Geometrically constrained growth factor concentration favors enrichment of goblet cells and mucus formation
Data files
Apr 14, 2026 version files 428.79 MB
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Figure2D.csv
4.36 KB
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Figure2E.csv
4.31 KB
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Figure2F.csv
496 B
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Figure2G.csv
494 B
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Figure3B.csv
1.29 KB
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Figure4B.csv
883 B
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Figure5BC.csv
951 B
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Figure5F.csv
425 B
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Figure6C.csv
17.78 KB
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Figure6D.csv
18.94 KB
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Figure7B.csv
1.06 KB
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FigureS3C.csv
1.04 KB
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FigureS3D.csv
9.44 KB
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README.md
15.88 KB
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Videos.zip
428.71 MB
Abstract
The colonic mucus composed of replenished gel-forming mucin secreted from goblet cells is of great importance for overall human health, and its better understanding could lead to the development of new drug targets in colon diseases and mucus defect therapies. In this dataset, we show the effect of the constrained geometry for WRN diffusion, on goblet cell differentiation and mucus formation. This advanced engineering approach focused on long-term culture to form a mucus layer barrier. The platform is an insert format with rehydrated collagen overlaying an impermeable film patterned with a geometrical array of micron-scale holes. This system enables a constrained geometry to preserve stem cell niches. The microniches possess enriched goblet cell generation and a thick, adhered mucus layer. Immunofluorescence-based assays were performed to quantitatively assess cell density, proliferation, migration, differentiation, mucus thickness, and the impact of bacteria and toxins on mucus-covered epithelial cells. The geometrical engineering approach to generate goblet cells and mucus protective barrier represents a significant innovation in developing microphysiological systems for emulating the gut epithelium for assaying self-renewal stem cells, goblet cell differentiation, mucus dynamics, and microbiota-mucus-host interactions.
Dataset DOI: 10.5061/dryad.95x69p8x9
Description of the data and file structure
This repository contains the data used in the analysis of in vitro cellular proliferation and differentiation, goblet cell characterization, mucus layer accumulation, and the ability of the formed mucus layer to protect intestinal epithelial cells from toxin insults and microbial invasion within a crypt matrix defined via WRN gradient diffusion. The datasets presents the data results from a variety of cell markers and staining profiles and is ideal for comparative studies, colon crypts characterization, mucus measurement, and polarization crypt studies.
Files and variables
File: Figure2G.csv
Description: The Figure2G.csv file contains information about the impact of the distance between the through holes (Gap 350 micrometers) on the formation of polarized planar crypts overtime (Days 8-23).
Variables
- Gap (Micrometer): Describes the center-to-center distance between two through-holes in the geometry of a 10×10 hole array on a photoresist epoxy film.
- Day (#): Describes the number of days the cells were cultured before fixation.
- Marker: This column contains information on cell identification marker EdU (5-ethynyl-2′-deoxyuridine) used to determine area coverage for stem/proliferative cells.
- Sample (Replicate #): This column contains the label of the replicate used for the analysis: Rep1, Rep2, and Rep3 (Replicate 1, Replicate 2, and Replicate 3).
- Coverage (%): Describes the percentage coverage of the EdU (5-ethynyl-2′-deoxyuridine) area for each replicate.
File: Figure7B.csv
Description: The Figure7B.csv file contains information about the impact of bacteria on colon crypts with and without a mucus layer. Data contains results of epithelial colon cells cocultured with LGG (Lactobacillus rhamnosus GG), and the coculture with S. aureus (Staphylococcus aureus) bacteria.
Variables
- Gap (Micrometer): Describes the center-to-center distance between two through-holes in the geometry of a 10×10 hole array on a photoresist epoxy film.
- Sample (Replicate #): This column contains the label of the replicate used for the analysis: Rep1, Rep2, and Rep3 (Replicate 1, Replicate 2, and Replicate 3).
- Mucus: This is classified as with mucus (+) or no mucus (-), which means that the cells generated (+) mucus during culture or did not generate it (-).
- Condition: This column contains information of the bacteria used for the co-culture or if there was no co-culture (control). Coculture with S. aureus (Staphylococcus aureus), Cocultured with LGG (Lactobacillus rhamnosus GG).
