. _:b38007552 . _:b572360846 . _:b572360854 . _:b572360890 . _:b38007553 . _:b572360875 . . . . _:b572360878 . _:b572360887 . . _:b572360853 . _:b572360976 . _:b572360852 . . _:b572360855 . _:b572360912 . . . _:b572360854 . _:b572360849 . . _:b38007536 "Together with methodology for low-input RNA-seq, MRAs potentiate screening of genetic mutants and drugs/small molecules at the genomic level >>32<<." . _:b572360848 . _:b38007501 . _:b572360851 . . _:b38007500 . _:b572360850 . _:b572360848 . _:b572360861 . _:b38007502 . _:b572360860 . _:b572360863 . _:b572360862 . . . . _:b572360857 . _:b572360908 . _:b38007551 "Epithelial cells were isolated from whole murine intestines, as previously described, with some modifications >>48<<. Briefly, intestines were opened longitudinally, rinsed in DPBS (Life Technologies, Grand Island, NY), minced, and incubated in 3mM EDTA (Sigma, St Louis, MO) in DPBS for 45min at 4\u00B0C with gentle agitation." . _:b572360927 . . _:b572360856 . _:b38007503 "Additionally, the field currently lacks a robust assay to study ISCs at the clonal level, a tool that has driven the understanding of stem cell niches in the hematopoietic system and mammary glands >>9<<, 10. Array-based technologies are emerging as a powerful method to study the functional characteristics of single and/or small numbers of stem cells in the hematopoietic system, and hold similar promise for epithelial tissues such as the" . . _:b572360859 . _:b572360858 . _:b38007500 "In vivo, ISCs are closely associated with Paneth cells (PCs), which function as niche cells and express soluble and insoluble factors associated with stemness, such as Wnt and Notch ligands 5, >>6<<. PCs have been shown to increase the efficiency of enteroid formation by ISCs in vitro 6. However, these studies relied on the co-culture of hundreds of ISCs with hundreds of PCs, and may not reflect physiological normal conditions in" . _:b572360869 . . _:b38007510 . . _:b572360868 . _:b38007497 "introduction" . _:b572360856 . . . _:b572360871 . _:b572360870 . _:b38007527 . "10.1038%2Fncb3104" . _:b38007515 . _:b572360865 . . . . . _:b572360864 . _:b572360867 . _:b38007540 _:b38007552 . _:b38007540 _:b38007553 . _:b38007509 . _:b572360880 . _:b572360866 . . _:b572360961 . _:b572360877 . . _:b572360945 . _:b572360876 . . _:b572360879 . _:b572360879 . _:b572360878 . _:b572360844 . _:b38007523 . _:b572360873 . _:b572360895 . . _:b38007522 . _:b572360872 . _:b572360875 . _:b38007530 "are enriched for the expression of transcripts associated with absorptive enterocytes (Sis), goblet cells (Muc2), PCs (Lyz, Defr-rs1), and endocrine cells (Chga, Chgb), consistent with a fully developed organoid phenotype (Fig. 6B,C) >>4<<." . _:b38007553 . . . _:b38007524 . _:b572360999 . _:b572360874 . . _:b572360978 . _:b572360903 . _:b572360885 . _:b572360963 . . _:b572360884 . _:b572360954 . . . _:b572360887 . _:b38007541 . _:b572360886 . _:b572360937 . _:b572360881 . . . . _:b572360962 . _:b572360880 . . . _:b572360883 . . _:b38007532 . _:b572360882 . _:b38007535 . _:b572360893 . . _:b38007534 . _:b572360892 . . . _:b38007528 "Early expression of Sis appears to be coincident with upregulation Hes1, a known driver of enterocyte fate, while Muc2 and Chga are upregulated at later time points, coincident with increased Atoh1 expression >>29<<, 30. By 10 days in culture, enteroids are enriched for the expression of transcripts associated with absorptive enterocytes (Sis), goblet cells (Muc2), PCs (Lyz, Defr-rs1), and endocrine cells (Chga, Chgb), consistent with a fully" . _:b38007526 . _:b572360895 . _:b572360957 . _:b572360975 . _:b572360894 . . _:b572360889 . _:b572360888 . _:b572360858 . _:b572360891 . . _:b572360890 . _:b572360864 . _:b572360901 . _:b572360900 . _:b572360881 . _:b572360899 . _:b38007545 . _:b572360903 . _:b572360902 . _:b572360966 . _:b572360897 . _:b572360896 . _:b572360899 . . _:b38007536 . _:b572360898 . _:b38007520 . _:b38007529 . . _:b572360909 . _:b38007521 . . _:b572360908 . _:b572360913 . . . _:b572360911 . _:b572360910 . . _:b572360905 . _:b38007519 "and large cell