Journal: Stem Cell Reports

Article Title: A Refined Culture System for Human Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Organoids

doi: 10.1016/j.stemcr.2017.11.004

Figure Lengend Snippet: Preparation of WRN CM by Lentiviral Infection (A and B) Wnt activities of recombinant hWNT3A or mWNT3A (A) and recombinant hRSPO1 or mRSPO1 (B). Activities in the absence of the hRSPO1 expression plasmid and recombinant proteins were considered as “1.” The assays were performed in three independent biological replicates. Data expressed as mean ± SEM. n.s., not significant (Student’s t test). (C–H) Mouse L cells stably expressing WRN were generated by lentiviral infection of each gene. (C–E) Wnt signaling activities of CM from L-WRN cells at densities of 2.8, 1.4, 0.7, and 0.35 × 10 6 cells/35-mm dish after 48 hr of culture (C); at a density of 1.4 × 10 6 cells/dish cultured between 24 and 96 hr (D); and at 1.4 × 10 6 cells/dish cultured for 72 hr in diluted CM or 300 ng/mL recombinant mWNT3A, with or without an hRSPO1 expression plasmid (E). Activities in the absence of WRN CM were considered as “1.” The assays were performed in three independent biological replicates. Data expressed as mean ± SEM. (F) Western blot analysis of the supernatants used in (C) undiluted (8 μL) and 4-fold diluted, and the recombinant proteins mWNT3A (1 and 0.3 ng), hRSPO1 (30 and 10 ng), and hNOG (10 and 3 ng) using anti-WNT3A, anti-RSPO1, and anti-NOG antibodies. (G) Images of human primary ileal organoids cultured in 25% WRN CM or the corresponding recombinant proteins for 7 days after a passage. Scale bars, 200 μm. (H) Wnt signaling activities of WRN CM (×1, ×1/3, ×1/10) stored in the deep freezer for 0, 6, or 12 months. The activities in the absence of CM were considered as “1.” The assay was performed in three independent biological replicates. Data expressed as mean ± SEM.

Article Snippet: When using Happy Cell ASM (Biocroi, Ireland), disrupted organoids were suspended with Happy Cell human organoid culture medium ( ) and seeded on ultra-low-attachment cell culture plate (Corning, 3473) (0.5 mL/well).

Techniques: Infection, Recombinant, Expressing, Plasmid Preparation, Stable Transfection, Generated, Cell Culture, Western Blot

Journal: Stem Cell Reports

Article Title: A Refined Culture System for Human Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Organoids

doi: 10.1016/j.stemcr.2017.11.004

Figure Lengend Snippet: Differentiation of TkDN4-M into Human Intestinal Organoids (A) Schematic procedures for intestinal organoid differentiation from human iPSC lines. (B) Images of the human iPSC line TkDN4-M that had been differentiated into spheroids (upper panels) and organoids (lower panels) with (right panels) or without (left panels) recombinant hWNT3A and human FGF2 (hFGF2). Arrowheads indicate spheroid- and organoid-like cell clumps. Scale bars, 200 μm. (C) Magnified images of the spheroids and organoids. Scale bars, 40 μm. (D) The numbers of spheroids per microscopic bright field in the presence or absence of WNT3A/FGF2 supplementation. The assays were performed in three independent experiments. Data are presented as mean ± SEM. ∗ p < 0.05, Student's t test. (E) Relative mRNA levels of SOX17 and CXCR4 in TkDN4-M cells differentiated into spheroids in the presence or absence of WNT3A/FGF2 supplementation were determined by qPCR and normalized to HPRT. The assay was performed in three independent biological replicates. Expression levels presented as mean ± SEM. ∗ p < 0.05, Student's t test. (F) Relative mRNA levels of the indicated genes in TkDN4-M cells during the course of differentiation (days 0, 3, and 7 [spheroids], and organoids) were determined by qPCR and normalized to HPRT . The highest expression levels of each gene in each differentiation stage were considered as “1.” The assays were performed in three independent biological replicates. Expression levels presented as mean ± SEM. (G) Images of TkDN4-M-derived organoids on day 7 after culture in 25% WRN CM, or the corresponding recombinant proteins. Scale bars, 200 μm. (H) Relative mRNA levels of LGR5 and VIL1 in TkDN4-M organoids cultured for short-term (passage 19) and long-term (passage 53) were determined by qPCR and normalized to HPRT . The assays were performed in three independent biological replicates. Expression levels presented as mean ± SEM. (I) Whole-mount images of TkDN4-M-derived organoids stained with phalloidin (blue), together with each indicated antibody. Scale bars, 50 μm.

