Fabrication of 3D engineered intestinal tissue producing abundant mucus by air–liquid interface culture using paper-based dual-layer scaffold

Crecia EF Soft Type 100 two-ply paper towel (115 × 218 mm in size: Nippon Paper Crecia, Tokyo, Japan) was used as a main paper material. Other paper materials, Kimwipe paper (S-200, Nippon Paper Crecia, Tokyo, Japan), copy paper (Askul Multipaper Super Economy+, Askul, Tokyo, Japan), and qualitative filter paper (No.2, ADVANTEC, Tokyo, Japan), were also used for control experiments. Gelatin powder from porcine skin (beMatrix™ gelatin LS-H, Nitta Gelatin, Osaka, Japan), 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP: Apollo Scientific, Stockport, UK), and 25% glutaraldehyde (GA: Fujifilm Wako Pure Chemical Corp., Osaka, Japan) for electrospinning microfibers were purchased commercially.

Caco-2 cell derived from a human colon cancer was obtained from RIKEN BRC (Tsukuba, Japan). The following commercially available reagents for cell culture were purchased and used: Dulbecco's modifled Eagle's medium (DMEM: Nacalai Tesque, Kyoto, Japan), non-essential amino acid solution (NEAA: Nacalai), penicillin–streptomycin (P/S) solution (Thermo Fisher Scientiflc, Waltham, MA, USA), fetal bovine serum (FBS: Biosera, Nuaillé, France), trypsin–ethylenediaminetetraacetic acid (Wako), Dulbecco's phosphate-buffered saline (PBS: Nacalai), and 4% paraformaldehyde (PFA) solution in PBS (Wako).

The ϕ12 mm Transwell-clear insert with 0.4 µm pore polyester membrane (1.12 cm² culture area) was purchased from Corning (NY, USA).

The paper-based dual-layer scaffold was fabricated as reported in the previous study [14]. Gelatin powder was dissolved in HFIP and stirred overnight to prepare 10 w/v% gelatin solution. The gelatin solution (3 ml) was loaded into a sterilized syringe (Terumo, Tokyo, Japan). The paper towel was folded in half on the long side and set on a drum collector (C-DR/D200W200, MECC, Fukuoka, Japan). Electrospinning was performed using an electrospinning equipment (NANON, MECC) under the following conditions (figure 1): 22 G syringe needle (Terumo), needle tip–collector distance 15 cm, drum rotating speed 100 rpm, syringe swing width 100 mm, swing speed 10 mm s⁻¹, feed rate 1.5 ml h⁻¹, applied voltage 18 kV, room temperature, and 50% humidity. The obtained dual-layer scaffolds were then exposed to the GA vapor overnight for crosslinking the gelatin to become insoluble in the aqueous cell culture medium. After drying in air for about 2 h, the substrate was heated at 100 °C for 1 h to inactivate unreacted GA. Insolubility of the dual-layer scaffold was examined by incubating in the culture medium at 37 °C for at most 14 d.

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The structures of the dual-layer scaffolds were observed by a scanning electron microscope (SEM: VE-9800, Keyence, Osaka, Japan), after Au sputtering at 35 mA for 0.5 min under vacuum condition (MSP-10, Vacuum Device, Ibaraki, Japan). The sample after the insolubility test was observed with the same pretreatment for the engineered tissue (see section 2.4). The diameter and porosity of the microfiber layer were measured and calculated by using DiameterJ plugin in ImageJ software (NIH, MD, USA).

Caco-2 cells for tissue fabrication were passaged using ϕ100 mm tissue culture polystyrene dishes and the culture medium composed of 88% DMEM, 10% FBS, 1% P/S, and 1% 1 × NEAA in the humidified 5% CO₂ atmosphere at 37 °C. The same medium, atmosphere, and temperature were employed in the ALI culture.

The paper-based dual-layer scaffolds were cut into 2 × 2 cm² pieces and sterilized by exposing both side to UV light for 15 min each. The scaffolds were pre-incubated overnight in 2.5 ml of the culture medium and transferred to a ϕ35 mm petri dish (Corning). 120 µl of the cell suspension (1.84 × 10⁶ cells ml⁻¹) was seeded to the scaffold and incubated for one day, and then 5.33 ml of the medium was added, and further incubation was conducted for another two days to allow Caco-2 cells to adhere well. In the ALI culture, the dual-layer scaffold with cells were just placed on top of a folded paper in a ϕ100 mm petri dish (Corning) filled with the culture medium up to the top of the folded paper (figure 2) and incubated for up to 21 d including the initial three days incubation. The medium was changed every two days. As a control experiment, submerged culture was conducted by placing the dual-layer scaffold directly on a ϕ100 mm petri dish containing the 20 ml medium after the initial three days incubation.

