucumi, E., Gomez-Sjoberg, R. & Fleming, R. Advantages and challenges of microfluidic cell culture in polydimethylsiloxane devices. Biosensors & Bioelectronics 63, 218-231, doi:10.1016 / j.bios.2014.07.029 (2015)), (ii) eliminating the need to prevent or reduce progeny production, e.g., on-chip. Size-based separation of offspring using a filter (Hulme, S. et al. Lifespan-on-a-chip: microfluidic chambers for performing lifelong observation of C. elegans. Lab on a Chip 10, 589-597, doi:10.1039 / b919265d (2010), Wen, H., Shi, W. & Qin, J. Multiparameter evaluation of the longevity in C. elegans under stress using an integrated microfluidic device. Biomedical Microdevices 14, 721-728 (2012), Xian, B. et al. WormFarm: a quantitative control and measurement device toward automated Caenorhabditis elegans aging analysis. Aging Cell 12, 398-409 (2013)), (ii) precise temporal control of the culture environment via the addition or removal of reagents (Wen, H., Shi, W. & Qin, J. Multiparameter evaluation of the longevity in C. elegans under stress using an integrated microfluidic device. Biomedical Microdevices 14, 721-728 (2012), Xian, B. et al. WormFarm: a quantitative control and measurement device toward automated Caenorhabditis elegans aginganalysis. Aging Cell 12, 398-409 (2013)), (iii ) an overall reduction in the number of aborted worms, and (iv) allowing for white light and fluorescent imaging. and the optical transparency of the device.

[0010] Despite the significant advantages of microfluidic-based approaches, previous work has been limited in three aspects. First, existing microfluidic devices for lifespan studies (Hulme, S. et al. Lifespan-on-a-chip: microfluidic chambers for performing lifelong observation of C. elegans. Lab on a Chip 10, 589-597, doi:10.1039 / b919265d (2010) and Wen, H., Shi, W. & Qin, J. Multiparameter evaluation of the longevity inC-elegans under stress using an integrated microfluidic device. BiomedicalMicrodevices 14, 721-728 (2012), Xian, B. et al. WormFarm: a quantitative control and measurement device toward automated Caenorhabditis elegans aginganalysis. Aging Cell 12, 398-409 (2013), Dong, L., Cornaglia, M., Lehnert, T. & Gijs, M. On-chip microfluidic biocommunicationassay for studying male-induced demise in C. elegans hermaphrodites. Lab on aChip 16, 4534-4545, doi:10.1039 / c6lc01005a (2016), Chung,K. et al. Microfluidic chamber arrays for whole-organism behavior-based chemical screening. Lab on a Chip 11, 3689-3697, doi:10.1039 / c1lc20400a (2011); Letizia, M. et al. Microfluidic-enabled phenotyping of a whole population of C. elegans worms over their embryonic and post-embryonic development at single-organism resolution. Microsystems & Nanoengineering 4(2018)) house animals in swimming chambers rather than crawling and therefore do not mimic the standard plate-like behavior of C. elegans populations. Housing worms in liquid culture for a significant portion of their lifespan induces changes in gene expression (Szewczyk, N. et al. Delayed development and lifespan extension as features of metabolic lifestyle alteration in C. elegans under dietary restriction. Journal of Experimental Biology 209, 4129-4139, doi:10.1242 / jeb.02492 (2006); Laranjeiro, R., Harinath, G., Burke, D., Braeckman, B. P. & Driscoll, M. Single swim sessions in C. elegans induce key features of mammalian exercises. BioMed Central Biology 15, doi:10.1186 / s12915-017-0368-4 (2017)).Additionally, mandatory swimming has been shown to induce fatigue and oxidative stress, outcomes that are not present in plate-grown animals (Laranjeiro, R., Harinath, G., Burke, D., Braeckman, B.P. & Driscoll, M. Single swim sessions in C. elegans induce key features of mammalian exercises. BioMed Central Biology 15, ). doi:10.1186 / s12915-017-0368-4 (2017), Chuang, H., Kuo,W., Lee, C., Chu, I. & Chen, C. Exercise in an electrotactic flow chamber ameliorates age-related degeneration in Caenorhabditis elegans. ScientificReports 6, doi:10.1038 / srep28064 (2016), Hartman, J. etal. Swimming Exercise and Transient Food Deprivation in Caenorhabditis elegansPromote Mitochondrial Maintenance and Protect Against Chemical-InducedMitotoxicity. Scientific Reports 8, doi:10.1038 / s41598-018-26552-9 (2018)). In contrast to swim chambers, several studies have reported micropillar chambers for C. elegans assays (Albrecht, D. & Bargmann, C. High-content Behavioral analysis of Caenorhabditis elegans in precise spatiotemporal chemical environments. Nature Methods 8, 599-605 (2011); Ai, X., Zhuo, W., Liang, Q., McGrath, P. & Lu, H. A high-throughput device for size-based separation of C. elegans developmental stages. Lab on a Chip 14, 1746-1752, doi:10.1039 / c3lc51334c (2014)) has not been constructed and validated for lifespan or aging studies. Second, most microfluidic studies have not integrated the different components required for aging assays into a compact system. These components include fluid delivery systems (e.g., pumps), illumination sources, and imaging hardware. This lack of integration leads to inefficiencies and reduced throughput for aging assays. Third, scoring of animal survival and movement is mostly manual, making analysis of large image datasets tedious. Additionally, the lack of a streamlined workflow for data analysis can introduce false positives and bias in the analysis.

[0011] Such a system is taught in European Patent Application No. 3209790, filed by Cornaglia, et al., entitled "Microfluidic Device, System and Method for the Study of Organisms." Briefly, the applicants are said to teach a microfluidic device for the culture, selection, and / or analysis of sample organisms, such as nematodes, as well as other biological entities, such as animal embryos. The device is said to include reservoirs, culture chambers, and a smart filtering system that allow for the selection of specific populations / specimens of sample organisms, thus enabling their long-term culture and phenotypic / behavioral analysis.

[0012] However, despite such devices, there remains a need for devices that allow for the long-term study of organisms such as nematodes, that more closely reflect their natural habitat, and that do not affect gene expression in the organism as prior art devices do. [Prior art documents] [Patent documents]

[0013] [Patent Document 1] European Patent Application No. 3209790 [Non-patent literature]