size (Fig. 3A). Importantly, all isolated cells expressed the PC-related gene Defcr-rs (Fig. 3B,C). Lgr5 was detected in some cells, consistent with recent reports on Lgr5 expression in a subset of PCs in vivo (Fig. 3C) >>19<<. Similarly, while all cells were negative for the enteroendocrine transcript Tac1, expression of Chga was observed in a single PC, consistent with reports of PC progenitors expressing this enteroendocrine marker (Fig." . . _:b572360904 . _:b572360888 . _:b572360907 . _:b572360876 . _:b572360938 . _:b572360946 . _:b572360906 . _:b38007526 "crypt-base columnar ISC markers Olfm4 and Smoc2 were strongly detected in single ISCs, a number of cells were negative for putative \u201C+4\u201D ISC markers Bmi1, Hopx, and Tert, contrary to studies conducted on populations of Lgr5high cells >>23<<-26. To test if this finding was reflective of a transcriptional response to ISC culture conditions, we compared gene expression profiles of single Lgr5high cells sorted directly into lysis buffer with those exposed to Matrigel culture" . _:b572360917 . _:b38007502 . . _:b572360916 . _:b572360919 . _:b572360988 . _:b572360918 . _:b38007533 . _:b572360913 . . _:b572360983 . _:b38007528 . _:b572360912 . _:b572360973 . _:b572360915 . . _:b572360847 . _:b572360914 . _:b38007498 "in vivo lineage tracing remains an important technique for the analysis of stem cell behavior, developments in primary stem cell culture have expanded the stem cell biologist's toolkit to include powerful, complementary in vitro assays >>1<<." . _:b572360925 . _:b572360884 . _:b572360924 . . . _:b572360861 . _:b572360927 . . _:b572360926 . . _:b572360933 . _:b572360921 . _:b38007497 _:b38007498 . _:b38007497 _:b38007499 . _:b38007497 _:b38007500 . _:b38007497 _:b38007501 . _:b572360920 . _:b38007497 _:b38007502 . . _:b38007497 _:b38007503 . _:b38007497 _:b38007504 . _:b38007497 _:b38007505 . _:b572360923 . _:b572360860 . _:b38007519 . _:b572360922 . _:b572360985 . _:b38007523 "Previous studies have suggested that cell-to-cell contact between ISCs and PCs may influence enteroid formation, but this has not been formally tested by comparison between touching and non-touching ISCs and PCs >>6<<. Using the same data generated in our ISC-PC co-culture experiments, we reanalyzed initial MRA contents to classify microwells by cell-to-cell contacts at t=0hr and correlated this status with enteroid formation outcome (Fig." . _:b572360956 . _:b572360933 . . _:b38007534 "PCs express soluble and insoluble ISC niche signaling components, including Wnt and Notch ligands 5, >>6<<. In the present study, we examine the impact of PC presence and contact with ISCs on enteroid formation." . _:b572360932 . . _:b38007520 "Similarly, while all cells were negative for the enteroendocrine transcript Tac1, expression of Chga was observed in a single PC, consistent with reports of PC progenitors expressing this enteroendocrine marker (Fig. 3C) >>19<<." . _:b572360935 . . _:b572360934 . _:b572360902 . _:b38007521 "Emerging evidence demonstrates that multiple intestinal progenitor cell populations are capable of dedifferentiating and functioning as ISCs in vitro and in vivo >>19<<-21. To address the possibility that enteroids might form from early progenitors in the PC population, 2,810 individual PCs were examined in subsequent in vitro experiments." . _:b38007541 "Sox9EGFP transgenic mice were originally generated by the GENSAT Brain Atlas Project, and are maintained on an outbred CD-1 background >>39<<. Lgr5EGFP-CreERT2 mice were obtained from Jackson Labs (stock number: 008875, Jackson Laboratory, Bar Harbor, ME) 40." . _:b572360929 . _:b572360851 . _:b572360928 . _:b572360931 . . . _:b572360844 "9"^^ . _:b572360847 "5"^^ . _:b572360845 "6"^^ . . _:b572360930 . _:b572360846 "5"^^ . . _:b572360986 . _:b572360941 . _:b572360940 . . _:b572360943 . _:b38007544 . _:b38007506 _:b38007528 . . _:b572360939 . _:b38007506 _:b38007529 . _:b38007506 _:b38007530 . _:b572360942 . _:b572360891 . _:b38007506 _:b38007524 . . _:b38007506 _:b38007525 . _:b38007506 _:b38007526 . _:b572360989 . _:b572360937 . _:b572360853 "4"^^ . _:b38007506 _:b38007527 . _:b38007506 _:b38007520 . _:b572360868 . _:b38007506 _:b38007521 . _:b38007506 _:b38007522 . _:b572360936 . _:b572360852 "4"^^ . _:b38007506 _:b38007523 . _:b38007540 "experimental procedures" . _:b572360939 . _:b572360855 "4"^^ . . _:b572360938 . _:b572360854 "4"^^ . _:b572360932 . _:b572360914 . _:b572360949 . _:b572360849 "4"^^ . _:b572360948 . _:b572360848 "4"^^ . . _:b572360951 . _:b572360851 "4"^^ . _:b572360950 . . _:b38007550 . _:b572360850 "4"^^ . . _:b572360861 "3"^^ . _:b572360945 . _:b572360982 . _:b572360866 . _:b572360860 "3"^^ . _:b572360944 . _:b572360916 . _:b572360917 . _:b38007506 _:b38007516 . . _:b572360918 . _:b38007506 _:b38007517 . _:b572360919 . _:b572360952 . _:b572360947 . _:b38007506 _:b38007518 . _:b572360863 "3"^^ . _:b572360912 . _:b38007506 _:b38007519 . _:b572360913 . _:b38007506 _:b38007512 . _:b572360914 . _:b38007532 "As an alternative approach, in vitro techniques that rely on the co-culture of isolated stem and niche cell populations have recently been used to assess the impact of individual niche components on stem cell behavior >>6<<, 31. However, these methods commonly rely on large numbers of cells, which may not reflect physiologically relevant niche behavior, and are not amenable to high-throughput studies. Here, we present an array-based platform that facilitates" . _:b38007506 _:b38007513 . _:b572360907 . _:b572360915 . _:b572360946 . _:b38007506 _:b38007514 . _:b572360862 "3"^^ . _:b572360924 . _:b38007503 . _:b38007506 _:b38007515 . _:b572360925 . _:b38007506 _:b38007508 . _:b572360926 . _:b38007506 _:b38007509 . _:b572360927 . _:b572360957 . _:b38007506 _:b38007510 . . _:b38007506 _:b38007511 . _:b572360857 "4"^^ . _:b572360920 . _:b572360921 . _:b572360871 . _:b572360922 . _:b572361001 . . _:b572360923 . _:b572360956 . _:b572360900 . _:b38007506 _:b38007507 . _:b572360856 "4"^^ . _:b572360901 . _:b572360902 . _:b572360903 . _:b572360959 . _:b572360896 . _:b572360859 "4"^^ . _:b572360897 . _:b572360898 . _:b572360899 . _:b572360958 . _:b572360908 . _:b572360858 "4"^^ . _:b572360909 . _:b572360910 . _:b572360893 . _:b572361002 . _:b572360953 . _:b572360869 "3"^^ . _:b572360911 . _:b572360904 . _:b572360905 . . _:b572360906 . _:b38007515 "Previous studies have isolated PCs by FACS of CD24High:SSCHigh populations >>6<<. However, CD24 is also expressed on ISCs, TAs, and enteroendocrine cells 3, 14, 16-18." . _:b572360918 . _:b572360907 . _:b38007509 "To facilitate tracking of isolated cells in MRAs, Sox9EGFP mice were crossed to CAGDsRed mice, which express the DsRed fluorescent transgene ubiquitously across all cell and tissue types (Fig. 1D) 3, >>13<<. Sox9EGFPlow:DsRed+ ISCs were plated in a single culture chamber of a two-chamber MRA, randomly seeded into microwells by centrifugation, and overlaid with Matrigel and ISC-supporting growth factors (Fig." . _:b572360952 . _:b572360868 "3"^^ . _:b572360948 . _:b572360949 . _:b572360950 . _:b572360951 . _:b572360955 . _:b572360871 "3"^^ . _:b572360846 . _:b572360944 . _:b572360945 . _:b572360946 . _:b38007549 "The SU-8 master was coated by octyltrichlorosilane using vapor deposition to render the surface of the master non-sticky to PDMS >>46<<. Clean glass slides (75 mm \u00D7 50 mm \u00D7 1 mm) were spin coated with a 15-\u03BCm thick layer of poly(acrylic acid) (PAA) by using 50 wt% solution and a spin speed of 2000 rpm, followed by a 1 hr bake on a 100\u00B0C hotplate to remove the water. The" . _:b572360947 . _:b572360954 . _:b572360870 "3"^^ . _:b572360956 . . _:b572360957 . _:b572360958 . _:b572360959 . _:b572360865 "3"^^ . . _:b572360952 . _:b572360953 . _:b572360954 . _:b572360955 . _:b572360864 "3"^^ . _:b572360932 . _:b572360933 . _:b572360934 . _:b38007529 "Early expression of Sis appears to be coincident with upregulation Hes1, a known driver of enterocyte fate, while Muc2 and Chga are upregulated at later time points, coincident with increased Atoh1 expression 29, >>30<<. By 10 days in culture, enteroids are enriched for the expression of transcripts associated with