Automated microfluidic system for analyzing lifespan and healthy lifespan 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 generally relates to the field of automated microfluidic systems for analyzing the lifespan and healthspan of organisms, for example, nematodes.

[0003] Statement of Federally Sponsored Research This invention was made with government support under Grant A G050503 awarded by the National Institutes of Health and Grant NNX15AL16G awarded by NASA. The government has certain rights in the invention. Background Art

[0004] Without limiting the scope of the present invention, the background of the invention is described herein in connection with 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 National Academy of Sciences 88, 5360-5363 (1991); Nicoccoli, 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 oral cavity ages, the socioeconomic burden caused by age-related diseases is enormous, making the development of treatments that promote healthy aging essential. C. elegans has a short lifespan (3-5 weeks) and shows remarkable genetic similarity to humans (approximately 38% orthologized by Shaye, D. & Greenwald, I. OrthoList: A Compendium of C. elegans Genes with Human Orthologs. Plos One 6, doi:10.1371 / journal.pone.0020085 (2011))) The signaling pathway It is preserved (Kenyon, CJ The genetics of aging. Nature 464, 504 - 512 (2010)) and is a powerful model organism for aging research. In addition, because the genome is fully 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 because of its excellent genetic plasticity (Kenyon, C. The plasticity of aging: Insights from long-livedmutants. 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 themolecular basis of plasticity. Genes & Development 22, 2149-2165, doi:10.1101 / gad.1701508 (2008), C-Elegance has become 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 genome 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 increased the number of possible methods for studying healthy aging using Caenorhabditis elegans.

[0006] Lifespan analysis has become a classic method for evaluating the effects of a wide variety of genes, proteins, and pharmaceutical compounds on aging and age-related diseases. However, conventional lifespan analysis is generally low-throughput and lacks the ability to analyze non-invasive health indicators. Aging assays generally involve animals crawling in nematode growth media (NGM). This is performed with Caenorhabditis elegans cultured on agar plates. During the breeding season, adults must be manually transferred to new plates to separate offspring from the original sample. To reduce the need for manual transfer, many laboratories maintain adult-only populations using a potent offspring inhibitor (2'-deoxy-5-fluorouridine, FUdR, 2'-deoxy-5-fluorouridine) (Mitchell, DH, Stiles, JW, Santelli, J. & Sanadi, DRS: Synchronous growth and aging of Caenorhabditis elegans in the presence of Fluorodeoxyuridine). 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 mediate diet-restriction-induced longevity in C-elegans. Nature 447, 545-+ (2007)). An alternative to this approach is to use germ-free 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 sterile mutants has led to a new technique for large-scale lifespan analysis in creeping Caenorhabditis elegans. Known as the Lifespan Machine (LSM), this technique allows for the analysis of populations of thousands of animals grown on FUdR-added agar, automatically capturing sequential images for scoring animal mortality and determining lifespan (Stroustrup, N. et al. The Caenorhabditis elegans Lifespan Machine. Nature Methods 10, 665-+ (2013)). The LSM technique provides insights 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 ageing. Nature 530, 103-+, doi:10.1038 / nature16550 (2016)), and has been useful in identifying compounds with robust longevity effects (Lucanic, M., Plummer, WT, Lithgow, GJ, Driscoll, M. & Phillips, PC Impact of genetic background and experimental reproducibilityon identifying chemical compounds with robust longevity effects. Naturecommunication 8, 14256 (2017)). Similarly, WorMotel technology has been used for longitudinal analysis of individuals in microfabricated well plates filled with agar. This becomes easier (Churgin, MA et al. Longitudinal imaging of Caenorhabditis elegans in a microfabricated device reveals variation inbehavioral 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. ADNA synthesis inhibitor is protective against proteotoxic stressors viamodulation of fertility pathways in Caenorhabditis elegans. Aging-Us 5, 759-769, doi:10.18632 / aging.100605 (2013)) and increases 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)) Several genotypes alter lifespan (Anderson, E. et al. C-elegans lifespan extension by osmotic stress requires FUdR, base ex Since it has been shown that cision repair, FOXO, and sirtuins can cause prolonged longevity in mutant nematodes, as demonstrated in 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), and 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), using FUdR in LSM and WorMotel technologies is problematic.

[0008] Furthermore, current technologies such as LSM and WorMotel lack the ability to study the effects of temporary environmental manipulation on lifespan. Such manipulations at user-defined time intervals are related to dietary restriction (Greer, E. & Brunet, A. Different dietary restriction regimens extend lifespan by both independent and overlapping genetic pathways in C-elegans. Aging Cell 8, 113-127 (2009)) and 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)) has been the focus of research. Currently, conventional research and high-level research The current lifespan technology lacks the ability to rapidly and reversibly manipulate environmental conditions, limiting its usefulness for survival analysis of animals exposed to specific environments.

[0009] In recent years, microfluidic approaches have begun to address the limitations of agar-based lifetime 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. Multiparameterevaluation 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 include (i) excellent permeability to oxygen and carbon dioxide, allowing 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)), and (ii) eliminating the need to prevent or reduce the production of offspring. 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. BiomedicalMicrodevices 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 by adding or removing reagents (Wen, H., Shi, W. & Qin, J. Multiparameter evaluation of the longevity in C-elegans understress 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 (iv) an overall reduction in the number of worms being terminated, and (iv) enabling white light and fluorescence imaging. This includes the optical transparency of the device.