[0014] [Non-Patent Document 1] Harman, D. The aging process: Major riskfactor for disease and death. Proceedings of National Academy of Science 88,5360-5363 (1991) [Non-patent document 2] Niccoli, T. & Partridge, L. Aging as aRisk Factor for Disease. Current Biology 22, R741-R752,doi:10.1016 / j.cub.2012.07.024 (2012) [Non-patent document 3] Farooqui, T. & Farooqui, AA Aging: An important factor for the pathogenesis of neurodegenerative diseases. Mechanismsof aging and development 130, 203-215 (2009) [Non-patent document 4] North, BJ & Sinclair, DA The intersection between aging and cardiovascular disease. Circulation research110, 1097-1108 (2012) [Non-patent document 5] White, MC et al. Age and cancer risk: apotentially modifiable relationship. American journal of preventive medicine46, S7-15 (2014) [Non-patent document 6] Shaye, D. & Greenwald, I. OrthoList: ACompendium of C. elegans Genes with Human Orthologs. Plos One 6,doi:10.1371 / journal.pone.0020085 (2011) [Non-Patent Document 7] Kenyon, CJ The genetics of aging. Nature464, 504 - 512 (2010) [Non-patent document 8] Consortium, C. e. S. & Consortium, C. eS Genome sequence of the nematode C-elegans: A platform for investigating biology. Science 282, 2012-2018, doi:10.1093 / glycob / cwi075 (1998) [Non-Patent Document 9] Kenyon, C. The plasticity of aging: Insightsfrom long-livedmutants. Cell 120, 449-460,doi:10.1016 / j.cell.2005.02.002|10.1016 / j.cell.2006.02.002 (2005) [Non-Patent Document 10] Fielenbach, N. & Antebi, A. C-elegansdauer formation and the molecular basis of plasticity. Genes & Development22, 2149-2165, doi:10.1101 / gad.1701508 (2008) [Non-Patent Document 11] Chalfie, M., Tu, Y., Euskirchen, G., Ward,WW & Prasher, DC Green fluorescent protein as a marker for gene-expression. Science 263, 802-805 (1994) [Non-Patent Document 12] Ran, F. et al. Genome engineering using theCRISPR-Cas9 system. Nature Protocols 8, 2281-2308, doi:10.1038 / nprot.2013.143(2013) [Non-Patent Document 13] Fire, A. et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811,doi:10.1038 / 35888 (1998) [Non-Patent Document 14] Mitchell, DH, Stiles, JW, Santelli, J. & Sanadi, DR Synchronous growth and aging of Caenorhabditis elegans in the presence of Fluorodeoxyuridine. The Journal of gerontology 34, 28-36 (1979) [Non-Patent Document 15] Gandhi, S., Santelli, J., Mitchell, DH, Stiles, JW &Sanadi, DR A simple method for maintaining large, aging populations ofCaenorhabditis elegans. Mechanisms of aging and development 12, 137-150 (1980) [Non-Patent Document 16] Bishop, N. & Guarente, L. Two neurons mediate diet-restriction-induced longevity in C-elegans. Nature 447, 545-+(2007) [Non-Patent Document 17] Vanvoorhies, W. Production of sperm reduces nematode life-span. Nature 360, 456-458, doi:10.1038 / 360456a0 (1992) [Non-Patent Document 18] Zhang, WB et al. Extended twilight amongisogenic c. elegans causes a disproportionate scaling between lifespan andhealth. Cell Systems 3, 333-345 (2016) [Non-Patent Document 19] Pittman, W., Sinha, D., Zhang, W., Kinser,H. & Pincus, ZA simple culture system for long-term imaging of individual C. elegans. Lab ona Chip 17, 3909-3920, doi:10.1039 / c7lc00916j (2017) [Non-Patent Document 20] Stroustrup, N. et al. The Caenorhabditiselegans Lifespan Machine. Nature Methods 10, 665-+ (2013) [Non-Patent Document 21] Stroustrup, N. et al. The temporal scaling of Caenorhabditis elegans aging. Nature 530, 103-+, doi:10.1038 / nature16550(2016) [Non-Patent Document 22] Lucanic, M., Plummer, WT, Lithgow, GJ,Driscoll, M. & Phillips, PC Impact of genetic background and experimental reproducibility on identifying chemical compounds with robustlongevity effects. Nature communication 8, 14256 (2017) [Non-Patent Document 23] Churgin, MA et al. Longitudinal imagingof Caenorhabditis elegans in a microfabricated device reveals variation in behavioral decline during aging. eLife 10 (2017) [Non-Patent Document 24] Anderson, E. et al. C-elegans lifespanextension by osmotic stress requires FUdR, base excision repair, FOXO, and sirtuins. Mechanisms of Aging and Development 154, 30-42 (2016) [Non-Patent Document 25] Angeli, S. et al. A DNA synthesis inhibitor is protective against proteotoxic stressors via modulation of fertility pathways in Caenorhabditis elegans. Aging-Us 5, 759-769, doi:10.18632 / aging.100605 (2013)

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[0015] In some embodiments of the present disclosure, a system for analyzing an animal includes a medium reservoir and a medium pump in fluid communication with the medium reservoir, a food reservoir and a food pump in fluid communication with the food reservoir, an input port in fluid communication with the medium pump and the food pump, and a microfluidic device in fluid communication with the input port, the microfluidic device comprising: (1) a micropipette for housing an animal; or (2) a microfluidic device including a plurality of micropillar chambers, each configured to allow one animal to crawl through, and a transparent exterior surface for at least illumination or imaging; an outlet port in fluid communication with the microfluidic device; a light source disposed outside the micropillar arena to illuminate the interior of the micropillar arena; an imager disposed outside the micropillar arena to image the interior of the micropillar arena; and a controller connected to the medium pump, the food pump, the microfluidic device, the light source, and the imager. In one embodiment, the microfluidic device includes an input flow distribution region in fluid communication with an input port, a micropillar arena in fluid communication with the input flow distribution region, the micropillars being distributed in a pattern configured to allow animals to crawl within the spaces between the micropillars and to retain adult animals while removing progeny animals, the micropillar arena including an organism loading port and a plurality of sieving channels to allow removal of progeny animals, and an outlet flow distribution region in fluid communication with the micropillar arena, which is in fluid communication with an outlet port. In another embodiment, each of the micropillars has a cross-section that includes a circular, oval, square, rectangular, or other polygonal cross-section, or some combination of these shapes. In another embodiment, the micropillar arena includes a distribution channel for introducing animals, the distribution channel including an inlet end and an outlet end, the inlet end being in fluid communication with the input port and the outlet end being in fluid communication with the outlet port, and a plurality of micropillar chambers distributed along and in fluid communication with the distribution channel, each micropillar chamber including a tapered neck for size-based selection and capture of one animal in each micropillar chamber at a sufficiently high fluid flow rate. In another aspect, each of the micropillar chambers has a cross-section that is circular, oval, square, rectangular, or other polygonal, or includes some combination of these shapes.In another embodiment, the analysis comprises at least one of recording animal movements, counting live and dead animals, studying animal behavior, or studying animal mobility. In another embodiment, the animal is at least a nematode of the genus Caenorhabditis. In another embodiment, the light source comprises a light emitting diode. In another embodiment, the imager is a digital imager.

[0016] In some embodiments of the present disclosure, a method of analyzing animals includes providing a plurality of animals to be analyzed; a medium reservoir and a medium pump in fluid communication with the medium reservoir; a food reservoir and a food pump in fluid communication with the food reservoir; an input port in fluid communication with the medium pump and the food pump; and a microfluidic device in fluid communication with the input port, wherein the microfluidic device is: (1) a micropillar arena for accommodating animals, the micropillar arena including a plurality of micropillars that allow the animals to crawl therethrough, the micropillar arena having a boundary of a shape that includes a circle, an oval, a square, a rectangle, or other polygonal shape, or some combination of these shapes; or (2) a micropillar chamber configured such that each micropillar chamber allows one animal to crawl therethrough. a micro-pillar arena configured to contain at least one micro-pillar chamber and a transparent exterior surface for at least illumination or imaging, an outlet port in fluid communication with the micro-fluidic device, a light source disposed outside the micro-pillar arena to illuminate the interior of the micro-pillar arena, an imager disposed outside the micro-pillar arena to image the interior of the micro-pillar arena, and a controller connected to a medium pump, a food pump, the micro-fluidic device, the light source, and the imager, illuminating the plurality of animals through the transparent exterior surface, imaging the plurality of animals through the transparent exterior surface to generate an image, and analyzing the image. In one aspect, the micro-fluidic device includes an input flow distribution region in fluid communication with the input port, and a pattern configured to allow animals to crawl within the spaces between the micro-pillars and to retain adult animals while removing progeny animals. The micropillar arena includes a micropillar arena in fluid communication with an input flow distribution area, the micropillar arena being fluidly connected to an input flow distribution area including a plurality of micropillars distributed in a line, an organism loading port, and a plurality of sieving channels to allow removal of progeny animals, and an outlet flow distribution area in fluid communication with the micropillar arena, which is in fluid communication with an outlet port. In another embodiment, each of the micropillars has a cross-section that includes a circular, elliptical, square, rectangular, or other polygonal cross-section, or some combination of these shapes. In another embodiment, the micropillar arena includes a distribution channel for introducing animals, the distribution channel including an inlet end and an outlet end, the inlet end fluidly communicating with the input port and the outlet end fluidly communicating with the outlet port, and a plurality of micropillar chambers are distributed along and in fluid communication with the distribution channel, each micropillar chamber including a tapered neck for size-based selection and capture of one animal in each micropillar chamber at a sufficiently high fluid flow rate. In another embodiment, each of the micropillar chambers has a cross-section that includes a circular, elliptical, square, rectangular, or other polygonal cross-section, or some combination of these shapes. In another embodiment, the analysis comprises at least one of recording animal movements, counting live and dead animals, studying animal behavior, or studying animal mobility. In another embodiment, the animal is at least a nematode of the genus Caenorhabditis. In another embodiment, the light source comprises a light emitting diode. In another embodiment, the imager is a digital imager.