absorptive enterocytes (Sis), goblet cells (Muc2), PCs (Lyz, Defr-rs1), and endocrine cells (Chga, Chgb), consistent with a fully developed" . _:b572360935 . _:b572360867 "3"^^ . _:b572360928 . _:b38007531 _:b38007532 . _:b572360929 . _:b572360865 . _:b38007531 _:b38007533 . _:b572360930 . _:b38007531 _:b38007534 . _:b572360866 "3"^^ . _:b572360931 . _:b38007531 _:b38007535 . _:b572360940 . _:b572360941 . _:b572360942 . _:b572360943 . _:b572360896 . _:b572360877 "3"^^ . _:b572360936 . _:b572360937 . _:b572361005 . . _:b572360938 . _:b572360939 . _:b572360953 . _:b572360876 "3"^^ . _:b572360852 . . _:b572360853 . _:b572360854 . _:b572360855 . _:b572360951 . _:b572360879 "3"^^ . _:b572360848 . _:b38007524 "Previous studies demonstrate significant enteroid movement and merger in vitro >>6<<. To further examine cell movement in the MRA platform, we performed time-lapse imaging of microwells." . _:b38007516 . _:b572360849 . _:b572360852 . _:b572360850 . _:b572360851 . _:b572360878 "3"^^ . _:b572360860 . _:b38007531 _:b38007536 . _:b572360861 . _:b38007531 _:b38007537 . _:b572360862 . _:b572360929 . _:b38007531 _:b38007538 . _:b572360873 "3"^^ . _:b572360863 . _:b38007531 _:b38007539 . _:b572360856 . _:b572360857 . _:b572360858 . _:b572360859 . _:b572360872 "3"^^ . _:b38007540 _:b38007541 . . _:b38007540 _:b38007542 . _:b572360875 "3"^^ . . _:b38007512 . _:b38007540 _:b38007543 . _:b572360874 "3"^^ . _:b572360844 . _:b38007540 _:b38007548 . _:b572360845 . _:b38007540 _:b38007549 . _:b572360885 "2"^^ . _:b572360846 . _:b38007540 _:b38007550 . _:b38007535 "These results support in vivo findings that stemness is most strongly correlated cells that exist in intimate contact with PCs, and provide insight to the functional role of PCs in maintaining stemness >>6<<. Importantly, the MRA platform was critical in testing dose and contact-dependency of PCs in a microscale format." . _:b572360980 . _:b572360847 . _:b38007540 _:b38007551 . . _:b38007540 _:b38007544 . _:b38007540 _:b38007545 . _:b572360884 "2"^^ . _:b38007540 _:b38007546 . _:b38007540 _:b38007547 . _:b572360884 . _:b572360862 . _:b572360885 . _:b38007543 "Lgr5EGFP-CreERT2 mice were genotyped by previously described PCR protocols >>40<<. All experiments were conducted on adult mice between 8 and 16 weeks of age." . _:b572360886 . _:b572360887 "2"^^ . _:b572360887 . _:b572360880 . _:b572360881 . . _:b572360886 "2"^^ . _:b572360882 . _:b572360883 . _:b572360892 . _:b38007511 . _:b572360893 . _:b572360894 . _:b572360895 . _:b572360881 "3"^^ . _:b572360888 . _:b572360889 . . _:b572360890 . _:b572360891 . _:b572360880 "3"^^ . _:b572360868 . _:b572360869 . _:b572360870 . . _:b572360871 . . _:b572360883 "3"^^ . _:b572360864 . _:b572360865 . _:b572360866 . _:b572360867 . _:b572360882 "3"^^ . _:b572360876 . _:b572360877 . . _:b572360878 . _:b572360893 "2"^^ . _:b572360879 . . _:b572360872 . _:b572360873 . _:b572360874 . _:b572361000 . _:b572360892 "2"^^ . _:b572360875 . _:b572360901 . _:b572360895 "2"^^ . _:b572360894 "2"^^ . . _:b572360889 "2"^^ . _:b572360974 . _:b572360886 . _:b572360888 "2"^^ . _:b572360906 . . _:b572360891 "2"^^ . _:b38007553 "FACS was conducted on a MoFloXDP (Beckman Coulter, Pasadena, CA) and Sox9EGFP and Lgr5EGFP cells were isolated as previously described 40, >>48<<, 49. Paneth cells were isolated by high expression of CD24, high side-scatter (SSC), and exclusion of CD45, with or without exclusion of Sox9EGFP (supplemental material, Fig." . _:b572360924 . . _:b572360890 "2"^^ . . _:b572360901 "2"^^ . . _:b572360900 "2"^^ . _:b572360903 "2"^^ . . _:b572360968 . . _:b572360902 "2"^^ . . _:b38007551 . _:b572360897 "2"^^ . . . _:b572360896 "2"^^ . . _:b572360899 "2"^^ . _:b572360964 . _:b572360934 . _:b572360872 . _:b572360898 "2"^^ . _:b572360984 . . _:b572360909 "2"^^ . . _:b572360930 . _:b572360908 "2"^^ . _:b572360911 "2"^^ . . . _:b572360910 "2"^^ . _:b38007500 . . _:b572360905 "2"^^ . . _:b38007501 . _:b572361006 . _:b572360904 "2"^^ . _:b38007502 . . _:b38007510 "tissue types (Fig. 1D) 3, 13. Sox9EGFPlow:DsRed+ ISCs were plated in a single culture chamber of a