[0010] Despite the significant advantages of microfluidic-based approaches, work to date has been limited in three respects. Firstly, 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), 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 chemicalscreening. Lab on a Chip 11, 3689-3697, doi:10.1039 / c1lc20400a (2011), Letizia, M. et al. Microfluidic-enabled phenotyping of a wholepopulation of C. elegans worms over their embryonic and post-embryonic development at single-organism resolution. Microsystems & Nanoengineering 4 (2018)) encapsulate animals in chambers that swim rather than crawl, and therefore do not mimic the standard plate-like animal behavior in C. elegans populations. By housing worms in liquid culture for a significant portion of their lifespan, alterations in gene expression are induced (Szewczyk, N. et al. Delayed development and lifespan extension asfeatures 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, BP & 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)).In addition, the mandatory swimming requirement has been shown to induce fatigue and oxidative stress, outcomes not present in animals grown on plates (Laranjeiro, R., Harinath, G., Burke, D., Braeckman, BP & 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 swimming chambers, several studies have reported micropillar chambers for sea elegans assays (Albrecht, D. & Bargmann, C. High-content While behavioral analysis of Caenorhabditis elegans in precise spatiotemporalchemical environments (Nature Methods 8, 599-605, 2011) and 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) have been conducted, none have been constructed and validated for lifetime or aging studies. Secondly, 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. Thirdly, scoring animal survival and movement is often manual, making the analysis of large image datasets cumbersome. In addition, the lack of a streamlined workflow for data analysis can lead to false positives and biases in the analysis.

[0011] Such a system is taught in European Patent Application No. 3209790, entitled "Microfluidic Device, System and Method for the Study of Organisms," filed by Cornaglia, et al. In short, these applicants are said to be teaching a microfluidic device for culturing, selecting, and / or analyzing sample organisms such as nematodes, as well as other biological entities such as animal embryos. The device is said to include a reservoir, culture chamber, and smart filtering system that enable the selection of specific populations / specimens of sample organisms, thus allowing for their long-term cultivation and phenotypic / behavioral analysis.

[0012] However, despite such devices, there remains a need for equipment that enables long-term study of organisms like nematodes, more accurately reflects their natural habitats, and does not affect gene expression in organisms like the devices of prior art. [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, B. J. & Sinclair, D. A. The intersection between aging and cardiovascular disease. Circulation research 110, 1097-1108 (2012) [Non-Patent Document 5] White, M. C. et al. Age and cancer risk: a potentially modifiable relationship. American journal of preventive medicine 46, S7-15 (2014) [Non-Patent Document 6] Shaye, D. & Greenwald, I. OrthoList: A Compendium of C. elegans Genes with Human Orthologs. Plos One 6, doi:10.1371 / journal.pone.0020085 (2011) [Non-Patent Document 7] Kenyon, C. J. The genetics of ageing. Nature 464, 504 - 512 (2010) [Non-Patent Document 8] 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) [Non-Patent Document 9] 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) [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 of Caenorhabditis 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) [Non-Patent Document 26] Aitlhadj, L. & Sturzenbaum, S. The use of FUdR can cause prolonged longevity in mutant nematodes. Mechanisms of Ageing and Development 131, 364-365 (2010) [Non-Patent Document 27] Van Raamsdonk, J. & Hekimi, S. FUdR causes a twofold increase in the lifespan of the mitochondrial mutant gas-1. Mechanisms of Ageing and Development 132, 519-521 (2011) [Non-Patent Document 28] Greer, E. & Brunet, A. Different dietary restriction regimens extend lifespan by both independent and overlapping genetic pathways in C-elegans. Aging Cell 8, 113-127 (2009) [Non-Patent Document 29] Kauffman, AL, Ashraf, JM,Corces-Zimmerman, MR, Landis, JN & Murphy, CT Insulin signalingand dietary restriction differentially influence the decline of learning andmemory with age. PLos Biology 8, e1000372 (2010) [Non-Patent Document 30] 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) [Non-Patent Document 31] Wen, H., Shi, W. & Qin, J.Multiparameter evaluation of the longevity in C-elegans under stress using anintegrated microfluidic device. Biomedical Microdevices 14, 721-728 (2012) [Non-Patent Document 32] 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) [Non-Patent Document 33] Xian, B. et al. WormFarm: a quantitative control and measurement device toward automated Caenorhabditis elegans aging analysis. Aging Cell12, 398-409 (2013)

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[0015] In some embodiments of the present disclosure, a system for analyzing animals comprises a culture medium reservoir and a culture medium pump in fluid communication with the culture medium reservoir, a food reservoir and a food pump in fluid communication with the food reservoir, an input port in fluid communication with the culture medium pump and the food pump, and a microfluidic device in fluid communication with the input port, wherein (1) a microfluidic device for housing animals (1) A micropillar arena comprising a plurality of micropillars that allow an animal to crawl through, and having a boundary of a shape that includes a circular, elliptical, square, rectangular, or other polygonal shape, or some combination thereof, or (2) a microfluidic device comprising a plurality of micropillar chambers, each micropillar chamber configured to allow one animal to crawl through, and a transparent outer surface for at least illumination or imaging, an outlet port that fluidly communicates with the microfluidic device, a light source positioned outside the micropillar arena to illuminate the 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 a culture medium pump, a food pump, the microfluidic device, the light source, and the imager. In one embodiment, the microfluidic device includes an input flow distribution region fluid-communicating with an input port, a micropillar arena fluid-communicating with the input flow distribution region, a micropillar arena fluid-communicating with the input flow distribution region, a micropillar arena fluid-communicating with the input flow distribution region, a micropillar arena fluid-communicating with the input flow distribution region, a micropillar arena fluid-communicating with the input flow distribution region, a micropillar arena fluid-communicating with the input flow distribution region, a micropillar arena fluid-communicating with the input flow distribution region, a micropillar arena fluid-communicating with the input flow distribution region, a micropillar arena fluid-communicating with the input port, a micropillar arena fluid-communicating with the input port, a micropillar arena fluid-communicating with the input port, a micropillar arena fluid-communicating with the input port, a micropillar arena fluid-communicating with the input port, a micropillar arena fluid-communicating with the input flow distribution region 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 thereof.In another embodiment, the analysis includes at least one of recording animal movements, counting living and dead animals, studying animal behavior, or studying animal mobility. In another embodiment, the animals are nematodes of at least the genus Caenorhabditis. In another embodiment, the light source includes light-emitting diodes. In another embodiment, the imager is a digital imager.