- DNA (%): This column contains information of the percentage coverage of the DNA (Deoxyribonucleic acid) area for each replica.
- PI (%): This column contains information of the percentage coverage of the dead cell marker, propidium iodide (PI) area for each replica.
- Coverage (%): Describes the percentage coverage of the dead cells by the total cells area for each replica.
File: Figure2F.csv
Description: The Figure2F.csv file contains information about the impact of the distance between the through holes (Gap 175 micrometers) on the formation of polarized planar crypts overtime (Days 8-23).
Variables
- Gap (Micrometer): Describes the center-to-center distance between two through-holes in the geometry of a 10×10 hole array on a photoresist epoxy film.
- Day (#): Describes the number of days the cells were cultured before fixation.
- Marker: This column contains information on cell identification marker EdU (5-ethynyl-2′-deoxyuridine) used to determine area coverage for stem/proliferative cells.
- Sample (Replicate #): This column contains the label of the replicate used for the analysis: Rep1, Rep2, and Rep3 (Replicate 1, Replicate 2, and Replicate 3).
- Coverage (%): Describes the percentage coverage of the EdU (5-ethynyl-2′-deoxyuridine) area for each replicate.
File: Figure2D.csv
Description: The Figure2D.csv file contains information about the impact of the distance between the through holes (Gap 175 micrometers) on the EdU zone diameter overtime (Days 8-23).
Variables
- Gap (Micrometer): Describes the center-to-center distance between two through-holes in the geometry of a 10×10 hole array on a photoresist epoxy film.
- Day (#): Describes the number of days the cells were cultured before fixation.
- Crypt (#): Describes the label of crypt that was analyzed in this condition. A total of 50 crypts were analyzed for each time.
- EdU zone diameter (micrometer): The length in micrometers of a line drawn from one side to the other of the area covered by EdU-positive cells within a single crypt.
File: Figure6D.csv
Description: The Figure6D.csv file contains information about the impact of the α-hemolysin toxin on integrin b4 from cells covered with and without a mucus layer.
Variables
- Gap: Describes the center-to-center distance between two through-holes in the geometry of a 10×10 hole array on a photoresist epoxy film.
- Mucus: This is classified as with mucus (+) or no mucus (-), which means that the cells generated (+) mucus during culture or did not generate it (-).
- Toxin: This column specifies the treatment with (+) or without (-) α-hemolysin toxin.
- Marker: This column specifies which cell marker (integrin b4) was analyzed after treatment with the toxin.
- Distance (micrometer): This column represents the intensity profile measured along a straight line (distance in micrometers).
- Replicate 1: This column contains information of the intensity profile for replicate 1
- Replicate 2: This column contains information of the intensity profile for replicate 2
- Replicate 3: This column contains information of the intensity profile for replicate 3
File: Figure4B.csv
Description: The Figure4B.csv file contains information about the goblet cell characterization by Mucins (Muc5AC, Muc1, Muc2), Lectin-based binding (LEL, LTL, WGA), antimicrobial peptide (RELM-β), and mucin maturation proteins (AGR2, TFF3).
Variables
- Gap (Micrometer): Describes the center-to-center distance between two through-holes in the geometry of a 10×10 hole array on a photoresist epoxy film.
- Marker: This column represent the marker of study: Muc5AC (Mucin 5AC), Muc1 (Mucin 1), Muc2 (Mucin 2), LEL (Lycopersicon esculentum lectin), LTL (Lotus tetragonolobus lectin), WGA (wheat germ agglutinin), RELM-β (Resistin-like molecule-β), AGR2 (Anterior Gradient 2), TFF3 (Trefoil factor 3).
- Sample (Replicate #): This column contains the label of the replicate used for the analysis: Rep1, Rep2, and Rep3 (Replicate 1, Replicate 2, and Replicate 3).
- Coverage (%): Describes the percentage coverage of the marker area for each replicate.
File: Figure6C.csv
Description: The Figure6C.csv file contains information about the impact of the α-hemolysin toxin on β-actin from cells covered with and without a mucus layer.
Variables
- Gap (Micrometer): Describes the center-to-center distance between two through-holes in the geometry of a 10×10 hole array on a photoresist epoxy film.