Article Snippet: When using Happy Cell ASM (Biocroi, Ireland), disrupted organoids were suspended with Happy Cell human organoid culture medium ( ) and seeded on ultra-low-attachment cell culture plate (Corning, 3473) (0.5 mL/well).

Techniques: Recombinant, Expressing, Derivative Assay, Cell Culture, Staining

Journal: Stem Cell Reports

Article Title: A Refined Culture System for Human Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Organoids

doi: 10.1016/j.stemcr.2017.11.004

Figure Lengend Snippet: Comparison of Human iPSC-Derived Organoids with Primary Intestinal Organoids (A) Images of organoids from the human primary ileum or transverse colon cultured with human organoid culture medium for 6 days. Scale bars, 200 μm. (B) Relative mRNA levels of the indicated genes in organoids derived from human primary ileum, colon, and iPSCs harvested at 6 days after passage. The relative mRNA levels were determined by qPCR and normalized to GAPDH . The assays were performed in three independent biological replicates. Data are presented as mean ± SEM.

Article Snippet: When using Happy Cell ASM (Biocroi, Ireland), disrupted organoids were suspended with Happy Cell human organoid culture medium ( ) and seeded on ultra-low-attachment cell culture plate (Corning, 3473) (0.5 mL/well).

Techniques: Derivative Assay, Cell Culture

Journal: Stem Cell Reports

Article Title: A Refined Culture System for Human Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Organoids

doi: 10.1016/j.stemcr.2017.11.004

Figure Lengend Snippet: Lentiviral Gene Transduction into Human iPSC-Derived Intestinal Organoids (A) Schematic representation of gene transduction into organoids. TkDN4-M-derived organoids cultured in Matrigel were disrupted by a 29G needle, seeded in collagen I-coated plates, and cultured for 4 days. After 2D cultures from organoids were infected by lentiviruses, cells (including IESCs) were trypsinized, harvested, and re-embedded in Matrigel to reform organoids. (B) The proportions of Venus + organoids per microscopic bright field after 7 days of infection with IRES-Venus lentivirus. “3D” and “2D” indicate lentiviral infection into organoids harvested from Matrigel and 2D-cultured cells in collagen I-coated plates, respectively. The assay was performed in three independent biological replicates. Data are presented as mean ± SEM. ∗ p < 0.05, Student's t test. (C) Bright-field and fluorescent images of TkDN4-M-derived organoids as in (B). Scale bars, 200 μm. (D) Regenerated organoid numbers per microscopic bright field followed by 2D cultures after 7 days of infection with or without IRES-Venus lentivirus. The assay was performed in three independent biological replicates. Data are presented as mean ± SEM. (E) Bright-field and fluorescent images of TkDN4-M-derived organoids 3 weeks after infection with Wnt3a -IRES-Venus lentivirus in 2D cultures. Scale bars, 200 μm. (F) Relative mRNA levels of exogenous Wnt3a and Wnt target genes ( LGR5 , AXIN2 , and EPHB3 ) in TkDN4-M-derived organoids infected with lentiviruses as in (E). The relative mRNA levels were determined by qPCR and normalized to GAPDH . The assay was performed in three independent biological replicates. Expression levels presented as mean ± SEM. ∗ p < 0.05, Student's t test. n.d., not detected.

Article Snippet: When using Happy Cell ASM (Biocroi, Ireland), disrupted organoids were suspended with Happy Cell human organoid culture medium ( ) and seeded on ultra-low-attachment cell culture plate (Corning, 3473) (0.5 mL/well).