two-chamber MRA, randomly seeded into microwells by centrifugation, and overlaid with Matrigel and ISC-supporting growth factors (Fig. 1E) >>6<<, 14. This resulted in a random distribution of ISCs across MRAs, with microwells containing one, multiple, or no ISCs (Fig 1F-H)." . _:b572360907 "2"^^ . _:b38007497 . . _:b38007542 "Lgr5EGFP-CreERT2 mice were obtained from Jackson Labs (stock number: 008875, Jackson Laboratory, Bar Harbor, ME) >>40<<. Male and female mice were used. For microwell array experiments requiring constitutive expression of dsRed in isolated ISCs, heterozygous Sox9EGFP mice were bred to homozygous CAGdsRed mice to produce Sox9EGFP:CAGdsRed offspring" . _:b572360992 . _:b38007503 . _:b572360906 "2"^^ . _:b572360915 . _:b572360928 . _:b572360917 "2"^^ . _:b38007506 . _:b572360916 "2"^^ . _:b38007497 . _:b572360923 . _:b572360936 . _:b572360922 . _:b38007498 . _:b572360919 "2"^^ . . . _:b38007499 . _:b572360918 "2"^^ . . _:b572360920 . _:b38007508 . _:b572360913 "2"^^ . _:b38007504 . _:b38007509 . _:b572360912 "2"^^ . _:b572360940 . . _:b38007510 . _:b572360915 "2"^^ . _:b38007537 "Array-based stem cell culture platforms are growing in use and present an efficient and cost-effective alternative to conventional cell culture >>33<<, 34. However, most platforms are not amenable to long-term cultures, such as required for the development of ISC-derived enteroids and other self-assembled, stem cell derived organoids 35-38." . _:b38007531 . _:b38007540 . _:b38007511 . _:b572360853 . _:b572360914 "2"^^ . . . _:b572360883 . _:b38007504 . _:b572360925 "2"^^ . _:b38007505 . _:b572360924 "2"^^ . . _:b38007537 . _:b38007539 . _:b572360927 "2"^^ . _:b38007506 . . _:b38007507 . . _:b572360926 "2"^^ . . _:b38007516 . _:b572360921 "2"^^ . _:b38007533 "As an alternative approach, in vitro techniques that rely on the co-culture of isolated stem and niche cell populations have recently been used to assess the impact of individual niche components on stem cell behavior 6, >>31<<. However, these methods commonly rely on large numbers of cells, which may not reflect physiologically relevant niche behavior, and are not amenable to high-throughput studies. Here, we present an array-based platform that facilitates the" . . _:b38007517 . _:b572360920 "2"^^ . _:b38007518 . _:b572360923 "2"^^ . _:b572360994 . . _:b38007519 . _:b572360922 "2"^^ . _:b572360993 . . _:b38007550 "This phenomenon is called discontinuous dewetting >>47<<. The array was then placed in a 95\u00B0C oven overnight to evaporate the GBL solvent, and pockets of polystyrene solution shrunk into solid polystyrene microrafts with a concave geometry (supplemental material, Fig." . _:b38007512 . _:b572360933 "2"^^ . _:b38007513 . _:b572360932 "2"^^ . _:b572360900 . _:b572360897 . _:b38007514 . _:b572360935 "2"^^ . _:b38007501 "PCs have been shown to increase the efficiency of enteroid formation by ISCs in vitro >>6<<. However, these studies relied on the co-culture of hundreds of ISCs with hundreds of PCs, and may not reflect physiological normal conditions in single intestinal crypts, where much smaller numbers of ISCs (\u223C15) and PCs (\u223C8) interact 6-8." . _:b38007515 . _:b572360934 "2"^^ . _:b38007524 . _:b572360929 "2"^^ . _:b38007525 . _:b572360928 "2"^^ . . _:b38007538 "Array-based stem cell culture platforms are growing in use and present an efficient and cost-effective alternative to conventional cell culture 33, >>34<<. However, most platforms are not amenable to long-term cultures, such as required for the development of ISC-derived enteroids and other self-assembled, stem cell derived organoids 35-38." . _:b38007513 "Conventional ISC cultures are capable of supporting enteroid growth for many weeks >>4<<. ISCs were maintained up to 8 weeks in MRAs, with retention of enteroids in their original microwells (Fig." . . _:b38007526 . . _:b572360931 "2"^^ . . . _:b572360925 . _:b38007527 . . _:b572360930 "2"^^ . . . . _:b38007520 . _:b572360941 "2"^^ . . . . _:b38007521 . _:b572360940 "2"^^ . . _:b38007517 . _:b572360869 . _:b38007522 . _:b572360943 "2"^^ . _:b38007527 "Subsequently, Dll1 expression initiates in a majority of enteroids at 48-hours, consistent with the emergence of secretory progenitor populations 21, >>28<<. Early expression of Sis appears to be coincident with upregulation Hes1, a known driver of enterocyte fate, while Muc2 and Chga are upregulated at later time points, coincident with increased Atoh1 expression 29, 30. By 10 days in" . _:b572360870 . _:b38007523 . _:b572360942 "2"^^ . _:b38007498 . _:b572360867 . _:b38007526 . . _:b38007532 . . _:b572360937 "2"^^ . _:b38007505 . _:b572360948 . _:b38007533 . _:b572360936 "2"^^ . _:b38007502 "However, these studies relied on the co-culture of hundreds of ISCs with hundreds of PCs, and may not reflect physiological normal conditions in single intestinal crypts, where much smaller numbers of ISCs (\u223C15) and PCs (\u223C8) interact >>6<<-8." . _:b38007505 "technologies are emerging as a powerful method to study the functional characteristics of single and/or small numbers of stem cells in the hematopoietic system, and hold similar promise for epithelial tissues such as the intestine >>11<<. In the present study, we describe a platform to study large numbers of single ISCs simultaneously, either at the clonal level or in the presence of niche cells." . _:b572360904 . _:b38007534 . . _:b572360969 . _:b572360939 "2"^^ . . _:b572360885 . _:b38007535 . _:b572360938 "2"^^ . . _:b38007528 . _:b572360949 "2"^^ . _:b38007529 . _:b38007545 "In ISC/PC co-culture experiments, growth factors, minus JAGGED-1 peptide and Y27632, were supplemented every two days, and media was changed every four days, as previously described >>42<<, 43." . . _:b572360948 "2"^^ . . _:b38007530 . _:b38007548 "An SU-8 master mold with an array of microposts was fabricated using standard photolithography with 100 \u03BCm thick SU-8 as described previously >>45<<. The SU-8 master was coated by octyltrichlorosilane using vapor deposition to render the surface of the master non-sticky to PDMS 46." . _:b572360951 "2"^^ . _:b38007507 "We hypothesized that previously described polydimethylsiloxane (PDMS)/polystyrene \u201Cmicroraft arrays\u201D (MRAs) could be utilized to isolate and culture single ISCs in three-dimensional ECM (Fig. 1A-C) >>12<<. Since ISCs require several days to develop into enteroids, MRAs had to be amenable to media changes 3, 4." . . _:b572360965 . _:b38007531 . _:b572360950 "2"^^ . _:b572360943 . _:b572360947 . _:b572360964 . _:b38007540 . _:b38007499 "Lgr5high or Sox9low ISCs are capable of forming \u201Centeroid\u201D structures in vitro, demonstrating multipotency and self-renewal >>2<<-4. In vivo, ISCs are closely associated with Paneth cells (PCs), which function as niche cells and express soluble and insoluble factors associated with stemness, such as Wnt and Notch ligands 5, 6." . _:b572360945 "2"^^ . . . _:b572360967 . _:b38007541 . _:b572360944 "2"^^ . _:b572360966 . _:b38007542 . . _:b572360971 . _:b572360947 "2"^^ . _:b572360873 . _:b38007543 . _:b38007522 "have speculated that PC-secreted WNTs are responsible for enhancing ISC growth in vitro, ISC-PC co-culture experiments were carried out in the absence of exogenous WNT, to avoid \u201Cmasking\u201D the potential impact of PCs on enteroid formation >>4<<-6. The GSK3\u03B2-inhibitor CHIR99021, a WNT agonist, was also excluded from co-culture experiments." . _:b572360991 . _:b572360961 . . _:b572360859 . _:b572360946 "2"^^ . _:b572360949 . _:b38007546 . . _:b38007536 . _:b572360960 . _:b572360957 "2"^^ . _:b572360963 . _:b38007552 "FACS was conducted on a MoFloXDP (Beckman Coulter, Pasadena, CA) and Sox9EGFP and Lgr5EGFP cells were isolated as previously described >>40<<, 48, 49. Paneth cells were isolated by high expression of CD24, high side-scatter (SSC), and exclusion of CD45, with or without exclusion of Sox9EGFP (supplemental material, Fig." . _:b38007537 . _:b572360981 . _:b572360956 "2"^^ . _:b572360962 . _:b38007538 . _:b572360959 "2"^^ . _:b572360973 . _:b38007539 . _:b572360958 "2"^^ . . _:b572360972 . _:b38007548 . _:b38007548 . _:b572361004 . _:b572360953 "2"^^ . . _:b572360975 . _:b38007549 . _:b572360952 "2"^^ . _:b572360974 . _:b38007550 . . _:b572360955 "2"^^ . _:b38007517 "However, CD24 is also expressed on ISCs, TAs, and enteroendocrine cells 3, 14, >>16<<-18. Since the experimental approach of MRA cultures examines events on a \u201Cper well\u201D basis, it was critical to refine isolation procedures for PCs to meet purity requirements of clonal and microscale co-cultures and avoid artifactual" . _:b572360931 . _:b38007551 . _:b572360969 . . _:b572360954 "2"^^ . _:b38007522 . _:b572360968 . _:b38007544 . _:b572360965 "2"^^ . _:b572360971 . _:b38007545 . _:b572360964 "2"^^ . _:b572360970 . _:b38007546 . . _:b572360967 "2"^^ . _:b572360981 . _:b38007547 . _:b38007512 "For biocompatibility experiments, we utilized high efficiency ISC culture methods that drive high Wnt and Notch signaling >>14<<. Tile scanning of the MRA in the dsRed wavelength immediately after plating and at 48hrs revealed that isolated ISCs had begun to produce primitive enteroids, indicative of biocompatibility (Fig. 1F-K)." . _:b572360966 "2"^^ . _:b572360998 . _:b572360980 . _:b572360961 "2"^^ . _:b572360995 . _:b572360983 . _:b572360960 "2"^^ . _:b572360935 . _:b572360982 . _:b38007506 "results" . _:b38007539 . _:b572360963 "2"^^ . . _:b572360977 . . _:b572360962 "2"^^ . "PMC0" . . _:b38007530 . _:b572360976 . _:b38007549 . _:b572360973 "2"^^ . . _:b572360979 . _:b572360972 "2"^^ . _:b572360980 . _:b572360981 . _:b572360978 . _:b572360982 . _:b572360975 "2"^^ . _:b572360983 . _:b38007546 "In ISC/PC co-culture experiments, growth factors, minus JAGGED-1 peptide and Y27632, were supplemented every two days, and media was changed every four days, as previously described 42, >>43<<." . _:b572360976 . _:b572360977 . _:b572360989 . . _:b572360974 "2"^^ . _:b572360978 . _:b572360979 . _:b572360988 . _:b572360989 . _:b572360988 . _:b572360990 . _:b572360969 "2"^^ . _:b572360991 . _:b572360984 . _:b572360985 . _:b572360991 . _:b572360986 . _:b572360968 "2"^^ . _:b572360987 . _:b572360964 . _:b572360965 . _:b572360990 . _:b572360966 . _:b572360971 "2"^^ . _:b572360967 . _:b572360960 . _:b572360961 . _:b572360985 . _:b572360962 . _:b38007518 "Previous characterization of the Sox9EGFP mouse model demonstrated that Sox9 is expressed at different levels in ISCs, progenitors, and enteroendocrine cells, but that the Sox9EGFP transgene is preferentially silenced in PCs >>7<<. We exploited this property to isolate a highly pure population of PCs by FACS exclusion of Sox9EGFP populations. PCs were FACS-isolated using CD24High:SSCHigh parameters, and the additional exclusion of all Sox9EGFP-positive cells" . _:b572360970 "2"^^ . _:b572360963 . _:b572360972 . _:b572360973 . _:b38007525 . _:b572360984 . _:b572360974 . _:b572360981 "2"^^ . _:b572360975 . _:b572360968 . _:b572360969 . _:b572360987 . _:b572360970 . _:b572360980 "2"^^ . _:b572360971 . _:b38007504 "Additionally, the field currently lacks a robust assay to study ISCs at the clonal level, a tool that has driven the understanding of stem cell niches in the hematopoietic system and mammary glands 9, >>10<<. Array-based technologies are emerging as a powerful method to study the functional characteristics of single and/or small numbers of stem cells in the hematopoietic system, and hold similar promise for epithelial tissues such as the" . _:b38007508 . . _:b572360986 . _:b572360983 "2"^^ . _:b572360997 . _:b572360982 "2"^^ . _:b38007538 . _:b572360996 . _:b572360977 "2"^^ . _:b572360999 . _:b572360976 "2"^^ . _:b38007508 "Since ISCs require several days to develop into enteroids, MRAs had to be amenable to media changes 3, >>4<<. To meet these requirements, polycarbonate cassettes, with dividers to create multiple media reservoirs, were bonded to MRAs (Fig. 1A,B, Supplementary Fig. 1H). Cassettes were fabricated with two or four culture chambers (\u223C2,500 or 5,000" . _:b572360996 . . _:b572360997 . _:b572360998 . _:b572360998 . _:b572360979 "2"^^ . _:b572360999 . _:b572360992 . _:b38007513 . _:b572360993 . _:b572360993 . . . _:b572360994 . _:b572360978 "2"^^ . _:b572360995 . _:b572361004 . . . _:b572361005 . _:b572360992 . . . _:b572360989 "2"^^ . _:b572361006 . _:b572361000 . _:b572361001 . _:b572360995 . _:b572361002 . _:b572360988 "2"^^ . _:b572361003 . _:b572360994 . _:b38007507 . _:b572360991 "2"^^ . . _:b572360996 . _:b572361005 . _:b572360849 . _:b572360990 "2"^^ . _:b572360905 . _:b572361004 . _:b572360985 "2"^^ . _:b38007539 . . . _:b572360984 "2"^^ . . _:b572360941 . _:b38007514 . _:b572361006 . _:b572360987 "2"^^ . . _:b572361001 . _:b572360986 "2"^^ . _:b572361000 . _:b572360997 "2"^^ . _:b572361003 . _:b572360996 "2"^^ . . _:b572361002 . _:b572360965 . _:b572360999 "2"^^ . _:b572360998 "2"^^ . . . _:b572360993 "2"^^ . . _:b38007526 . _:b38007547 "Scanned images were stitched into a single composite image using the open source image analysis suite FIJI >>44<< and then segmented into address-associated individual well images using an algorithm, \u201CSegmenter." . _:b572360992 "2"^^ . _:b572360877 . _:b572360995 "2"^^ . _:b572360987 . _:b572360994 "2"^^ . _:b572360850 . . _:b572361005 "2"^^ . _:b38007499 . _:b572361004 "2"^^ . _:b572360959 . _:b572360863 . _:b572360894 . _:b572361006 "2"^^ . _:b572360874 . _:b572360892 . _:b572361001 "2"^^ . _:b38007516 "However, CD24 is also expressed on ISCs, TAs, and enteroendocrine cells 3, >>14<<, 16-18. Since the experimental approach of MRA cultures examines events on a \u201Cper well\u201D basis, it was critical to refine isolation procedures for PCs to meet purity requirements of clonal and microscale co-cultures and avoid artifactual" . . _:b572360958 . _:b572360898 . _:b572360944 . _:b572361000 "2"^^ . . _:b572361003 "2"^^ . _:b572361002 "2"^^ . . _:b38007544 "growth factors, with some modification, at the time of plating: 15\u03BCM JAGGED-1 peptide (AnaSpec, San Jose, CA), 750ng/mL EGF (R&D, Minneapolis, MN), 100ng/mL NOGGIN (Peprotech, Rocky Hill, NJ), and 500nM LY2157299 (Selleck Chemicals) >>41<<. Initial media in validation experiments contained 2.5\u03BCM CHIR99021 (Selleck Chemicals) and 2.5\u03BCM Thiazovivin (Selleck Chemicals)." . . . . _:b572360960 . . . . _:b38007531 "discussion" . _:b572361003 . _:b572360997 . _:b38007514 "To achieve this, we developed an image analysis computational pipeline (Fig. 2; Supplementary Methods) >>15<<." . . _:b572360979 . _:b572360926 . . . _:b38007539 "However, most platforms are not amenable to long-term cultures, such as required for the development of ISC-derived enteroids and other self-assembled, stem cell derived organoids >>35<<-38. MRAs facilitate the culture of thousands of primary stem cells over many days and weeks as well as high throughput reconstitution of the stem cell niche at physiologically relevant cell numbers. The power of the MRA platform is" . . _:b38007539 . _:b572360889 . _:b572360955 . _:b572360845 . . _:b572360950 . _:b38007552 . _:b572360882 . . . _:b38007543 . _:b572360970 . _:b572360919 . _:b38007518 . _:b38007542 . . . _:b572360977 . . _:b572360972 . _:b38007547 . _:b572360911 . . _:b572360921 . _:b38007526 . _:b572360910 . . . _:b572360855 . _:b572360917 . _:b572360990 . _:b572360916 . . _:b572360909 . _:b38007499 . . _:b572360942 . _:b572360857 . . . . _:b572360967 . _:b38007502 . _:b38007511 "types (Fig. 1D) 3, 13. Sox9EGFPlow:DsRed+ ISCs were plated in a single culture chamber of a two-chamber MRA, randomly seeded into microwells by centrifugation, and overlaid with Matrigel and ISC-supporting growth factors (Fig. 1E) 6, >>14<<. This resulted in a random distribution of ISCs across MRAs, with microwells containing one, multiple, or no ISCs (Fig 1F-H)." . _:b38007525 "single ISC and enteroid retrieval for downstream analysis, MRAs were modified so that standard polystyrene rafts at the bottom of each microwell were replaced with magnetized rafts, as recently described (Supplementary Fig. 5A) >>12<<, 22. A raft release device was fitted to a 10X objective to liberate the rafts from the PDMS wells, and a magnetic wand was used to retrieve rafts for transfer to RNA lysis buffer (Supplementary Fig." . _:b572360845 . . _:b38007521 . _:b572360844 . . _:b572360847 . .