- Mucus: This is classified as with mucus (+) or no mucus (-), which means that the cells generated (+) mucus during culture or did not generate it (-).
- Toxin: This column specifies the treatment with (+) or without (-) α-hemolysin toxin.
- Marker: This column specifies which cell marker (β-actin) was analyzed after treatment with the toxin.
- Distance (micrometer): This column represents the intensity profile measured along a straight line (distance in micrometers).
- Replicate 1: This column contains information of the intensity profile for replicate 1
- Replicate 2: This column contains information of the intensity profile for replicate 2
- Replicate 3: This column contains information of the intensity profile for replicate 3
File: Figure3B.csv
Description: The Figure3B.csv file contains information about the impact of the distance between the through holes on the cell markers: Muc2 (Mucin 2), ChgA (Chromogranin A), ALP (Alkaline phosphatase), KRT20 (Keratin 20), EdU (5-ethynyl-2′-deoxyuridine), DNA (deoxyribonucleic acid), ZO-1 (Zonula occludens-1), OCLN (Occludin).
Variables
- Gap (Micrometer): Describes the center-to-center distance between two through-holes in the geometry of a 10×10 hole array on a photoresist epoxy film.
- Marker: Muc2 (Mucin 2), ChgA (Chromogranin A), ALP (Alkaline phosphatase), KRT20 (Keratin 20), EdU (5-ethynyl-2′-deoxyuridine), DNA (deoxyribonucleic acid), ZO-1 (Zonula occludens-1), OCLN (Occludin).
- Sample (Replicate #): This column contains the label of the replicate used for the analysis: Rep1, Rep2, and Rep3 (Replicate 1, Replicate 2, and Replicate 3).
- Coverage (%): Describe the percentage coverage of the DNA, EdU, KRT20, Muc2, ALP, ChgA, ZO-1, and OCLN area for each replica.
File: Figure5F.csv
Description: The Figure5F.csv file contains information about the mucus thickness build up over time.
Variables
- Gap (Micrometer): Describes the center-to-center distance between two through-holes in the geometry of a 10×10 hole array on a photoresist epoxy film.
- Day (#): Describes the number of days the cells were cultured before fixation.
- Marker: Contains information of the marker Muc2 (mucin 2) used for mucus identification and measurement.
- Sample (#): This column contains the label of the replicate used for the analysis: Rep1, Rep2, and Rep3 (Replicate 1, Replicate 2, and Replicate 3).
- Thickness (Micrometer): This column contains data of the distance between the DNA of the cells and the fluorescent red beads. This distance represents the thickness of the mucus secreted and accumulated.
File: Figure2E.csv
Description: The Figure2E.csv file contains information about the impact of the distance between the through holes (Gap 350 micrometers) on the EdU zone diameter overtime (Days 8-23).
Variables
- Gap (Micrometer): Describes the center-to-center distance between two through-holes in the geometry of a 10×10 hole array on a photoresist epoxy film.
- Day (#): Describes the number of days the cells were cultured before fixation.
- Crypt (#): Describes the label of crypt that was analyzed in this condition. A total of 50 crypts were analyzed for each time.
- EdU zone diameter (Micrometer): The length in micrometers of a line drawn from one side to the other of the area covered by EdU-positive cells within a single crypt.
File: Figure5BC.csv
Description: The Figure5BC.csv file contains information about the Muc2 (mucin 2) and DNA (deoxyribonucleic acid) coverage area representing the presence of goblet cells.
Variables
- Gap (Micrometers): Describes the center-to-center distance between two through-holes in the geometry of a 10×10 hole array on a photoresist epoxy film.
- Sample (#): This column contains the label of the replicate used for the analysis: Rep1, Rep2, and Rep3 (Replicate 1, Replicate 2, and Replicate 3).
- Day (#): Describes the number of days the cells were cultured before fixation.
- Marker: Contains information of the marker Muc2 (mucin 2) or DNA (deoxyribonucleic acid) used.
- Coverage (%): Describe the percentage coverage of the DNA, or Muc2 area for each replicate.
File: FigureS3D.csv
Description: The FigureS3D.csv contains data that represents a predicted WNT (Wnt-3a protein) concentration determined in the geometry of a 10 holes in a row as time-dependent in a simulation through the 40 µm collagen-filled holes and a top layer of 10 µm collagen.