[0016] In some embodiments of the present disclosure, a method for analyzing animals comprises the steps of providing a plurality of animals to be analyzed; a culture medium reservoir and a culture medium pump in fluid communication with the culture medium reservoir; a food reservoir and a food pump in fluid communication with the food reservoir; an input port in fluid communication with the culture medium pump and the food pump; and a microfluidic device in fluid communication with the input port, wherein (1) a micropillar arena for housing animals, comprising a plurality of micropillars allowing animals to crawl in there, and having a boundary of a shape including circular, elliptical, square, rectangular, or other polygonal shape, or some combination thereof; or (2) each micropillar chamber allowing one animal to crawl in there The disclosed method includes loading multiple animals into an analyzer comprising: a microfluidic device comprising a plurality of micropillar chambers and a transparent outer surface for at least illumination or imaging; an outlet port in fluid communication with the microfluidic device; a light source positioned outside the micropillar arena to illuminate the 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 a culture medium pump, a food pump, the microfluidic device, the light source, and the imager; illuminating the multiple animals through the transparent outer surface; imaging the multiple animals through the transparent outer surface to generate an image; and analyzing the image. In one embodiment, the microfluidic device comprises an input flow distribution region in fluid communication with an input port and a pattern configured to allow animals to crawl in the space between micropillars and to retain adult animals while removing offspring animals. The system includes a micropillar arena fluid-communicating with an input flow distribution region, which includes a micropillar arena fluid-communicating with an input flow distribution region, which includes a micropillar arena fluid-communicating with an outlet port, and a micropillar arena fluid-communicating with an outlet port. In another embodiment, each micropillar has a cross-section including a circular, elliptical, square, rectangular, or other polygonal cross-section, or some combination of these shapes. In another embodiment, the micropillar arena includes an inlet end and an outlet end, which includes a distribution channel for introducing animals, the inlet end fluid-communicating with an input port and the outlet end fluid-communicating with an outlet port, and a plurality of micropillar chambers are distributed along the distribution channel fluid-communicating with it, and each micropillar chamber includes a tapered neck for selecting and capturing one animal on size in each micropillar chamber at a sufficiently high fluid flow rate. In another embodiment, each micropillar chamber has a cross-section including a circular, elliptical, square, rectangular, or other polygonal cross-section, or some combination of these shapes. In another embodiment, the analysis includes at least one of recording animal movements, counting living and dead animals, studying animal behavior, or studying animal mobility. In another embodiment, the animals are nematodes of at least the genus Caenorhabditis. In another embodiment, the light source includes light-emitting diodes. In another embodiment, the imager is a digital imager.

[0017] In some embodiments of the present disclosure, a system for analyzing nematodes comprises a culture reservoir and a culture pump fluidly communicating with the culture reservoir, a food reservoir and a food pump fluidly communicating with the food reservoir, an input port fluidly communicating with the culture pump and the food pump, and a microfluidic device fluidly communicating with the input port, wherein (1) a micropillar arena for containing nematodes, comprising a plurality of micropillars that allow nematodes to crawl therein, and having a boundary of shape including circular, elliptical, square, rectangular, or other polygonal shapes, or some combination thereof, or (2) a microfluidic device comprising a plurality of micropillar chambers, each configured to allow one nematode to crawl in it, and a transparent outer surface for at least illumination or imaging; an outlet port in fluid communication with the microfluidic device; a light source located outside the micropillar arena to illuminate the inside of the micropillar arena; an imager located outside the micropillar arena to image the inside of the micropillar arena; and a controller connected to a culture medium pump, a food pump, the microfluidic device, the light source, and the imager. In one embodiment, the microfluidic device comprises an input flow distribution region in fluid communication with an input port; a micropillar arena in fluid communication with the input flow distribution region, wherein a plurality of micropillars are distributed in a pattern configured to allow nematodes to crawl in the space between micropillars and to retain adult nematodes while removing progeny nematodes, and includes a nematode loading port and a plurality of sieve channels that enable removal of progeny nematodes; and an outlet flow distribution region in fluid communication with a micropillar arena in fluid communication with an outlet port. In another embodiment, each micropillar 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 an inlet end and an outlet end, the inlet end being in fluid communication with an input port and the outlet end being in fluid communication with an outlet port, and includes a distribution channel for introducing nematodes, the inlet end being in fluid communication with an input port and the outlet end being in fluid communication with an outlet port, and a plurality of micropillar chambers are distributed along the distribution channel in fluid communication with it, each micropillar chamber includes a tapered neck for selecting and capturing one nematode on size in each micropillar chamber, and is configured to allow one nematode to crawl inside it. In another embodiment, each micropillar chamber has a cross-section including a circular, elliptical, square, rectangular, or other polygonal cross-section, or some combination of these shapes. In another embodiment, the analysis includes at least one of recording the movement of nematodes, counting live and dead nematodes, studying the behavior of nematodes, or studying the mobility of nematodes. In another embodiment, the nematodes are at least Caenorhabditis (Caenorha). It belongs to the genus *Bditis*. In another embodiment, the light source includes a light-emitting diode. In another embodiment, the imager is a digital imager. [Brief explanation of the drawing]