[0017] In some embodiments of the present disclosure, a system for analyzing nematodes includes a medium reservoir and a medium pump in fluid communication with the medium reservoir, a food reservoir and a food pump in fluid communication with the food reservoir, an input port in fluid communication with the medium pump and the food pump, and a microfluidic device in fluid communication with the input port, the microfluidic device including: (1) a micropillar arena for housing nematodes, the micropillar arena including a plurality of micropillars that allow nematodes to crawl therethrough, and having a boundary of a shape that includes a circle, an oval, a square, a rectangle, or other polygonal shape, or some combination of these shapes; is disclosed as including (2) a microfluidic device including a plurality of micropillar chambers, each micropillar chamber configured to allow one nematode to crawl therethrough, and a transparent exterior surface for at least illumination or imaging; an outlet port in fluid communication with the microfluidic device; a light source disposed outside the micropillar arena to illuminate the interior of the micropillar arena; an imager disposed outside the micropillar arena to image the interior of the micropillar arena; and a controller connected to a medium pump, a food pump, the microfluidic device, the light source, and the imager. In one aspect, the microfluidic device includes an input flow distribution region in fluid communication with the input port, a micropillar arena in fluid communication with the input flow distribution region, the micropillar arena having a plurality of micropillars distributed in a pattern configured to allow nematodes to crawl within the spaces between the micropillars and to retain adult nematodes while removing progeny nematodes, and including a nematode loading port and a plurality of sieve channels to allow removal of progeny nematodes; and an outlet flow distribution region in fluid communication with the micropillar arena, which is in fluid communication with the outlet port. In another embodiment, each of the micropillars has a cross-section that is circular, elliptical, square, rectangular, or other polygonal, or includes some combination of these shapes.In another embodiment, the micropillar arena includes a distribution channel for introducing nematodes, the distribution channel including an inlet end and an outlet end, the inlet end being in fluid communication with the input port and the outlet end being in fluid communication with the outlet port, and a plurality of micropillar chambers are distributed along and in fluid communication with the distribution channel, each micropillar chamber including a tapered neck for size-based selection and capture of one nematode in each micropillar chamber, configured to allow one nematode to crawl therethrough. In another embodiment, each of the micropillar chambers has a cross-section that includes a circular, oval, square, rectangular, or other polygonal cross-section, or some combination of these shapes. In another embodiment, the analysis includes at least one of recording nematode movement, counting live and dead nematodes, studying nematode behavior, or studying nematode mobility. In another embodiment, the nematodes are at least Caenorhabditis (Caenorha). In another embodiment, the light source comprises a light emitting diode. In another embodiment, the imager is a digital imager. [Brief explanation of the drawings]

[0018] For a more complete understanding of the features and advantages of the present invention, reference should now be made to the detailed description of the invention taken in conjunction with the accompanying drawings. [Figure 1] 1 shows an example of a description and components of the NemaLife machine of the present invention. The main components of the NemaLife unit are: (i) a housing for the fluidic and electrical modules, (ii) an iPOD holder, (iii) an iPOD for imaging, (iv) a slot for a microfluidic device, which is illuminated from the side, (v) a user interface display for controlling the unit, and (vi) fluid reservoirs for sterilizing the unit and washing / feeding animals. [Figure 2A] ~ [Figure 2G]The detailed design of microfluidic device I is shown. (Figure 2A) A microfluidic device for culturing C. elegans. The device is filled with blue food dye. The rectangular area represented by the black dashed line is the micropillar arena that houses the crawling animals. The flow distribution zone, inlet / outlet ports, sieve channel, and animal loading ports on both sides of the micropillar arena are highlighted. (Figure 2B) A micropillar arena loaded with approximately 60 adult animals and the food source Escherichia coli (E. coli) OP50. (Figure 2C) A portion of the sieve channel contains a series of evenly spaced rectangular blocks that prevent adults from escaping the micropillar arena but allow progeny to be washed away. (Figure 2D) A top view of the micropillar arena (device I). All dimensions are in μm. (Figure 2E) and (Figure 2F) Architecture of the pillars and sieve channel. Pillars of 100 μm height and 70 μm diameter hang from the ceiling, evenly spaced (160 μm) apart in a square grid. Sieve channels are created by repeating rectangular blocks of 1000 μm x 200 μm with 35 μm spacing between them. The pillars are arranged in a square grid. (Figure 2G) Detailed design of the flow distribution zone. [Figure 3A] ~ [Figure 3I]Further details of the microfluidic device for housing and culturing individuals are shown. (Figure 3A) The device contains 3 x 10 circular micropillar chambers (filled with green food dye) connected by a distribution channel that can accommodate individuals. Black dashed arrows indicate the distribution channel, air purge, inlet, micropillar chamber, and outlet. (Figure 3B) A magnified view of a portion of the microfluidic device showing three chambers. Black arrows indicate the direction of fluid flow through the distribution channel and through the chambers. A single chamber is shown in the image (red dashed border). Each 3 mm diameter chamber is connected to the upstream channel (red dashed arrow) via a narrow tapered neck / arm (black arrow) and to the downstream channel via a sieve channel (black dashed arrow). A magnified view of an animal crawling within the chamber is shown next to it. Scale bar: 1 mm. (Figure 3C) Schematic of the animal loading protocol using the cartridge method. A cartridge is prepared by aspirating 200 μL of worm solution (yellow) into a tube, followed by buffer solution (blue). (Figure 3D) Capture of an animal in the neck of the chamber by introducing a plug of worm solution (50-100 worms / mL) at a flow rate of 5 ml / h for 90 seconds. Each arrow indicates a captured animal. (Figure 3E) The captured worm is pushed into the chamber by introducing buffer solution behind the worm solution into the cartridge at 15 ml / h for 15 seconds. (Figure 3F) Top view of the entire device. All dimensions are in μm. (Figure 3G)-(Figure 3I) Detailed design of the sieve channel, tapered loading neck, fluid inlet, and air purge port. [Figure 4A] ~ [Figure 4E] The detailed design of the NemaLife Machine is shown. (FIG. 4A) Isometric view of the housing for the NemaLife Machine (drawn to scale). This can include (FIG. 4B) the top cover plate, (FIG. 4C) the bottom chassis, (FIG. 4D) the iPOD holder - top view, and (FIG. 4E) the iPOD holder (top view and 3D view). [Figure 5]Figure 1 shows a flow chart of the system workflow for the NemaLife machine. The imaging and fluidics systems are controlled via a microcontroller, with steps displayed on an LCD display located on the top cover plate of the machine. The microcontroller receives user commands from the LCD display. [Figure 6] NemaLife operational workflow. Animals hatched and grown on agar plates are transferred to micropillar arenas at the L4-young adult stage. Daily, the microfluidic device is washed to separate the offspring, the animals are fed, and they are imaged by iPOD. Images are analyzed with the image processing software NemaCode to generate lifespan curves. [Figure 7A] ~ [Figure 7B] Figure 7A and Figure 7B(i)-(v) show the workflow for automated image analysis for live / dead scoring. (Figure 7A) Image analysis workflow for tracking worm objects in images for lifespan and healthspan assays. (Figure 7B) Graphical user interface of NemaCode image analysis software. This software allows users to independently navigate between tabs to confirm and validate the analysis. Figure 7B(i)-(v) show the main steps of the image analysis software: (i) image rotation and cropping of the region of interest (worm culture arena only), (ii) illumination correction and thresholding, (iii) segmentation, (iv) worm object detection and validation, and (v) annotation of dead and alive. [Figure 8A] ~ [Figure 8E]Optimization of parameters for the NemaLife Machine protocol. (Figure 8A) Number of progeny remaining in microfluidic device I as a function of pump wash flow rate. (Figure 8B) Lifespan of wild-type (N2 isolate) and strain IG274, in which the nlp-29p::GFP reporter was induced in the epidermis at 20°C with a pump flow of 7.5 mL / min. (Figure 8C) Lifespan of wild-type (N2 isolate) C. elegans fed four different diets (bacterial concentrations) once daily at 20°C. (Figure 8D) Lifespan of wild-type (N2 isolate) C. elegans fed once daily (24-hour intervals) and twice daily (12-hour intervals) at 20°C. P-value (12-hour interval vs. 24-hour interval) = 0.92. (Figure 8E) Batch-to-batch variation in lifespan of wild-type (N2 isolate) C. elegans at 20.54 ≤ n ≤ 67 in the NemaLife system. [Figure 9] Evaluation of mutant lifespan in the NemaLife Machine. Lifespan of mutants age-1 (TJ1052), eat-2 (DA1113), daf-16 (GR1307), and skn-1 (EU35) compared to wild type (N2 isolate). N=113 for wild type, 122 for age-1, 97 for eat-2, 119 for daf-16, and 121 for skn-1. The table below shows the median and maximum lifespan for each of the strains studied. [Figure 10A] ~ [Figure 10C] Healthspan assessment of wild-type C. elegans cultured in NemaLife. Animals were grouped into high, medium, and low mobility groups by tracking them in 90-second videos. Data are shown for (Figure 10A) 0-10 second (Figure 10B) 50-60 second (Figure 10C) and 80-90 second video segments. [Figure 11] Determination of progeny production of animals cultured in the NemaLife Machine. Progeny were counted by collecting lavage fluid over a 4-day period in both devices and compared with data from animals raised on agar plates. DETAILED DESCRIPTION OF THE INVENTION