Techniques: Transduction, Derivative Assay, Cell Culture, Infection, Expressing

Journal: Stem Cell Reports

Article Title: A Refined Culture System for Human Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Organoids

doi: 10.1016/j.stemcr.2017.11.004

Figure Lengend Snippet: Growth of Human iPSC-Derived Organoids by Suspension Culture Using Happy Cell ASM (A) Time-lapse bright-field images of TkDN4-M-derived organoids cultured in Matrigel, Happy Cell ASM, or FCeM, each supplemented with 25% WRN CM and requisite ingredients for human organoid culture. Scale bars, 200 μm. (B) Whole-mount images of TkDN4-M-derived organoids cultured in Happy Cell culture medium were stained with phalloidin (blue), together with each indicated antibody. Scale bars, 50 μm. (C and D) Relative mRNA levels of the indicated genes in TkDN4-M-derived organoids cultured in Matrigel (C) or Happy Cell ASM (D) for 5 days and then exposed to each pre-stimulation medium containing the indicated concentrations of recombinant human IL-22 for 2 days. The relative mRNA levels were determined by qPCR and normalized to GAPDH . The assays were performed in three independent biological replicates. Data presented as mean ± SEM.

Article Snippet: When using Happy Cell ASM (Biocroi, Ireland), disrupted organoids were suspended with Happy Cell human organoid culture medium ( ) and seeded on ultra-low-attachment cell culture plate (Corning, 3473) (0.5 mL/well).

Techniques: Derivative Assay, Cell Culture, Staining, Recombinant

Journal: Stem Cell Reports

Article Title: A Refined Culture System for Human Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Organoids

doi: 10.1016/j.stemcr.2017.11.004

Figure Lengend Snippet: Characterization of Human iPSC-Derived Organoids Cultured in Happy Cell ASM (A) Relative mRNA levels of the indicated genes in TkDN4-M-derived organoids cultured with Matrigel (open bars) or Happy Cell ASM (closed bars) plus 25% WRN CM and requisite ingredients for human organoid culture after one (P1) or two (P2) passages in each culture condition. The relative mRNA levels were determined by qPCR and normalized to GAPDH . The assays were performed in three independent biological replicates. Data presented as mean ± SEM. ∗ p < 0.05, Student's t test. (B) Relative mRNA levels of the indicated genes in TkDN4-M-derived organoids cultured in Happy Cell ASM with or without 300 ng/mL recombinant mWNT3A for 6 days. The relative mRNA levels were determined by qPCR and normalized to GAPDH . The assays were performed in three independent biological replicates. Data presented as mean ± SEM. ∗ p < 0.05, Student's t test. (C) Whole-mount images of TkDN4-M-derived organoids cultured in Happy Cell ASM with or without 300 ng/mL recombinant mWNT3A after staining with phalloidin (blue) and anti-MKI67 antibodies. Scale bars, 50 μm.

Article Snippet: When using Happy Cell ASM (Biocroi, Ireland), disrupted organoids were suspended with Happy Cell human organoid culture medium ( ) and seeded on ultra-low-attachment cell culture plate (Corning, 3473) (0.5 mL/well).

Techniques: Derivative Assay, Cell Culture, Recombinant, Staining

Journal: bioRxiv

Article Title: 3D observation of large-scale subcellular dynamics in vivo at the millisecond scale

doi: 10.1101/672584

Figure Lengend Snippet: a , The system works in epi-fluorescence mode. A large volume within the 3D imaging range is excited simultaneously by a highly-inclined illumination and collected by the DAOSLIMIT. The inhomogeneous distribution of the refractive index in multicellular specimens produces strong spatially nonuniform aberrations at the back-pupil plane, which can be segmented for correction with adaptive optics. For illustration, light from different sub-apertures is labelled with different colours. A microlens array is inserted at the image plane for parallel acquisition of multiplexed phase-space measurements, whose resolution is further enhanced by the scanning process with a two-dimensional galvo scanning system. During reconstruction, we first realign the pixels from the raw data into the high-resolution multiplexed phase-space. Then, a mutual iterative tomography algorithm is employed to obtain the high-resolution volume with pixel-wise wavefront corrections. b , The multiplexed phase-space measurements can be synthesized for 3D reconstruction with the digital beam propagation. However, the sample-induced aberration will result in a distorted focus. We can digitally shift the sub-aperture point spread function (PSF), akin to applying a correction wavefront estimated during the volume reconstruction, to create a perfect focus. Both the spatial overlap induced by scanning and the frequency aliasing induced by the small aperture of each microlens facilitate the incoherent synthetic aperture during the 3D reconstruction, up to the diffraction limit of the whole objective’s NA.