[0018] For a more complete understanding of the features and advantages of the present invention, a detailed description of the invention is provided here with reference to the accompanying figures. [Figure 1] The present invention provides a description of the NemaLife machine and an example of its components. The main components of the NemaLife unit are: (i) a housing for fluid and electrical modules, (ii) an iPod holder, (iii) an iPod for imaging, (iv) a slot for a microfluidic device, the microfluidic device is illuminated from the side, (v) a user interface display for controlling the unit, and (vi) a fluid reservoir for sterilizing the unit and washing / feeding animals. [Figure 2A] ~ [Figure 2G]Detailed design of microfluidic apparatus I is shown. (Figure 2A) Microfluidic apparatus for culturing C. elegans. This apparatus is filled with blue food coloring. The rectangular area represented by the black dashed line is the micropillar arena for housing the crawling animals. Flow distribution zones, inlet / outlet ports, sieve channels and animal loading ports on both sides of the micropillar arena are highlighted. (Figure 2B) Micropillar arena loaded with approximately 60 adult animals and the food source E. coli OP50. (Figure 2C) Part of the sieve channel includes a series of equally spaced rectangular blocks that prevent adults from escaping the micropillar arena but allow offspring to be washed away. (Figure 2D) Top view of the micropillar arena (apparatus I). All dimensions are in μm. (Figure 2E) and (Figure 2F) Architecture of the pillars and sieve channels. Columns with a height of 100 μm and a diameter of 70 μm are suspended from the ceiling at equal intervals (160 μm) within a rectangular grid. A sieve channel is created by repeating rectangular blocks of 1000 μm × 200 μm with a gap (35 μm) between them. The columns are arranged within the rectangular grid. (Figure 2G) Detailed design of the flow distribution zone. [Figure 3A] ~ [Figure 3I]Further details of the microfluidic apparatus for containing and culturing organisms are shown. (Figure 3A) This apparatus includes 3 × 10 circular micropillar chambers (filled with green food coloring) connected in a distribution channel capable of containing organisms. Black dashed arrows indicate the distribution channel, air purge, inlet, micropillar chambers, and outlet. (Figure 3B) Enlarged view of a portion of the microfluidic apparatus showing three chambers. Black arrows indicate the direction of fluid flow through the distribution channel and through the chambers. The image shows a single chamber (red dashed boundary). Each 3 mm diameter chamber is connected to an upstream channel (red dashed arrow) via a narrow tapered neck / arm (black arrow) and a downstream channel via a sieve channel (black dashed arrow). An enlarged view of an animal crawling inside the chamber is shown next to it. Scale bar: 1 mm. (Figure 3C) Schematic diagram of the animal loading protocol using the cartridge method. Cartridges are prepared by aspirating 200 μL of worm solution (yellow) into a tube, followed by buffer (blue). (Figure 3D) Capture of animals at the neck of the chamber by introducing a plug of worm solution (50-100 worms / mL) at a flow rate of 5 ml / hour for 90 seconds. Each arrow indicates a captured animal. (Figure 3E) Captured worms are pushed into the chamber by introducing buffer behind the worm solution into the cartridge at a flow rate of 15 ml / hour for 15 seconds. (Figure 3F) Top view of the entire apparatus. All dimensions are in μm. (Figures 3G)-(Figure 3I) Detailed design of the sieve channel, tapered loading neck, fluid inlet and air purge port. [Figure 4A] ~ [Figure 4E] Detailed design of the NemaLife Machine is shown. (Figure 4A) Isometric view of the housing for the NemaLife Machine (drawn to scale). This may include (Figure 4B) the top cover plate, (Figure 4C) the bottom chassis, (Figure 4D) the iPod holder - top view, and (Figure 4E) the iPod holder (top view and 3D view). [Figure 5]This is a flowchart of the NemaLife machine's system workflow. The imaging and fluid systems are controlled via a microcontroller, and the steps are displayed on an LCD display located on the machine's top cover plate. The microcontroller receives user commands from the LCD display. [Figure 6] This is the NemaLife operational workflow. Animals hatched and grown on agar plates are transferred to a micropillar arena at the L4-juvenile adult stage. The microfluidic apparatus is cleaned daily to separate offspring, the animals are fed, and then imaged using an iPod. The images are analyzed using the image processing software NemaCode to generate lifespan curves. [Figure 7A] ~ [Figure 7B] Figures 7A and 7B(i)-(v) illustrate the workflow for automated image analysis for viability / death scoring. (Figure 7A) Image analysis workflow for tracking worm objects in images for lifespan and healthy lifespan assays. (Figure 7B) Graphical user interface of NemaCode image analysis software. This software allows the user to independently navigate between tabs to review and validate the analysis. Figures 7B(i)-(v) illustrate the key steps of the image analysis software: (i) rotation and cropping of the image of the region of interest (worm culture arena only), (ii) illumination correction and thresholding, (iii) segmentation, (iv) detection and validation of worm objects, and (v) annotation of death and survival. [Figure 8A] ~ [Figure 8E]Optimization of parameters for the NemaLife Machine protocol. (Figure 8A) Number of offspring remaining in microfluidic device I as a function of pump flush flow rate. (Figure 8B) Lifespan of wild-type (N2 isolate) and IG274 strain induced on the epidermis with nlp-29p::GFP reporter at 20°C with a pump flow of 7.5 mL / min. (Figure 8C) Lifespan of wild-type (N2 isolate) C. elegans fed once daily with four different diets (bacterial concentrations) at 20°C. (Figure 8D) Lifespan of wild-type (N2 isolate) C. elegans with once daily (24-hour intervals) and twice daily (12-hour intervals) feeding at 20°C. P-value (12-hour intervals vs. 24-hour intervals) = 0.92. (Figure 8E) Batch-to-batch variability of lifespan of wild-type (N2 isolate) C. elegans in the NemaLife system for 20.54 ≤ n ≤ 67. [Figure 9] This report presents the evaluation of mutant lifespans in the NemaLife Machine. Lifespans of mutants age-1 (TJ1052), eat-2 (DA1113), daf-16 (GR1307), and skn-1 (EU35) are compared to the 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 lifespans for each of the studied strains. [Figure 10A] ~ [Figure 10C] This shows the assessment of the healthy lifespan 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. (Figure 10A) Data is shown for video segments from 0 to 10 seconds, (Figure 10B) 50 to 60 seconds, and (Figure 10C) 80 to 90 seconds. [Figure 11] This study demonstrates the determination of offspring production from animals cultured in the NemaLife Machine. Offspring were counted by collecting the washing fluid in two devices over a four-day period and compared with data from animals cultured on agar plates. [Modes for carrying out the invention]

[0019] While various embodiments of the present invention will be discussed in detail below, it should be understood that the present 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 methods of constructing and using the present invention and do not limit the scope of the invention.