[0019] While the making and using of various embodiments of the invention are discussed in detail below, it should be understood that the invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not limit the scope of the invention.

[0020] To facilitate the understanding of the present invention, several terms are defined below. Terms used herein have the meanings commonly understood by one of ordinary skill in the areas relevant to the present invention. Terms such as "a," "an," and "the" are not intended to refer to a singular entity only, but include general classes of which specific examples can be used to illustrate. While terms in this specification are used to describe specific embodiments of the present invention, their usage does not limit the present invention except as outlined in the claims.

[0021] Currently, research on nematodes involves laborious collection and transfer of animals on culture plates to study them over their lifespan. Exposing animals to different environments over time is also difficult. Scoring is done manually, which can introduce bias. The integrated system and microfluidic culture device of the present invention solves all these problems. (As used herein, "animal" includes, but is not limited to, organisms such as nematodes, fish, and tadpoles.)

[0022] Caenorhabditis elegans is a powerful animal model for aging research. Standard longevity assays on agar plates involve the laborious task of harvesting and transferring animals to prevent young offspring from contaminating age-synchronized adult populations. Large-scale studies avoid offspring contamination using progeny-blocking drugs or sterile mutants, but such manipulations alter adult physiology and the impact of reproduction on normal aging. Furthermore, some agar-growth-based technology platforms, such as automated lifespan machines, do not allow for the easy addition or removal of reagents such as food or drugs after the initiation of a study. We have developed an automated microfluidic system called the NemaLife Machine (NLM) that addresses the current limitations of plate-based aging assays. The NLM device integrates (1) a microfluidic device and flow control system for culturing C. elegans with programmed washing and food delivery of progeny, (2) a lighting and smart device imaging system for recording the movement of animals or groups of individuals, and (3) data analysis software for scoring live / dead animals, their behavior, and their mobility. The machine has a footprint of 1 ft. 2 The NLM system is compact and easy to integrate with other microfluidic devices and can be operated via an onboard interactive display. We developed an operational workflow to evaluate various system parameters and reliably generate lifespan and healthspan data for C. elegans. Furthermore, the device was validated in classical aging mutant and dietary restriction longevity studies. Overall, the NLM system's ability to generate reliable lifespan and physiological data highlights the potential of this automated machine for gene and drug screening, as well as fundamental investigations into C. elegans lifespan / healthspan.

[0023] We have now developed a novel technology, called the NemaLife Machine (NLM), for studying aging in C. elegans that addresses the limitations of agar-based studies and current microfluidic systems. As shown in Figure 1, our device is a compact, benchtop, integrated platform that incorporates all the functionality required for lifespan studies in C. elegans. The main components of the NemaLife unit are: (i) a housing for the fluidic and electrical modules; (ii) an iPOD holder; (iii) an iPOD for imaging; and (iv) a microfluidic system for imaging. ) a slot for the microfluidic device. The microfluidic device is illuminated from the side and contains (v) a user interface display for controlling the unit, and (vi) fluid reservoirs for sterilizing the unit and washing / feeding the animals. At the core of the NLM is an optimized micropillar arena, which allows animals to adopt a crawling progression similar to that of worms on agar plates, while also acting as a screen to hold adults and prevent fluid-induced damage when removing offspring. The microfluidic device is connected to a programmable pump that washes and delivers food to the offspring according to a user-defined sequence. LED-based lighting and an iPod™ smart device are integrated into the system to capture video of the animals crawling within the micropillar arena. Custom-written The MATLAB software enables users to analyze images and score them for live / dead animals and mobility. This new technology will accelerate gene and drug screening and further advance our understanding of healthy aging at the molecular, cellular, and tissue levels.

[0024] Microfluidic devices for lifelong culture and observation. At the core of NLM are microfluidic devices that enable culture and lifelong observation. Here, we developed two PDMS-based microfluidic devices. Device I allows the lifelong study of a group of animals (50-100 per chamber), while Device II allows the lifelong study of individual animals. The designs of the two devices are discussed below.