Article Snippet: Human 3D cerebral organoids were generated and maintained in human 3D cerebral organoids medium (Cat # HopCell-3DM-001, Hopstem Bioengineering) by Hopstem Bioengineering according to the manufacturer's protocols.

Techniques: Fluorescence, Imaging, Tomography, Synthesized

Journal: bioRxiv

Article Title: 3D observation of large-scale subcellular dynamics in vivo at the millisecond scale

doi: 10.1101/672584

Figure Lengend Snippet: a , Orthogonal MIPs from 16-μm-thick slabs of the focal stack captured by WFM, including 90 axial slices at 200-nm steps. The sample used here is a HeLa cell labelled with actin (green) and nuclei (blue). The Fourier transforms of the MIPs are shown on the right to indicate the degrees of information recovery in low-SNR conditions. The yellow dashed circle corresponds to the spatial frequency of the Abbe diffraction limit for comparison. b , Results reconstructed by applying 3D RL deconvolution to the focal stack in a, with enhanced SNR and contrast. c , Results obtained by traditional LFM with 3D deconvolution, showing much lower spatial resolution and reconstruction artefacts close to the native object plane. d , Results reconstructed by DAOSLIMIT with a 67% overlap ratio (corresponding to 3 × 3 lateral shifts), indicating higher SNR and resolution especially in the x-z and y-z planes (notably only 9 images are used for 3D reconstruction, 10 times less than WFM). Scale bar: 20 μm.

Article Snippet: Human 3D cerebral organoids were generated and maintained in human 3D cerebral organoids medium (Cat # HopCell-3DM-001, Hopstem Bioengineering) by Hopstem Bioengineering according to the manufacturer's protocols.

Techniques:

Journal: bioRxiv

Article Title: 3D observation of large-scale subcellular dynamics in vivo at the millisecond scale

doi: 10.1101/672584

Figure Lengend Snippet: a , Colour-coded MIP of the 3D volume reconstructed by sLFM without DAO. Different colours encode different depths. Scale bar: 10 μm. b , Colour-coded MIP of the 3D volume reconstructed by DAOSLIMIT. Scale bar: 10 μm. c , Different corrected wavefronts applied to different areas across the field of view in a. d , Orthogonal MIPs from 2-μm-thick slabs of the selected area in a, with a cross-section profile illustrating the resolution loss from aberration. The white arrow points to a structure blurred by the aberration. Scale bar: 2 μm. e , Orthogonal MIPs from 2-μm-thick slabs of the selected area in b, with a cross-section profile illustrating the diffraction-limited performance with DAO. The average correction wavefront of the selected region is shown in the inset at the unit of wavelength. The white arrow points to the ring structure resolved after DAO. Scale bar: 2 μm. f - g , Colour-coded MIPs of the selected areas in b at different time stamps marked at the bottom row. The arrow in f indicates a fast-moving mitochondrion in 3D, whereas the arrow in g indicates a mitochondrial fusion process happening in 3D. Scale bar: 1 μm. h , The distributions of the average speed and tracking length with the same tracking algorithm applied to 3D videos without DAO, 2D videos with DAO and 3D videos with DAO. More accurate speed can be estimated in 3D, whereas the SNR and resolution enhancements by DAO facilitate longer tracking duration and more mitochondria under tracking.

Article Snippet: Human 3D cerebral organoids were generated and maintained in human 3D cerebral organoids medium (Cat # HopCell-3DM-001, Hopstem Bioengineering) by Hopstem Bioengineering according to the manufacturer's protocols.