[0020] To facilitate understanding of the present invention, several terms are defined below. The terms used herein have meanings that would be commonly understood by those skilled in the art in the field relating to the present invention. Terms such as "a," "an," and "the" are not intended to refer only to singular entities, but include a general category for which specific examples may be used as illustrations. The terms used herein are used to describe specific embodiments of the present invention, but their use is not intended to limit the present invention except as outlined in the claims.

[0021] Currently, nematode research involves the cumbersome collection and transfer of animals from culture plates to study them throughout their life cycle. Exposing animals to different environments over time is also difficult. Scoring is done manually, which can introduce bias. The integrated system and microfluidic culture apparatus of the present invention solve 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 studies. Standard longevity assays on agar plates involve the cumbersome task of collecting and transferring animals to prevent young offspring from contaminating an age-synchronized adult population. Large-scale studies avoid offspring contamination using offspring-blocking agents or sterile mutants, but such manipulation alters the physiological function of adults and changes the reproductive impact on normal aging. Furthermore, some agar growth-based technology platforms, such as automated longevity machines, do not allow for the easy addition / removal of reagents such as food or drugs after the start of the study. We have developed an automated microfluidic system called the NemaLife Machine (NLM) to address the current limitations of plate-based aging assays. The NLM device integrates (1) a microfluidic apparatus and flow control system for cultivating C. elegans by programming the washing of offspring and the delivery of food, (2) a lighting and smart device imaging system for recording the movement of animal or individual populations, and (3) data analysis software for scoring living / dead animals, their behavior and their mobility. The machine has a footprint of 1 ft 2 It is compact, easily integrates with other microfluidic devices, and can be operated via an onboard interactive display. The inventors evaluated various system parameters and developed an operational workflow that reliably generates lifespan and healthy lifespan data for C. elegans. Furthermore, the device has been validated in classical aging variant and diet-restricted longevity studies. Overall, the NLM system's ability to produce reliable lifespan and physiological data highlights the potential of this automated machine for gene and drug screening, and for fundamental investigations into lifespan / healthy lifespan in C. elegans.

[0023] The inventors have now developed a novel technology called the NemaLife Machine (NLM) for aging research in C. elegans, addressing the limitations of agar-based research and current microfluidic systems. As shown in Figure 1, the apparatus of the present invention is a compact benchtop integrated platform incorporating all the functionality required for lifelong research of C. elegans. The main components of the NemaLife unit are (i) housing for fluid and electrical modules, (ii) iPOD holder, (iii) iPOD for imaging, (iv) ) is a slot for a microfluidic device. The microfluidic device is illuminated from the side and has (v) a user interface display for controlling the unit, and (vi) a fluid reservoir for sterilizing the unit and washing / feeding animals. At the heart of the NLM is an optimized micropillar arena, which also functions as a screen to hold adults and prevent fluid-induced damage when removing offspring, while allowing animals to employ a crawling progression similar to worms on an agar plate. This microfluidic device is connected to a programmable pump that washes offspring and delivers food according to a user-defined sequence. LED-based lighting and an iPod® smart device are integrated into the system to capture video of animals crawling inside the micropillar arena. Custom described MATLAB software will enable users to analyze images and score them based on whether the animals are alive / dead and their 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 apparatus for lifelong culture and observation. At the core of NLM is a microfluidic apparatus that enables culture and lifelong observation. Here, the inventors have developed two PDMS-based microfluidic apparatuses. Apparatus I enables lifelong study of animal populations (50-100 animals per chamber), while Apparatus II enables lifelong study of individual animals. The designs of the two apparatuses are discussed below.

[0025] Apparatus I. As shown in Figures 2A and 2B, the microfluidic apparatus 20 includes a large rectangular micropillar arena 22, a flow distribution zone 24, and sieve channels 28a, b, and c. Animals are introduced into the apparatus through an animal loading port 30, and the animals are washed and fed through inlet ports 32 and outlet ports 34. The arena contains an array of micropillars arranged as a square grid. The sieve channels allow for the washing away of juveniles and eggs, but retain adult populations (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 and a height of 100 μm, with a distance of 160 μm between the centers of the pillars (or a gap of 90 μm between the pillars). The sieve channel is 1 mm long and 100 μm wide, separated by a gap of 35 μm. The flow distribution zone includes a triangular section with two sides having a length of 12.2 mm and a third side having 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] Apparatus II. As shown in Figure 3A, this microfluidic apparatus includes a series of micropillar chambers capable of accommodating individuals. The apparatus design includes inlet / outlet ports and side channels for purging air. Animals flow through distribution channels, with some animals bypassing the distribution channels and entering the micropillar chambers (Figure 3B, top). The inlet of the micropillar chamber has a tapered neck that allows for selection and capture based on the size of a single animal (Figure 3B, bottom left), followed by forced injection into the chamber (Figure 3B, bottom right).