[0025] Device I. As shown in Figures 2A and 2B, microfluidic device 20 includes a large rectangular micropillar arena 22, a flow distribution zone 24, and sieving channels 28a, b, and c. Animal loading port 30 allows animals to be introduced into the device, and inlet port 32 and outlet port 34 allow the animals to be washed and fed. The arena includes an array of micropillars arranged in a square lattice. The sieving channels allow for the washing away of juveniles and eggs while retaining the adult population (Figure 2C). The flow distribution zone ensures that the flow introduced during washing / feeding is evenly distributed throughout the micropillar chamber.

[0026] The geometric dimensions of each section are shown in Figures 2D-2G. Importantly, the micropillar chamber is 40 mm long and 17.1 mm wide (Figure 2D). As shown in Figure 2E, the pillars have a diameter of 70 μm, a height of 100 μm, and a center-to-center spacing between pillars of 160 μm (or a gap between pillars of 90 μm). The sieve channels are 1 mm long and 100 μm wide, separated by a 35 μm gap. The flow distribution zone comprises a triangular section with two sides measuring 12.2 mm and a third side the same length as the width of the micropillar chamber (Figure 2D). The shape of the distributor in the flow distribution zone is shown in Figure 2G.

[0027] Device II. As shown in Figure 3A, this microfluidic device contains a series of micropillar chambers capable of housing individuals. The device design includes inlet / outlet ports and side channels for purging air. Animals flow through the distribution channels, with some animals bypassing the distribution channels and entering the micropillar chambers (Figure 3B, top). The inlets of the micropillar chambers have tapered necks that allow for size-based selection and capture of single animals (Figure 3B, bottom left) followed by forced injection into the chamber (Figure 3B, bottom right).

[0028] The experimental protocol for capturing individuals in the chamber is based on a unique loading method. As shown in Figure 3C, using continuous suction, the tube is filled with growth medium at the front (200 µL) and rear (600-800 µL) ends, interspersed with animal solution (20-200 µL). In the first step, the cartridge is pumped at a low flow rate of 5 mL / h, allowing the animals to be captured in the neck of the micropillar chamber (Figure 3D), while the offspring are washed out through the sieve channel. In the second step, a high flow rate of 25 mL / h is used to push the captured animals into the chamber. The cartridge method also helps to eliminate excessive animal intrusion into the chamber.

[0029] The geometric dimensions of this device are shown in Figures 3F-3I. The flow channel is 250 µm wide. The micropillar chamber is 3 mm in diameter, and the pillar characteristics are the same as in Device I. The sieve channel has rectangular blocks 700 µm long and 100 µm wide. The gap between the blocks is 35 µm. The tapered neck begins at 350 µm and narrows to 25 µm over a length of 2300 µm.

[0030] Description of the NemaLife machine. The NLM contains fluidics, imaging, and microcontroller systems integrated into a compact benchtop unit, as shown in Figure 4A. This unit contains two rectangular boxes. The top cover plate of the larger box (Figure 4B) houses slots that hold two microfluidic devices, and the bottom chassis (Figure 4C) houses the LED array, pump, fan, and printed circuit board. The smaller rectangular box houses an iPod for imaging (Figures 4D, 4E). This iPod holder is designed to achieve an optimal focal plane for acquiring video of animals in the micropillar arena. Video acquisition was typically performed at 10–30 fps with a resolution of 1920 × 1080 pixels and a field of view of 54 × 30 mm.

[0031] An operation chart of the system is shown in Figure 5. We designed the NLM to accommodate two microfluidic devices 50a, 50b, allowing the user to operate both devices with a single operation of the unit. Two peristaltic pumps 52a, 52b are integrated, fluidly connecting a medium reservoir 54 and a food reservoir 56 to the pumps 52a, 52b, through the two microfluidic devices 50a, 50b, and to a waste reservoir 58, one used for washing the offspring / eggs and the other used for feeding. Check valves are used to direct fluid from the single pump (52a, 52b) into the two microfluidic devices 50a, 50b. A microcontroller 60 is designed to operate both pumps 52a, 52b, the LED array 62, and the display 64. The display 64 can be an LCD display and can provide an interactive menu with detailed instructions, allowing the user to interface with the machine and perform experimental workflows.

[0032] Workflow. The workflow for performing aging experiments in NLM, shown in Figure 6, involves the following steps: (i) Sterilize the microfluidic device with 70% ethanol, followed by rinsing with DI water. (ii) Load animals from age-synchronized culture plates into microfluidic devices I and II. Animals are typically L4-young adults. (iii) 3.5 per chip. Washing and feeding are performed daily on the NLM for 60-120 seconds at a wash flow rate of ~7.5 mL / hr. Feeding is performed for 2-5 seconds at a similar flow rate range. Images are acquired both during the wash and at the end of the feeding cycle. Video acquisition is performed for 30-90 seconds at a frame rate of 10-30 fps. Videos are stored on an iPod or in the cloud for later retrieval for image processing and data analysis.

[0033] Data analysis software. NemaCode is a big winner of "worm-movies" This is a GUI-driven application for automating the detection of C. elegans from datasets. The application is compiled in MATLAB and relies on the MATLAB Compiler Runtime (MCR) environment for analysis. The application design follows a model-view structure where "models" execute functions in a global namespace that can be called by "views" (UI based on MATLAB's App Designer). Each model function is delegated an independent task by the GUI, either data processing (upload / export), analysis, or result validation using callbacks and event handlers.

[0034] The analysis steps in the application workflow (see Figure 7A) can be listed as 1) image preprocessing, 2) object detection, 3) worm filtering, 4) live / dead calculation, and 5) movement calculation.

[0035] In step (1), automatic ROI (Region of Interest) cropping with alignment correction is performed. The auto cropping function proceeds by applying a median intensity filter to reduce noise, and fits a square pulse function to the image intensity at three vertical positions. The midpoint of the ascending region of the fit is at the top and This provides the location of the bottom wall. After fitting, the wall slope is calculated from the vertical extent, and the image is rotated to align the device with the horizontal image axis. Next, the side walls are determined as peaks in the intensity gradient around the horizontal centerline, and an ROI is cropped based on the known side walls, top, and bottom of the device (see blue dashed line in Figure 7B, i). Step (1) reduces the image size to the ROI, accelerating subsequent analysis.

[0036] In step (2), the program performs object detection on the cropped image by intensity-based discrimination and multi-scale feature detection to highlight and segment worm-like structures. First, the image cleaning function performs a high-pass filter followed by a low-pass filter and morphological dilation to remove the contrast. The filter parameters are set by an external user through a GUI depending on the illumination level and pixel resolution. Second, the work of Frangi et al. Multiscale feature detection based on (optionally) is used to highlight worm-like structures with diverse width and length scales (see Figure 7B, ii).

[0037] The contrast image is then binarized by a thresholding function based on an adaptive threshold calculated based on local first-order statistics and image size, such that objects are assigned a value of 1 while the background is assigned a value of 0 (see Figure 7B, iii). In addition, the user is provided with options for morphological dilation / erosion of objects, as well as the ability to impose raw cutoffs on minimum and maximum object size (total number of pixels comprising an object), regardless of object morphology. The GUI provides users with visual readouts to verify the output of the image cleaning and thresholding functions.

[0038] In step (3), the morphology of the object region is considered to filter the worms from other objects that could not be separated based on the intensity-based approach in step (2). Used within the property function, length (head-to-tail distance in pixels), area (total pixels), width (area / perimeter), and three-dimensionality (area / (convex area)) are estimated as object metrics (Figure 7B, iv). Metrics from the object property function are used to establish minimum and maximum filtering constraints for filtering worms from other objects. The user is provided with a visual readout in the GUI to validate the output of step (3) by graphing various worm metrics and showing annotated images of the ROI (see Figure 7B, iv). Step (3) therefore provides an additional layer of separation to reduce false positives in worm detection by considering the area, three-dimensionality, length, and width metrics of the worm.

