Biochemical and rheological analysis of human colonic culture mucus reveals similarity to gut mucus

Abstract

The goal of this project was to validate the functional relevance and utility of mucus produced by an in vitro intestinal cell culture model. This is facilitated by the need to physiologically replicate both healthy and abnormal mucus conditions from native intestinal tissue, where mucus properties have been connected to intestinal disease models. Mucus harvested from colonic cell cultures derived from healthy donors was compared to mucus collected from surgically resected, noninflamed transverse colon tissue. The rheological and biochemical properties of these mucus samples were compared using oscillational rheometry, particle-tracking microrheology, multiangle laser light scattering, refractometry, and immunohistochemical imaging. An air-liquid interface culture of primary human colonic epithelial cells generated a continuous monolayer with an attached mucus layer that displayed increasing weight percent (wt%) of solids over 1 week (1.3 ± 0.5% at 2 days vs. 2.4 ± 0.3% at 7 days). The full range of mucus concentrations (0.9-3.3%) observed during culture was comparable to that displayed by ex vivo mucus (1.3-1.9%). Bulk rheological measurements displayed similar wt%-based complex viscosities between in vitro and ex vivo mucus, with the complex viscosity of both systems increasing with wt% of solids. Particle-tracking microrheology showed higher complex viscosities for ex vivo mucus samples than in vitro mucus which was explained by a greater fraction of water present in in vitro mucus than ex vivo, i.e., in vitro mucus is more heterogeneous than ex vivo. Refractometry, multiangle laser light scattering, and immunostaining showed increased mucus complex size in ex vivo mucus compared with in vitro mucus, which may have been due to the admixture of mucus and cellular debris during ex vivo mucus collection. The air-liquid interface culture system produced intestinal mucus with similar composition and rheology to native human gut mucus, providing a platform to analyze pathological differences in intestinal mucus.

Figure 1

Schematic of ALI + VIP culture of colonic cells followed by mucus collection. (a) Cells (green) are seeded on a microporous insert (gray) with expansion medium (orange) in both the apical and basal reservoirs. (b) Cells are grown into a confluent monolayer over the course of 3–5 days. (c) After confluence is achieved, both the apical and basal media are aspirated. Differentiation medium with VIP (red) is placed in the basal reservoir underneath the microporous insert. No medium is added to the apical reservoir. A hydrated continuous mucus layer (yellow) forms on the surface of the epithelial cells. (d) After 2–7 days of culture, the mucus layer is collected.

Figure 2

Properties of the VIP + ALI epithelial monolayers. (a) TEER was measured across confluent layers of colonic epithelial cells (n = 3), cultured on microporous inserts. Additionally, a microporous insert with no cells (submerged in PBS) was measured as a control. (b) Mucus was collected from monolayers (n = 3 per day) over 2–7 days and the weight percentage of solids was measured. (c) The pH of the apical and basal reservoirs of cell monolayers was measured (n = 9). ^∗p < 0.05.

Figure 3

Bulk rheology of mucus samples. In vitro mucus samples grown from three separate transverse colon donors were collected from 2 to 7 days for each sample. Similarly, ex vivo mucus samples were collected from five separate samples of surgical resection tissue from noninflamed transverse colons. These samples were measured in 40 μL aliquots using cone-and-plate oscillational rheometry, measuring the storage modulus (a) and loss modulus (b) of the mucus. To demonstrate gelation, which is indicated when G′ > G′′, G′/G′′ was plotted for each sample (c). The complex viscosity at 1 Hz was calculated and plotted for each sample against the weight percentage of solids (d). Power laws are plotted for both in vitro and ex vivo data for (a), (b), and (d).

Figure 4

Mucus microrheology via microbead tracking. Collected mucus samples from both in vitro and ex vivo donors were tested for weight percentage of solids, and samples within the range of 1.4–2.0% solids were separated into 40 μL aliquots. These aliquots were loaded with fluorescent beads, and the movement of these beads was tracked for 30 s, with 30 videos/image sequences per sample. (a) Representative trajectories of individual beads from in vitro mucus comprised of 1% solids and 3% solids were tracked and plotted in the x-y plane. (b) Complex viscosities were calculated from the average MSD of all beads in the in vitro and ex vivo mucus samples. These viscosity measurements record average η of individual tracked beads for a given ω, across all videos tracked via PTMR for a given sample. (c) The log of the complex viscosity at 1 Hz of each individual bead in each set of samples was calculated, and these values were plotted based on their frequency of occurrence. These histograms displayed two distinct peaks, one close to the viscosity of water, and the other representing beads stuck in mucus complexes via our PTMR tracking software. (d) The frequency-dependent range of complex viscosities was calculated and plotted only for beads embedded in a mucus complex.

Figure 5

Biochemical properties of mucus samples via size-exclusion chromatography with multiangle laser light-scattering and refractometry (SEC-MALS). Mucus was separated by size exclusion chromatography and detected by multiangle light scattering, measuring the total mass of mucins in the sample (a), average molar mass (b), and average radius of gyration (c). ^∗p < 0.05; ^∗∗∗p < 0.001.

Figure 6

Staining of DNA and MUC2 in in vitro and ex vivo samples. Confocal microscopy images were acquired of the in vitro apical washings and ex vivo mucosal scrapings after staining DNA (blue) and MUC2 (red). Scale bar in all images represents 50 μm.

Figure 7

Quantifying the complex size and surface coverage of in vivo and ex vivo. DNA and MUC2 in mucus complexes were stained and their size quantified. Representative images of this result are shown for both in vitro (a) and ex vivo (b) samples. The mean size of these complexes in μm² was calculated (c), and the average surface coverage of mucus over the entire image relative to the background was calculated (d). The number of cells in each image included in this data set was also calculated from the presence of DNA clusters within each image (e). Scale bar for all images is 50 μm. Statistical significance was detected in 7C and 7D (p < 0.01 for each) but not in 7D (p = 0.13).