Techniques:

Journal: bioRxiv

Article Title: 3D observation of large-scale subcellular dynamics in vivo at the millisecond scale

doi: 10.1101/672584

Figure Lengend Snippet: a , Orthogonal MIPs of the 3D membrane structures reconstructed by WFM with 3D deconvolution (left panel) and DAOSLIMIT (right panel). b , 100 Hz 3D imaging of vesicle dynamics and potential vesicle trafficking in one zebrafish epithelial cell in the gastrulation stage (Supplementary Video 2). The colour-coded MIP of the volume at 0 s is shown in the left panel with different colours corresponding to different depths. The 3D tracking trajectories of every vesicle are shown in the right panel. c , The colour-coded MIP of the in vivo membrane dynamics reconstructed without DAO and time-loop algorithms are shown on the left, whereas that reconstructed with DAO, and time-loop algorithms is shown on the right, indicating improvement in both SNR and resolution (Supplementary Video 3). d , The effectiveness of the time-weighted algorithm on the compensation of the sacrificed temporal resolution resulting from lateral scanning (Supplementary Video 3). Orthogonal MIPs across 5-μm-thick slabs of the selected area in c are displayed with different time stamps marked at the bottom row. The results without the time-weighted algorithm are shown on the first row, and results with the time-weighted algorithm are shown on the second row, which eliminates the motion blur of a moving vesicle (white arrow) with the same spatial resolution. ( e-g ) Orthogonal MIPs of the selected areas in c with different time stamps marked at the bottom row (Supplementary Video 4). Dynamic filopodia retraction processes can be observed in e and g. A cell division process showed gradual membrane enrichment dynamics at the boundary of daughter cells in f. h – i , The colour-coded MIP of the 3D membrane dynamics in vivo at 10 Hz. Different zoom-ins with orthogonal MIPs in h show the 3D movements of a migrasome and a clear cell migration in 3D at high spatiotemporal resolution (Supplementary Video 6). High-speed fluctuations of the filopodia membrane can be observed in i, including filopodia retraction process and migrasome movements (Supplementary Video 7). Scale bar: 10 μm.

Article Snippet: Human 3D cerebral organoids were generated and maintained in human 3D cerebral organoids medium (Cat # HopCell-3DM-001, Hopstem Bioengineering) by Hopstem Bioengineering according to the manufacturer's protocols.

Techniques: Imaging, In Vivo, Migration

Journal: bioRxiv

Article Title: 3D observation of large-scale subcellular dynamics in vivo at the millisecond scale

doi: 10.1101/672584

Figure Lengend Snippet: a , Colour-coded MIP of the GCamp6s-labelled human 3D cerebral organoids obtained by DAOSLIMIT. Different colours represent different depths. b , Temporal-coded MIP of the selected area in a for the start time (the time point when the signal reaches 10% of the maximum intensity). Different colours correspond to the start points of the spontaneous calcium response, and the intensity represents the MIP of the temporal trace standard deviation for each voxel. Spontaneous 3D calcium propagation evoked from the intersection of two neurons can be clearly observed. c , Temporal-coded MIP of the selected area in a, illustrating the rise time (the time required to increase from 20% to 80% of the maximum intensity). Different colours correspond to the rise time of the spontaneous calcium response, and the intensity represents the MIP of the temporal trace standard deviation for each voxel. d , Orthogonal MIPs of the selected areas in a with different time stamps marked at the bottom row (Supplementary Video 9). The video was captured at 30 Hz. e , The temporal traces (ΔF/F 0 ) of the ROIs labelled in a. f , Colour-coded MIP of the Drosophila larval Cho neurons with the jGCaMP7s indicator. Different colours represent different depths. g, Temporal-coded MIP for start time. h , Temporal-coded MIP for rise time. i , The temporal traces (ΔF/F 0 ) of the ROIs labelled in f. The red arrow indicates the time point when we applied the 500 Hz sound stimulus. j, Orthogonal MIPs of the selected areas in f with different time stamps marked at the bottom row (Supplementary Video 10). The video was captured at 100 Hz. Scale bar: 10 μm.