[0039] After the final morphology-based detection in step (3), live / dead calculation is performed in step (4) by estimating the degree of movement for each worm in two frames using a live / dead counter function. First, the user selects and skips an appropriate number of frames to properly capture and represent the worm's movement, arriving at consecutive frames 1 and 2. Next, the location of the worm's body (binarized object coordinates) as an image region in frame 1 is used to evaluate the change in the same image region (same coordinates) in frame 2. This change is calculated as Δ = TotalPixels_Region1_Frame1 - TotalPixels_Region1_Frame2. Using a sensitivity criterion in the form of number_of_pixels, each worm is considered alive if the pixel change is Δ > number_of_pixels. The number of frames to skip and the sensitivity criteria depend on the acquisition frame rate and image resolution. Live / dead calculations are repeated for each worm across all consecutive selected frames (with skips) to calculate live and dead statistics. The results are compiled in the form of a table recording frame-by-frame measurements of worm size, body characteristics, and live / dead status. Additionally, the user is presented with graphs showing the number of live worms, total number of worms, and other statistics. A visual readout is also provided in the GUI, allowing the user to scroll through the processed frames to visualize the results of the assay, and worm movies are displayed with blue and red markers overlaid on the worm's center of mass to indicate live versus dead status (Figure 7B, v).

[0040] In step (5), movement calculations are performed by tracking the worm. Worm tracking is performed by skipping frames based on user input, allowing for movement of at least one worm length in consecutive frames, e.g., frame 1 and frame 2. A circular neighborhood (N) is then assigned around each worm's center of mass in frame 1, with a radius Rn equal to the maximum physiologically possible worm movement during the skipped frame. Next, the worms in the neighborhood regions from frame 1 are superimposed on frame 2 to identify the worms contained in these regions in frame 2. Worms found in frame 2 through this juxtaposition are considered neighbors of the worm from frame 1. Knowing the pixel displacement of the center of mass between neighbors, the video acquisition rate, and the skipped frame number, worm speed is estimated. In the case of conflicts due to the collocation of multiple neighbors, we calculated a similarity index based on a least-squares-type estimation of the worm shape metric (from step (3)) between the parent worm from frame 1 and its possible neighbors in frame 2. The smallest value of the similarity index is assumed to be the most similar, and therefore most likely, neighbor. The worm movement calculation is similarly implemented over multiple frames to yield Lagrangian tracks.

[0041] These steps (1-5) together therefore integrate the analysis as a seamless and scalable process, from preprocessing the uploaded raw data to estimating survival and movement. In implementing the analysis algorithm, we have enabled a sequential processing version for a single video, as well as a parallel processing version capable of processing multiple videos for high throughput.

[0042] We first evaluated the optimal pump flow rate for washing progeny from the microfluidic device. We tested flow rates of 3.5, 4.5, and 7.5 mL / min per chip with a 90-second wash time. The results, shown in Figure 8A, indicate that 7.5 mL / min is the optimal flow rate for removing progeny. We also evaluated whether this flow rate would cause cuticle damage in worms by using strain IG274, which carries the reporter nlp-29p::GFP in the cuticle. As shown in Figure 8B, we did not observe any difference in the lifespan of this strain compared to the wild-type strain, indicating that a flow rate of 7.5 mL / hr does not cause cuticle damage.

[0043] Next, we varied the feeding dose from 0.1 to 100 mg / mL per day. The lifespan curves shown in Figure 8C indicate that 0.1 mg / mL was too low for animal survival, while 1 mg / mL extended lifespan. However, similar lifespan data were obtained at 10 to 100 mg / mL. Therefore, feeding at 10 to 100 mg / mL is the optimal food concentration for achieving lifespan data in C. elegans. We also tested whether feeding frequency made a difference. As shown in Figure 8D, feeding at 100 mg / mL once or twice daily did not result in any difference in the lifespan curves. In Figure 8E, we demonstrate the reproducibility of the lifespan curves by overlaying data sets from nine independent experiments. We found that the variability in median and maximum lifespans was 5.3% and 7.4%, respectively.

[0044] To further test the overall system, we performed experiments on mutants that have been well characterized for their longevity (Figure 9). We found that, as expected, age-1, eat-2, and skn-1 exhibited an extension of lifespan, while daf-16 exhibited a shortening of lifespan. .

[0045] Lifespan Assessment. Video recording of animals in the micropillar arena with an iPod provides the ability to assess their healthspan using measures of movement and reproduction. We tracked the movement of wild-type animals and grouped them into high-, medium-, and low-mobility groups (Figures 10A-10C). Animals that moved more than their body length in 10 seconds were classified as high-mobility worms, while those that moved less than their body length in 10 seconds were classified as medium-mobility. Animals that moved very little in 10 seconds were considered to belong to the low-mobility group. We performed this group analysis by tracking movement during the first 0-10 seconds, 50-60 seconds, and 80-90 seconds of the acquired video. We observed that the proportion of animals exhibiting high and medium mobility declined with age. The onset of mobility decline was rapid after 7 days.

[0046] A unique feature of the NemaLife Machine is that it is possible to collect fluid from the nematode arena during the washing step. As a result, progeny can be collected and used to assess lifetime reproductive fitness in different strains. We collected the washed fluid and counted the number of progeny per animal. The data in Figure 11 show that the number of progeny from animals reared in the NemaLife Machine was greater than those reared on standard agar plates. This result demonstrates the health of animals in the microfluidic device environment and that the washed fluid can be collected to determine the reproductive potential of a population of animals.

[0047] We have successfully demonstrated that C. elegans can be effectively maintained in the NemaLife microfluidic device throughout their lifespan without the use of chemicals (such as progeny-blocking drugs, antibacterial or antifungal compounds) in an environment that mimics longevity on agar plates. The micropillars in the microfluidic device allow the animals to maintain their natural crawling progression, eliminating stress such as swim-induced fatigue. Both individual and populations of crawling animals can be studied throughout their lifespan. The main advantages of the NemaLife Machine are: Lifelong culture without animal transfer ·Swimming stress-free plate-shaped animal behavior Automated scoring of animal activity and survival Throughput: 24 assays / hour / user / machine Collecting progeny for downstream assays Video archiving for retrospective analysis is.

[0048] The above advantages offered by the NemaLife Machine will be extremely important in a wide range of applications, including drug screening, toxicology testing, genetic screening, behavioral phenotyping and disease research.

[0049] Worm culture. All animals were cultured on 60 mm Petri dishes containing nematode growth medium (NGM) at 20°C before being loaded into the microfluidic chamber. The NGM-filled Petri dishes were seeded with 300–400 μL of Escherichia coli OP50 bacteria and incubated at 20°C for 48 hours. To synchronize age, 20–25 pregnant adults were placed on the seeded plates and allowed to lay eggs for 3–4 hours. After egg laying, the animals were removed from the plates, and the plates with eggs were incubated for 60–72 hours. The day of egg laying was scored as day 0.