[0028] The experimental protocol for capturing individuals within the chamber is based on a unique loading method. As shown in Figure 3C, using continuous aspiration, the tube is filled with growth medium in the front (200 μL) and rear (600–800 μL) sections, with animal solution (20–200 μL) scattered throughout. In the first step, the cartridge is injected by a pump at a low flow rate of 5 mL / hour, allowing animals to be captured at the neck of the micropillar chamber (Figure 3D), while offspring are washed away through the sieve channel. In the second step, the captured animals are pushed into the chamber using a high flow rate of 25 mL / hour. The cartridge system also helps to prevent excessive intrusion of animals into the chamber.

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

[0030] Description of the NemaLife machine. The NLM includes a fluid, imaging, and microcontroller system integrated into a compact benchtop unit as shown in Figure 4A. This unit includes two rectangular boxes. The top cover plate of the larger box (Figure 4B) houses slots for holding 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 the optimal focal plane for acquiring animal video in a micropillar arena. Video acquisition was typically performed at 10-30 fps with a resolution of 1920 x 1080 pixels and a field of view of 54 x 30 mm.

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

[0032] Workflow. The workflow for performing an aging experiment in NLM, as shown in Figure 6, involves the following steps: (i) Sterilize the microfluidic apparatus with 70% ethanol, followed by rinsing with DI water. (ii) Load animals from age-synchronized culture plates into microfluidic apparatus I and II. The age of the animals is typically L4 - juvenile adult. (iii) 3.5 per tip Washing and feeding are performed daily on the NLM for 60-120 seconds at a washing flow rate of ~7.5 mL / hour. Feeding is performed for 2-5 seconds at a similar flow rate range. Images are taken both during washing and at the end of the feeding cycle. Video is acquired for 30-90 seconds at a frame rate of 10-30 fps. The video is saved on an iPod or in the cloud and retrieved later for image processing and data analysis.

[0033] Data analysis software. NemaCode is a major player in "worm-movies". This is a GUI-driven application for automating the detection of C-elements 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 invoked by "views" (UI based on MATLAB's App Designer). Each model function is delegated an independent task by the GUI to either data processing (upload / export), analysis, or validation of results 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) life / dead calculation, and 5) movement calculation.

[0035] In step (1), automatic ROI (Region of Interest) trimming with alignment correction is performed. The auto cropping function proceeds by applying an intermediate intensity filter to reduce noise and fits a rectangular pulse function to the image intensity at three vertical positions. The midpoint of the rise region of the fit is the peak and The position of the bottom wall is provided. After fitting, the wall inclination is calculated from the vertical range, 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 the ROI is trimmed based on the fact that the side walls, top, and bottom of the device are known (see the blue dashed line in Figure 7B, i). Step (1) reduces the image size to the ROI, accelerating the subsequent analysis.

[0036] In step (2), the program performs object detection on the cropped image by intensity-based discrimination and multiscale 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 extension, and control The last is increased to adjust the lighting. Filter parameters are set by an external user via a GUI, depending on the lighting level and pixel resolution. Next, the study by Frangi et al. Using multiscale feature detection based on (optional) the model, we highlight worm-like structures with diverse width and length scales (see Figure 7B, ii).

[0037] Next, the contrast image is binarized by a thresholding function based on an adaptive threshold calculated based on local primary statistics and image size, so that objects are assigned a value of 1 while the background is assigned 0 (see Figure 7B, iii). In addition, the user is provided with options for morphological expansion / erosion of objects, and the ability to impose raw cutoffs to minimum and maximum object sizes (total number of pixels constituting an object), regardless of the object's morphology. The user is provided with visual readings in the GUI 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 worm from other objects that could not be separated based on the intensity-based approach in step (2). MATLAB's regionprops function is used for the object The length (head-to-tail distance in pixels), area (total pixels), width (area / perimeter), and three-dimensionality (area / (convex area)) are estimated as object metrics using the property function (Figure 7B, iv). The 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 GUI with visual readings to verify the output of step (3) by graphing the various worm metrics and showing an annotated image of the ROI (see Figure 7B, iv). Step (3) thus 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] Following the final morphological detection in step (3), the live / dead calculation is performed in step (4) by estimating the degree of motion for each worm in two frames using a live / dead counterfunction. First, the user selects and skips an appropriate number of frames to arrive at consecutive frames 1 and 2, so that the worm's motion can be properly captured and represented. Next, the change in the same image region (same coordinates) in frame 2 is evaluated using the position of the worm's body as an image region (coordinates of the binarized object) in frame 1. This change is calculated as Δ = TotalPixels_Region1_Frame1 - TotalPixels_Region1_Frame2. Using a sensitivity criterion of the form number_of_pixels, each worm is deemed live if the change in pixels is Δ > number_of_pixels. Classify as alive. The number of frames to skip and the sensitivity criteria depend on the acquisition frame rate and image resolution. Calculate the survival / death status for each worm by repeating the survival / death calculation across all consecutive selected frames (with skips). The results are compiled into a table recording the worm's size, physical characteristics, and frame-by-frame measurements of survival / death determination. In addition, the user is presented with graphs showing the number of live worms, the total number of worms, and other statistics. The GUI also provides a visual reading tool for the user to scroll through processed frames to visualize the assay results, and worm movies are displayed with blue and red markers superimposed on the worm's center of gravity to indicate the live versus dead status (Figure 7B, v).

[0040] In step (5), the movement is calculated by tracking the worm. Worm tracking is performed by skipping frames based on user input so that at least one worm length of movement is possible in consecutive frames, e.g., frame 1 and frame 2. A circular neighborhood (N) is then assigned around the centroid of each worm in frame 1 such that the radius Rn is equal to the maximum physiologically possible worm movement during the period of the skipped frames. Next, the worms in the neighborhood region from frame 1 are superimposed onto 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. The worm velocity is estimated given the pixel displacement of the centroid between neighbors, the video acquisition rate, and the number of skipped frames. In the case of competition due to the collocation of multiple neighbors, the inventors calculated a similarity index based on a least-squares estimation of the worm shape metric (from step (3)) between the parent worm from frame 1 and the possible neighbors in frame 2. The minimum similarity index is assumed to be the most similar, and therefore most likely, neighbor. The worm's movement calculation is implemented similarly across multiple frames to generate a Lagrangian track.