Article Snippet: Human 3D cerebral organoids were generated and maintained in human 3D cerebral organoids medium (Cat # HopCell-3DM-001, Hopstem Bioengineering) by Hopstem Bioengineering according to the manufacturer's protocols.

Techniques: Standard Deviation

Journal: bioRxiv

Article Title: Fluorescence Lifetime Imaging Microscopy (FLIM) visualizes internalization and biological impact of nanoplastics in live intestinal organoids

doi: 10.1101/2025.01.27.635069

Figure Lengend Snippet: A: Representative 3D confocal images of WGA-Alexa Fluor 488 and phalloidin–Texas Red (F-actin labeling) co-stained organoids with apical-out (AO), apical-basal (AB) and basal-out (BO) topology. Scale bar is 100 µm. B: Large field mosaic scan of PFA-fixed pig intestinal organoids after 18 h of polarity reversion, co-stained with fluorescent WGA and phalloidin. Left: representative images of AO, AB and BO organoids, indicated on mosaic image ( right ). Scale bar is 100 µm. C: Comparison of the yield of AO, AB and BO topology, estimated with WGA staining and F-actin labeling, respectively (AO organoids: 93.1% / 95.5%, BO organoids 0.53% / 0.55%, analyzed from 4 mosaic scanned images, see table ST3). D: Total quantification of topology, observed with WGA and Nile Red labeling for polarity reverted organoids: AO with lipid droplets (AO LDs) and AO without lipid droplets (AO no-LDs), AB and BO, quantified from mosaic scanned image. E: Lipid droplets display a characteristic distribution in AO organoids, contrasting with BO. F: Co-staining with WGA and Nile Red reveals organoid structure in apical-basal organoid (AB). Scale bar is 50 µm.

Article Snippet: Lipidure TM -CM5206 (AMS.52000034GB1G, Amsbio, UK), Matrigel growth factor-reduced (734-0269, VWR, Belgium), human intestinal organoid growth medium (STEMCELL technologies, 06010, Belgium), mouse intestinal organoid growth medium (STEMCELL technologies, 06005, Belgium), DMEM high glucose GlutaMax TM Supplement media (61965026, Gibco, Belgium), 0.5 M EDTA solution (15575020, Invitrogen, Belgium), 24-well Tissue Culture plates (734-2325, VWR, Belgium), sodium valproate (P4543, Sigma-Aldrich,Ireland), CHIR99021 (SML1046, Sigma-Aldrich,Ireland), PBS (18912-014, Gibco, Belgium).

Techniques: Labeling, Staining, Comparison

Journal: bioRxiv

Article Title: Fluorescence Lifetime Imaging Microscopy (FLIM) visualizes internalization and biological impact of nanoplastics in live intestinal organoids

doi: 10.1101/2025.01.27.635069

Figure Lengend Snippet: A: Scheme of experimental workflow. B: Representative confocal fluorescence images and corresponding phasor plots of the pig small intestinal organoid incubated with NP A-D (10 µg/ mL, 18 h), co-stained with Nile Red. NP A, B and D displayed signals on both fluorescent intensity images and phasor plots, whereas type C showed no signal, similar to control. C: Different types of uptake of NP type D uptake (10 µg/mL NPD, 20% laser power) into organoids, co-stained with WGA, with respect to the topology and size. Left: partial NP D uptake in AO, showing high pixel signal on a phasor plot. Middle: organoid size-dependent NP D uptake in AO. ROI 1: enhanced NP D pixel signal in a small AO, ROI 2: reduced NP D pixel signal in larger organoid. Right: Homogeneous NP D distribution in BO with strong signal on a phasor plot. D: Topology-dependent uptake of NP D (magenta) in apical-basal organoid (AB), co-stained with WGA (green) and Nile Red (yellow). Left: 3D reconstruction shows distinct NP D distribution in AO and BO regions. Right: representative fluorescence image of AB organoid taken from 3D reconstruction. AO and BO regions defined by WGA and Nile Red signals, with corresponding NP type D phasor FLIM plots of these regions (bottom right).