[0050] Fabrication and preparation of microfluidic devices. All microfluidic devices were fabricated in poly(dimethyl)siloxane (PDMS) using soft lithography ( McDonald, JC et al. Fabrication of microfluidic systems in poly(dimethylsiloxane). Electrophoresis 21, 27–40 (2000)). As previously described, a mold was fabricated using SU-8 photolithography to achieve a chamber height of approximately 100 μm and a micropillar height of approximately 75 μm (Rahman, M. et al. NemaFlex: A microfluidics-based technology for measurement of muscular strength of C. elegans. Lab on a chip 18, 2187–2201, doi:10.1039 / c8lc00103k (2018)). A 4–6 mm thick layer of PDMS (Sylgard 184A and B, 1:10 by weight, Dow Corning) was cast into the mold, and inlet / outlet holes were drilled using a 1 mm punch. The PDMS device was then irreversibly bonded to a glass surface and made hydrophilic by plasma treatment (Harrick Plasma). Before using the device in lifespan / healthspan experiments, the interior of the device was sterilized by filling it with 70% ethanol for 5 minutes. The device was then rinsed 4-5 times with liquid NGM solution. The device was then treated with 5 wt% Pluronic F127 (Sigma-Aldrich) for 30 minutes to prevent protein and bacterial accumulation (Xian, B. et al. WormFarm: a quantitative control and measurement device toward automated Caenorhabditis elegans aging analysis. Aging Cell 12, 398-409 (2013)). Additionally, Therefore, the Pluronic treatment also helps remove any trapped air bubbles. After incubation, excess Pluronic was removed by washing with liquid NGM. Pluronic-treated devices were stored in a moist Petri dish at 20 °C for immediate use or at 4 °C for future use.

[0051] Food preparation. Escherichia coli OP50 was used as the bacterial food source for worms grown on NGM medium and maintained in the device. Unless otherwise stated, approximately 10 9 A 100 mg / mL bacterial suspension in liquid NGM solution corresponding to bacteria / mL was used for the lifespan assay. E. coli OP50 was grown overnight at 37°C in standard LB medium. A 100 mg / mL bacterial suspension was prepared by centrifuging 500 mL of overnight bacterial culture and resuspending the pellet in liquid NGM. The concentrated OP50 was stored at 4°C for up to one week for later use.

[0052] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, the compositions of the invention can be used to achieve the methods of the invention.

[0053] It will be understood that the specific embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.

[0054] All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0055] The use of the word "a" or "an" in the claims and / or specification when used in conjunction with the word "comprising" can mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more than one." The use of the word "or" in the claims is used to mean "and / or" unless expressly indicated to refer to alternatives only, or those alternatives are not mutually exclusive, although the present disclosure supports a definition that refers only to alternatives and "and / or." Throughout this application, the term "about" is used to mean that a value will not exceed the limits set forth in the present specification unless the method used to determine that value is sufficient. is used to indicate that the measurement includes the inherent error variation of the device or the variation that exists between study subjects.

[0056] As used in this specification and claims, the words "comprising" (and all forms of comprising, such as "comprise" and "comprises"), "having" (and all forms of having, such as "have" and "has"), "including" (and all forms of including, such as "includes" and "include"), or "containing" (and all forms of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. In any of the embodiments of the compositions and methods provided herein, "comprising" can be replaced with "consisting essentially of" or "consisting of." As used herein, the phrase "consisting essentially of" includes the specified integers or steps, as well as any other elements or methods that are specifically included. It is required that the invention not be materially affected in its characteristics or functionality. As used herein, the term "consisting" means that the entirety of a stated integer (e.g., a feature, element, characteristic, property, method / process step or limitation) is not materially affected in any way. definition) or integer (e.g., feature, element, characteristic, property, method / process step) It is used to indicate the presence of only a group (group or restriction).

[0057] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and, if order is important in the particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, combinations including repeats of one or more items or terms are expressly included, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, etc. One of ordinary skill in the art will understand that there is typically no limit to the number of items or terms in any combination, unless otherwise apparent from the context.

[0058] As used herein, without limitation, approximating terms such as "about," "substantial," or "substantially" refer to a condition that, when so modified, is understood not necessarily to be absolute or complete, but that would be considered by one of ordinary skill in the art to be close enough to warrant specifying the condition as existing. The degree to which the description can vary will depend on how large a change can occur and still allow one of ordinary skill in the art to recognize the modified feature as still possessing the desired characteristics and capabilities of the unmodified feature. Generally, but subject to the preceding discussion, numerical values ​​herein modified by approximating terms such as "about" can vary by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12, or 15% from the stated value.

[0059] All of the compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations can be applied to the compositions and / or methods, and in the steps or sequence of steps of the methods, described herein without departing from the concept, spirit, and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.

[0060] To assist the Patent Office, and any reader of any patent that may issue on this application, in interpreting the claims appended hereto, Applicant wishes to note that none of the appended claims intends for paragraph 6 of 35 U.S.C. 112(f), 35 U.S.C. 112(f), or equivalents thereof, as they exist on the filing date of this application, to apply to any of the appended claims, unless the words "means for" or "step for" are expressly used in a particular claim.

[0061] For each claim, each dependent claim may depend from both the independent claim and each of the previous dependent claims for each and every claim, so long as the previous claim provides appropriate antecedent for a term or element of the claim.