[0041] These steps (1-5) together integrate the analysis as a seamless and scalable process, from preprocessing uploaded raw data to estimating survival and movement. In implementing the analysis algorithm, the inventors 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] The inventors first evaluated the optimal flow rate of a pump for washing offspring from a microfluidic device. They tested flow rates of 3.5, 4.5, and 7.5 mL / min per tip with a washing time of 90 seconds. The results shown in Figure 8A indicate that 7.5 mL / min is the optimal flow rate for removing offspring. The inventors also evaluated whether this flow rate causes cuticle damage to worms by using strain IG274, which has the reporter nlp-29p::GFP in its epidermis. As shown in Figure 8B, the inventors observed no difference in the lifespan of this strain compared to the wild type, which indicates that a flow rate of 7.5 mL / min does not cause cuticle damage.

[0043] Next, the inventors varied the feeding dose from 0.1 to 100 mg / mL per day. The lifespan curve shown in Figure 8C indicates that 0.1 mg / mL was too low for animal survival, while 1 mg / mL extended lifespan. However, similar lifespan data was obtained at 10 to 100 mg / mL. Therefore, a food administration of 10 to 100 mg / mL is the optimal food concentration for achieving the lifespan data in C. elegans. The inventors also tested whether feeding frequency made a difference. As shown in Figure 8D, feeding at 100 mg / mL once or twice a day made no difference in the lifespan curve. In Figure 8E, the inventors demonstrate reproducibility in the lifespan curve by overlaying data sets from nine independent studies. The inventors found that the variability of the median and maximum lifespan was 5.3% and 7.4%, respectively.

[0044] To further test the entire system, the inventors ran mutants that had been well-characterized for their longevity (Figure 9). As expected, the inventors found that age-1, eat-2, and skn-1 showed extended lifespan, while daf-16 showed shortened lifespan. .

[0045] Lifespan assessment. Video recording of animals in a micropillar arena using an iPod provides the ability to assess the healthy lifespan of animals using measures of movement and regeneration. The inventors 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. The inventors performed analysis of these groups by tracking the movement in the first 0-10 seconds, 50-60 seconds, and 80-90 seconds of the acquired video. The inventors observed that the proportion of animals exhibiting high and medium mobility decreased with age. The onset of the decrease in mobility was rapid after 7 days.

[0046] A unique feature of the NemaLife Machine is its ability to collect fluid from the nematode arena during the washing step. As a result, offspring can be collected and used to evaluate reproductive fitness throughout the lifespan of different strains. The inventors collected the washed fluid and counted the number of offspring per animal. The data in Figure 11 shows that the number of offspring from animals housed in the NemaLife Machine is greater than that from animals housed on standard agar plates. This result indicates that the animals are healthy in the microfluidic environment and that the collected washed fluid can be used to determine the reproductive capacity of an animal population.

[0047] The inventors have successfully demonstrated that *C. elegans* can be effectively maintained throughout its lifespan in a NemaLife microfluidic apparatus without the use of chemicals (such as progeny blockers, antibacterial agents, or antifungal compounds) in an environment that replicates longevity on an agar plate. Micropillars in the microfluidic apparatus allow animals to maintain their natural crawling progression, eliminating stress such as fatigue induced by swimming. Both individual and population crawling animals can be studied throughout their lifespan. The main advantages of the NemaLife Machine are: • Lifetime culture without animal transfer • The stress-free behavior of plate-shaped animals that swim • Automated scoring of animal activity and survival • Throughput: 24 assays / hour / user / machine • Collect offspring for downstream assays • Video archives for retrospective analysis That is the case.

[0048] The advantages offered by the NemaLife Machine are crucial in a wide range of applications, including drug screening, toxicological testing, genetic screening, behavioral phenotypic analysis, and disease investigation.

[0049] Worm culture. All animals were cultured on 60 mm Petri dishes containing Nematode Growth Medium (NGM) at 20°C and then loaded into a microfluidic chamber. 300-400 μL of bacteria, Escherichia coli OP50, was seeded into Petri dishes filled with NGM 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 containing the eggs were incubated for 60-72 hours. The egg-laying day was counted as day 0.

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

[0051] Food preparation. E. coli OP50 was used as a bacterial food source for worms grown on NGM culture medium and maintained in the apparatus. Unless otherwise noted, approximately 10 9 A 100 mg / mL bacterial suspension in liquid NGM solution corresponding to the bacteria / mL was used in the lifetime assay. E. coli OP50 was grown overnight in standard LB medium at 37°C. The 100 mg / mL bacterial suspension was prepared by centrifugation of 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] Any embodiment discussed herein can be implemented with respect to any method, kit, reagent, or composition of the present invention, and vice versa. Furthermore, the methods of the present invention can be achieved using the compositions of the present invention.

[0053] It will be understood that the specific embodiments described herein are presented as examples, not as limitations of the invention. The main features of the invention can be used in various embodiments without departing from the scope of the invention. Those skilled in the art will be able to recognize or confirm numerous equivalents to the specific procedures described herein by means of simple routine experimentation. Such equivalents are considered to be within the scope of the invention and are covered by the claims.

[0054] All publications and patent applications described herein represent the level of skill of those skilled in the art to which the present invention relates. All publications and patent applications are incorporated herein by reference to the same extent as each individual publication or patent application is specifically and individually indicated as being incorporated by reference.