Variables
- Gap (micrometers): Describes the center-to-center distance between two through-holes in the geometry of a 10 holes in a row in a two-dimensional simulation.
- Length (micrometers) : Distance in micrometers from the first hole to the 10th hole in the row.
- Predicted WNT (Wnt-3a protein) concentration (nM): Results of the computationally simulated WRN (Wnt-3a, R-spondin, and Noggin proteins) concentration, from basal to luminal diffusion through each hole in the row.
File: FigureS3C.csv
Description: The FigureS3D.csv contains data that represents a predicted WNT (Wnt-3a protein) concentration determined in the first hole of the 10 holes row simulation.
Variables
- Gap (micrometers): Describes the center-to-center distance between two through-holes in the geometry of a 10 holes in a row in a two-dimensional simulation.
- Length (micrometers): Distance in micrometers from side to side of the first hole.
- Predicted WNT (Wnt-3a protein) concentration (nM): Results of the computationally simulated WRN (Wnt-3a, R-spondin, and Noggin proteins) concentration, from basal to luminal diffusion through a single hole.
File: Videos.zip
Description: The Videos.zip folder contains 5 files:
3D-reconstruction2-Crypts with mucus layer.mp4
3D-reconstruction-Crypts with mucus layer.mp4
Brightfield-Array with mucus.avi
WNT Diffusion simulation-175 Micrometer Gap.gif
WNT Diffusion simulation-350 Micrometer Gap.gif
File: 3D-reconstruction2-Crypts with mucus layer.mp4
Description: This video shows the amount of mucus accumulated over 23 days of culture. The video displays cell nuclei in blue (DNA), the secreted and accumulated mucus in yellow (Muc2), and fluorescent beads in red. Different angles and imaging styles are used to visualize the mucosal layer and the Muc2-positive goblet cells.
File: 3D-reconstruction-Crypts with mucus layer.mp4
Description: This video shows the amount of mucus accumulated over 18 days of culture. The video displays cell nuclei in blue (DNA), the secreted and accumulated mucus in yellow (Muc2), and fluorescent beads in red. Different angles and imaging styles are used to visualize the mucosal layer and the Muc2-positive goblet cells.
File: Brightfield-Array with mucus.avi
Description: This video shows a 10x10 array of crypts in in vivo culture, using brightfield microscopy to observe cellular growth and the viscous layer coating them.
File: WNT Diffusion simulation-175 Micrometer Gap.gif
Description: This animated image shows a simulation of the diffusion of WNT (Wnt-3a protein) from the basal medium toward the luminal side, through an orifice 70 µm in diameter and 40 µm in depth. The concentration prediction is consistent with the formation of a concentration gradient (WNT concentration: basal 1 nM—red—; luminal 0 nM—blue—) established over the time interval of 0 to 720 seconds.
File: WNT Diffusion simulation-350 Micrometer Gap.gif
Description: This animated image shows a simulation of the diffusion of WNT (Wnt-3a protein) from the basal medium toward the luminal side, through an orifice 70 µm in diameter and 40 µm in depth. The concentration prediction is consistent with the formation of a concentration gradient (WNT concentration: basal 1 nM—red—; luminal 0 nM—blue—) established over the time interval of 0 to 720 seconds.
Code/software
Files .cvs can be opened using Excel or free desktop software such as CSViewer or Modern CSV, or import files directly into Google Sheets.
Files .mp4, .gif, and .avi can be opened using media players like VLC Media Player, Windows Media Player, or QuickTime.
Access information
Other publicly accessible locations of the data:
- No other publicly accessible locations are used.
Data was derived from the following sources:
- No other sources were used.
Data was collected using confocal microscopy on fixed or live imaged samples; consult the main publication for full details.
Equipment: A confocal laser scanning microscope (Olympus, Fluoview FV3000) was used to acquire the data used for this study.
No custom code was used to generate or process the data described in this data descriptor.
Processing Information: Images were processed using ImageJ Fiji or Imaris (Bitplane, Switzerland). ROI were manually cured and measure.
Usage Notes: The data can be used for further analysis with the methods described in the associated publication.