[0062] (References) 1 Harman, D. The aging process: Major risk factor for disease and death. Proceedings of National Academy of Science 88, 5360-5363 (1991) 2 Niccoli, T. & Partridge, L. Ageing as a Risk Factor for Disease. CurrentBiology 22, R741-R752, doi:10.1016 / j.cub.2012.07.024 (2012) 3 Farooqui, T. & Farooqui, A. A. Aging: An important factor for thepathogenesis of neurodegenerative diseases. Mechanisms of aging and development130, 203-215 (2009) 4 North, B. J. & Sinclair, D. A. The intersection between aging andcardiovascular disease. Circulation research 110, 1097-1108 (2012) 5 White, M. C. et al. Age and cancer risk: a potentially modifiablerelationship. American journal of preventive medicine 46, S7-15 (2014) 6 Shaye, D. & Greenwald, I. OrthoList: A Compendium of C. elegans Geneswith Human Orthologs. Plos One 6, doi:10.1371 / journal.pone.0020085 (2011) 7 Kenyon, C. J. The genetics of ageing. Nature 464, 504 - 512 (2010) 8 Consortium, C. e. S. & Consortium, C. e. S. Genome sequence of thenematode C-elegans: A platform for investigating biology. Science 282,2012-2018, doi:10.1093 / glycob / cwi075 (1998) 9 Kenyon, C. The plasticity of aging: Insights from long-lived mutants. Cell120, 449-460, doi:10.1016 / j.cell.2005.02.002|10.1016 / j.cell.2006.02.002 (2005) 10 Fielenbach, N. & Antebi, A. C-elegans dauer formation and the molecularbasis of plasticity. Genes & Development 22, 2149-2165,doi:10.1101 / gad.1701508 (2008) 11 Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W. & Prasher, D. C. Greenfluorescent protein as a marker for gene-expression. Science 263, 802-805(1994) 12 Ran, F. et al. Genome engineering using the CRISPR-Cas9 system. NatureProtocols 8, 2281-2308, doi:10.1038 / nprot.2013.143 (2013) 13 Fire, A. et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811, doi:10.1038 / 35888 (1998) 14 Mitchell, D. H., Stiles, J. W., Santelli, J. & Sanadi, D. R. Synchronousgrowth and aging of Caenorhabditis elegans in the presence of Fluorodeoxyuridine. The Journal of gerontology 34, 28-36 (1979) 15 Gandhi, S., Santelli, J., Mitchell, D. H., Stiles, J. W. & Sanadi, D. R.A simple method for maintaining large, aging populations of Caenorhabditis elegans. Mechanisms of aging and development 12, 137-150 (1980) 16 Bishop, N. & Guarente, L. Two neurons mediate diet-restriction-inducedlongevity in C-elegans. Nature 447, 545-+ (2007) 17 Vanvoorhies, W. Production of sperm reduces nematode life-span. Nature 360, 456-458, doi:10.1038 / 360456a0 (1992) 18 Zhang, W. B. et al. Extended twilight among isogenic c. elegans causes adisproportionate scaling between lifespan and health. Cell Systems 3, 333-345(2016) 19 Pittman, W., Sinha, D., Zhang, W., Kinser, H. & Pincus, Z. A simpleculture system for long-term imaging of individual C. elegans. Lab on a Chip 17,3909-3920, doi:10.1039 / c7lc00916j (2017) 20 Stroustrup, N. et al. The Caenorhabditis elegans Lifespan Machine. NatureMethods 10, 665-+ (2013) 21 Stroustrup, N. et al. The temporal scaling of Caenorhabditis elegans ageing.Nature 530, 103-+, doi:10.1038 / nature16550 (2016) 22 Lucanic, M., Plummer, W. T., Lithgow, G. J., Driscoll, M. & Phillips, P.C. Impact of genetic background and experimental reproducibility on identifyingchemical compounds with robust longevity effects. Nature communication 8, 14256(2017) 23 Churgin, M. A. et al. Longitudinal imaging of Caenorhabditis elegans in amicrofabricated device reveals variation in behavioral decline during aging.eLife 10 (2017) 24 Anderson, E. et al. C-elegans lifespan extension by osmotic stress requires FUdR, base excision repair, FOXO, and sirtuins. Mechanisms of Ageing andDevelopment 154, 30-42 (2016) 25 Angeli, S. et al. A DNA synthesis inhibitor is protective againstproteotoxic stressors via modulation of fertility pathways in Caenorhabditis elegans. Aging-Us 5, 759-769, doi:10.18632 / aging.100605 (2013) 26 Aitlhadj, L. & Sturzenbaum, S. The use of FUdR can cause prolongedlongevity in mutant nematodes. Mechanisms of Ageing and Development 131,364-365 (2010) 27 Van Raamsdonk, J. & Hekimi, S. FUdR causes a twofold increase in thelifespan of the mitochondrial mutant gas-1. Mechanisms of Ageing andDevelopment 132, 519-521 (2011) 28 Greer, E. & Brunet, A. Different dietary restriction regimens extendlifespan by both independent and overlapping genetic pathways in C-elegans.Aging Cell 8, 113-127 (2009) 29 Kauffman, A. L., Ashraf, J. M., Corces-Zimmerman, M. R., Landis, J. N. &Murphy, C. T. Insulin signaling and dietary restriction differentiallyinfluence the decline of learning and memory with age. PLos Biology 8, e1000372(2010) 30 Hulme, S. et al. Lifespan-on-a-chip: microfluidic chambers for performinglifelong observation of C. elegans. Lab on a Chip 10, 589-597,doi:10.1039 / b919265d (2010) 31 Wen, H., Shi, W. & Qin, J. Multiparameter evaluation of the longevity inC-elegans under stress using an integrated microfluidic device. Biomedical Microdevices 14, 721-728 (2012) 32 Wen, H., Yu, Y., Zhu, G., Jiang, L. & Qin, J. A droplet microchip withsubstance exchange capability for the developmental study of C-elegans. Lab ona Chip 15, 1905-1911 (2015) 33 Xian, B. et al. WormFarm: a quantitative control and measurement devicetoward automated Caenorhabditis elegans aging analysis. Aging Cell 12, 398-409(2013) 34 Dong, L., Cornaglia, M., Lehnert, T. & Gijs, M. On-chip microfluidic biocommunication assay for studying male-induced demise in C. elegans hermaphrodites. Lab on a Chip 16, 4534-4545, doi:10.1039 / c6lc01005a (2016) 35 Halldorsson, S., Lucumi, E., Gomez-Sjoberg, R. & Fleming, R. Advantagesand challenges of microfluidic cell culture in polydimethylsiloxane devices.Biosensors & Bioelectronics 63, 218-231, doi:10.1016 / j.bios.2014.07.029(2015) 36 Chung, K. et al. Microfluidic chamber arrays for whole-organismbehavior-based chemical screening. Lab on a Chip 11, 3689-3697,doi:10.1039 / c1lc20400a (2011) 37 Letizia, M. et al. Microfluidic-enabled phenotyping of a whole population ofC. elegans worms over their embryonic and post-embryonic development atsingle-organism resolution. Microsystems & Nanoengineering 4 (2018) 38 Szewczyk, N. et al. Delayed development and lifespan extension as featuresof metabolic lifestyle alteration in C-elegans under dietary restriction.Journal of Experimental Biology 209, 4129-4139, doi:10.1242 / jeb.02492 (2006) 39 Laranjeiro, R., Harinath, G., Burke, D., Braeckman, B. P. & Driscoll, M.Single swim sessions in C. elegans induce key features of mammalian exercises.BioMed Central Biology 15, doi:10.1186 / s12915-017-0368-4 (2017) 40 Chuang, H., Kuo, W., Lee, C., Chu, I. & Chen, C. Exercise in anelectrotactic flow chamber ameliorates age-related degeneration inCaenorhabditis elegans. Scientific Reports 6, doi:10.1038 / srep28064 (2016) 41 Hartman, J. et al. Swimming Exercise and Transient Food Deprivation inCaenorhabditis elegans Promote Mitochondrial Maintenance and Protect AgainstChemical-Induced Mitotoxicity. Scientific Reports 8,doi:10.1038 / s41598-018-26552-9 (2018) 42 Albrecht, D. & Bargmann, C. High-content behavioral analysis ofCaenorhabditis elegans in precise spatiotemporal chemical environments. NatureMethods 8, 599-605 (2011) 43 Ai, X., Zhuo, W., Liang, Q., McGrath, P. & Lu, H. A high-throughputdevice for size based separation of C-elegans developmental stages. Lab on aChip 14, 1746-1752, doi:10.1039 / c3lc51334c (2014) 44 McDonald, J. C. et al. Fabrication of microfluidic systems inpoly(dimethylsiloxane). Electrophoresis 21, 27-40 (2000) 45 Rahman, M. et al. NemaFlex: A microfluidics-based technology for measurementof muscular strength of C. elegans. Lab on a chip 18, 2187-2201, doi:DOI:10.1039 / c8lc00103k (2018)

Claims

[Claim 1] 1. A system for analyzing an animal, comprising: a medium reservoir and a medium pump in fluid communication with said medium reservoir; a food reservoir and a food pump in fluid communication with the food reservoir; an input port in fluid communication with the medium pump and the food pump; a microfluidic device in fluid communication with the input port, (1) A micropillar arena for housing the animal, the micropillar arena comprising a plurality of micropillars that allow the animal to crawl through, the micropillar arena having a boundary of a shape that includes a circle, an oval, a square, a rectangle, or other polygonal shape, or some combination of these shapes; or (2) a plurality of micropillar chambers, each configured to allow one animal to crawl therethrough; At least a transparent outer surface for lighting or imaging. the microfluidic device comprising: an outlet port in fluid communication with the microfluidic device; a light source disposed outside the micropillar arena to illuminate the interior of the micropillar arena; an imager disposed outside the micropillar arena to image the interior of the micropillar arena; and a controller connected to the medium pump, the food pump, the microfluidic device, the light source, and the imager; The system comprising:

Images