Article Snippet: Lipidure TM -CM5206 (AMS.52000034GB1G, Amsbio, UK), Matrigel growth factor-reduced (734-0269, VWR, Belgium), human intestinal organoid growth medium (STEMCELL technologies, 06010, Belgium), mouse intestinal organoid growth medium (STEMCELL technologies, 06005, Belgium), DMEM high glucose GlutaMax TM Supplement media (61965026, Gibco, Belgium), 0.5 M EDTA solution (15575020, Invitrogen, Belgium), 24-well Tissue Culture plates (734-2325, VWR, Belgium), sodium valproate (P4543, Sigma-Aldrich,Ireland), CHIR99021 (SML1046, Sigma-Aldrich,Ireland), PBS (18912-014, Gibco, Belgium).

Techniques: Fluorescence, Incubation, Staining, Control

Journal: bioRxiv

Article Title: Fluorescence Lifetime Imaging Microscopy (FLIM) visualizes internalization and biological impact of nanoplastics in live intestinal organoids

doi: 10.1101/2025.01.27.635069

Figure Lengend Snippet: A, B: Representative images of NP D uptake (10 µg/mL, 18 h) in pig intestinal organoids in respect to their apical-basal topology with high loading in both AO and BO (A) and high loading in BO and zero at AO (B). Scale bar is 50 µm. C, D: Comparison of NP D uptake in AO vs. BO organoids as a function of loading concentration (0-1 µg/mL, left panel and 1-50 µg/mL, right panel) on a widefield fluorescence microscope (intensity-based approach, C), and confocal FLIM microscope (phasor FLIM event counting approach, D). FLIM events were counted from the phasor plots reconstructed in the napari phasor plugin from the exported list of G and S coordinates. Results of one of the two independent experimental replicates are shown. Both C and D data were produced from the same samples. The box charts represent 25, median and 75 percentiles with dots corresponding to individual intensity or event count square ROI square ROI normalized values. Attribution of organoids to AO and BO-topology groups was done based on WGA staining. BO group also includes AB organoids. Statistical comparison between AO and BO groups over a range of loading concentrations was performed by Mann-Whitney test (lines represent detected statistical difference, p <0.05).

Article Snippet: Lipidure TM -CM5206 (AMS.52000034GB1G, Amsbio, UK), Matrigel growth factor-reduced (734-0269, VWR, Belgium), human intestinal organoid growth medium (STEMCELL technologies, 06010, Belgium), mouse intestinal organoid growth medium (STEMCELL technologies, 06005, Belgium), DMEM high glucose GlutaMax TM Supplement media (61965026, Gibco, Belgium), 0.5 M EDTA solution (15575020, Invitrogen, Belgium), 24-well Tissue Culture plates (734-2325, VWR, Belgium), sodium valproate (P4543, Sigma-Aldrich,Ireland), CHIR99021 (SML1046, Sigma-Aldrich,Ireland), PBS (18912-014, Gibco, Belgium).

Techniques: Comparison, Concentration Assay, Fluorescence, Microscopy, Produced, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Mucin MUC5B levels are reduced in the colonic epithelia of ulcerative colitis patients

doi: 10.1101/2024.02.21.581373

Figure Lengend Snippet: (A) Scoring system to analyze MUC5B proteins in intestinal tissues. Positively stained cells were quantified per field of view under 10X magnification. At least five fields of view were assessed for each tissue and staining patterns were scored as follows: 0 (negative or very low numbers of positive cells, 0-10%), 1 (low, 10-25%), 2 (moderate, 25-50%) or 3 (high, >50%). (B) Immunohistochemical analysis of MUC5B in an inflamed colonic segment from a UC patient. (C) Quantification of MUC5B staining in colons from non-IBD controls (n=12) and in inflamed colons from UC patients (n=28). Error bars represent SEM, * P < 0.05.

Article Snippet: The plates were incubated at 37°C for 20 min to allow the domes to solidify after which 750 μl of human intestinal organoid growth media (Human IntestiCult Organoid Growth Medium, hIOGM, StemCell Technologies, 06010) was added along with 10 μM ROCK inhibitor (Y-27632, Miltenyi, 130-106-138).

Techniques: Staining, Immunohistochemical staining