[0055] The use of the words "a" or "an" in the claims and / or specification in conjunction with the term "comprising" may mean "one," which also coincides with the meanings of "one or more," "at least one," and "one or more than one." The use of the term "or" in the claims is used to mean "and / or" unless explicitly indicated to refer only to substitutes, or unless these substitutes are mutually exclusive, although this disclosure supports a definition that refers only to substitutes and "and / or." Throughout this application, the term "about" means that a certain value is used by this method to determine that value. It is used to indicate that the variation includes inherent errors in the device or variations that exist between the subjects of study.

[0056] When used herein and in the claims, “comprising” (and any form of “comprising,” such as “comprise” and “comprises”), “having” (and any form of “having,” such as “have” and “has”), “including” (and any form of “including,” such as “includes” and “include”), or “containing” (and any form of “containing,” such as “contains” and “contain”) are inclusive, or open-ended, and do not exclude additional, undescribed elements or method steps. In any embodiment of the components and methods provided herein, “comprising” may be replaced with “consisting essentially of” or “consisting of.” When used herein, the phrase “consisting essentially of” includes a specified integer or step, and also includes a particular It is required that the modifications do not substantially affect the features or functions of the claimed invention. As used herein, the term "consisting" refers to the described whole (integer) (e.g., features, elements, characteristics, methods / process steps or limitations). (definite) or complete (integer) (e.g., feature, element, characteristic, property, method / process step) It is used to indicate the existence of only the group (or limited group).

[0057] As used herein, the term “or any combination thereof” refers to all permutations and combinations of the enumerated items preceding the term. For example, “A, B, C, or any combination thereof” is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB, where the order is important in the particular context. Continuing this example, combinations containing repetitions of one or more items or terms are explicitly included, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, etc. A person skilled in the art will understand that, unless otherwise evident from the context, there is generally no limit to the number of items or terms in any combination.

[0058] When used herein, approximate terms such as “about,” “substantial,” or “substantially” refer to a state that, when modified in this way, is understood not necessarily absolute or complete, but which would be considered to be close enough to a person skilled in the art to guarantee that the state exists. The degree to which the description may vary will depend on how large the change is, yet still enough to allow a person skilled in the art to recognize the modified feature as still possessing the required characteristics and capabilities of the unmodified feature. Generally, but subject to the preceding discussion, numerical values ​​in this specification modified by approximate terms such as “about” may vary by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12, or 15% from the stated value.

[0059] All of the structures and / or methods disclosed and claimed herein can be fabricated and performed without undue experimentation in light of this disclosure. While the structures and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that modifications can be applied to the structures and / or methods described herein, and to the steps or sequences of steps of the methods, without departing from the concept, spirit and scope of the invention. All such similar alternatives and modifications that are 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 issued in connection with this application in interpreting the claims attached herein, the applicant would like to note that, as of the filing date of this application, none of the attached claims are intended to apply paragraph 6 of paragraph (f) of § 112 of the United States Patent Act, unless the words “means for” or “steps for” are expressly used in a particular claim.

[0061] For each claim, each dependent claim may be dependent on both the independent claim and each of the preceding dependent claims for each of the claims, insofar as the preceding claim provides an appropriate antecedent for the terms or elements of the claim.

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Claims

1. A system for analyzing nematodes, A culture medium reservoir and a culture medium pump that is in fluid communication with the culture medium reservoir; A food reservoir and a food pump that is in fluid communication with the food reservoir; An inlet port that communicates fluid with the culture medium pump and the food pump; A microfluidic device that communicates fluidly with the aforementioned inlet port, (1) A micropillar arena for housing the nematodes, comprising a plurality of micropillars that allow the nematodes to crawl therein, wherein the microfluidic device further comprises a first plurality of sieve channels between the inlet port and the micropillar arena and a second plurality of sieve channels between the micropillar arena and the outlet port, wherein the first and second plurality of sieve channels are configured to allow the passage of progeny nematodes and eggs, but not the passage of adult nematodes; and (2) A plurality of micropillar chambers, each configured to allow one nematode to crawl through; and At least a transparent outer surface for illumination or imaging; The microfluidic apparatus comprising: An outlet port that communicates fluid with the aforementioned microfluidic device; A light source positioned outside the micropillar arena to illuminate the interior of the micropillar arena; An imager positioned outside the micropillar arena to image the inside of the micropillar arena; and The culture medium pump, the food pump, the microfluidic device, the light source, and the controller connected to the imager; A system that includes these features.

2. The aforementioned microfluidic device An input flow distribution region that communicates with the aforementioned inlet port; A micropillar arena having fluid communication with the input flow distribution region, wherein the plurality of micropillars are distributed in a pattern configured to allow the nematodes to crawl in the space between the micropillars and to retain adult nematodes while removing progeny nematodes; and An outlet flow distribution region that is in fluid communication with the micropillar arena that is in fluid communication with the outlet port; or A distribution channel for introducing the nematodes, comprising an inlet end and an outlet end, wherein the inlet end is in fluid communication with the inlet port, the outlet end is in fluid communication with the outlet port, and the plurality of micropillar chambers are distributed along the distribution channel in fluid communication with it, and each micropillar chamber is provided with a tapered neck for selecting and capturing one nematode in each micropillar chamber based on size at a sufficiently high fluid flow rate, the distribution channel The system according to claim 1, comprising:

3. The system according to claim 2, wherein each of the micropillars has a cross-section including a circular, elliptical, square, rectangular, or other polygonal cross-section, or some combination of these shapes.

4. The system according to claim 1, further comprising analyzing at least one of the following: recording the movement of the nematodes, counting the living and dead nematodes, studying the behavior of the nematodes, or studying the mobility of the nematodes.

5. The system according to claim 1, wherein the nematode is at least a nematode of the genus Caenorhabditis.

6. The system according to claim 1, wherein the light source comprises a light-emitting diode and the imager is a digital imager.

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