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The ALI culture and the submerged culture using Transwell were also performed as control experiments. 0.5 ml of the cell suspension (1.24 × 10⁵ cells ml⁻¹) was seeded to an insert membrane and cultured for one day. Next, 0.5 ml of the culture medium was added to a bottom well and cultured for another two days for sufficient cell adhesion. For the ALI culture in Transwell, 2.0 ml of the medium was added to the bottom well only. For the submerged culture, 0.5 ml and 1.5 ml of the medium were added to the insert and the bottom well, respectively.

The samples cultured under each condition for 5, 7, 10, 12, and 21 d in total were fixed in 4% PFA and washed with 1 × PBS. Sample preparation and observation for SEM and confocal laser scanning microscopy (CLSM: FV1000, Olympus, Tokyo, Japan) were conducted as described elsewhere [14]. For CLSM observation, actin filaments and nuclei were fluorescently stained with Alexa Fluor 568 Phalloidin and Hoechst 33 342 (Thermo Fisher). Immunofluorescent staining of tight junction protein, zonula occludens-1 (ZO-1) [7], was performed using ZO-1 (D6L1E) rabbit monoclonal antibody and Alexa Fluor 488 anti-rabbit IgG (Cell Signaling Technology, MA, USA).

Cross-sectional images of the 3D engineered intestinal tissues (day 12) were obtained by histological observation with Hematoxylin and Eosin (HE) staining and transmission electron microscope (TEM) observation. The samples for histological observation were prepared by a standard method and observed using an optical microscope (TS100, Nikon, Tokyo, Japan) [18]. For TEM observation (H-7600, Hitachi, Tokyo, Japan), ultra-thin sections of the resin embedded tissues were stained with uranyl acetate and lead hydroxide.

Alanyl aminopeptidase (ANPEP) activities of the engineered intestinal tissues were measured as follows. The culture media of the tissues on day 5, 7, 10, 12, and 21 were replaced with the culture medium supplemented with 1.5 mM L-alanine 4-nitroanilide hydrochloride (L-Ala-NA) as a substrate. The volume of the culture medium was 0.4 ml per 1 cm² of the scaffold plus additional 100 µl for sampling at t = 0 h; total volumes were 1.7 ml for the dual-layer scaffold (4 cm²) and 548 µl for Transwell (1.12 cm²). All the tissues including those fabricated by the ALI culture were immersed in the medium with L-Ala-NA and incubated. For compensating substrate adsorption to the dual-layer scaffold, the intact 1 cm² scaffold was also added to the Transwell samples.

At t = 0 and t = 2 h, 100 µl of each culture medium was transferred to a 96-well plate and absorbance at 405 nm was measured to determine the concentration of p-nitroanilide produced by the enzymatic reaction (PowerScan HT, DS Pharma Biomedical, Osaka, Japan). The ANPEP activity was calculated using the formula (1)

$$\begin{align}U{ } = \frac{{\Delta {\text{Abs}} \cdot V}}{{\varepsilon \cdot l \cdot V^{^{\prime}} \cdot t}}{ }\end{align} \tag{ 1 }$$

U (µmol min⁻¹ l⁻¹): enzyme activity per unit culture area, ΔAbs: Abs_{t = 2h}–Abs_{t = 0h}, V: volume of the medium containing L-Ala-NA per unit culture area (0.4 ml), V': volume of the sample solution (0.1 ml), : molar absorption coefficient (9450 M⁻¹ cm⁻¹) [19], l: optical path length (0.294 cm), t: reaction time (120 min).

The activities of CYP3A4 in the engineered intestinal tissues were measured using P450-Glo^TM CYP3A4 assay and screening system (V9001, Promega, Madison, WI, USA). The culture media of the tissues on day 5, 7, 10, 12, and 21 were replaced with the culture medium supplemented with Luciferin-IPA solution (diluted to 1/1000 in v/v). The volume of the culture medium was 0.4 ml per 1 cm² of the scaffold: 1.6 ml for the dual-layer scaffold and 448 µl for Transwell. The intact 1 cm² scaffold was added to the Transwell samples as in the aminopeptidase assay. After the reaction at 37 °C for 1 h, 25 µl of the medium was transferred to the 96-well plate, and 25 µl of Reconstituted Luciferin Detection Reagent was added to quantify the luciferin produced by CYP3A4. The reaction was continued at room temperature for 20 min, and then luminescence intensity was measured using a microplate luminometer (GloMax96, Promega) and expressed in the relative light unit (RLU). The values of the experiments for each scaffold without cells were subtracted as background.

The tissues on day 5, 7, 10, 12, and 21 of culture were fixed with 4% PFA. After washing with 1 × PBS, the mucus of the tissues was stained with aqueous solution containing 0.1% alcian blue and 3% acetic acid at room temperature for 12 h. The volume of the staining solution was 0.4 ml per 1 cm² of the scaffold: 1.6 ml for the dual-layer scaffold and 448 µl for Transwell. After washing with 1 × PBS, the paper layer of the dual-layer scaffold was peeled off for easy observation with an optical microscope. The images of 10 different fields of view were acquired under the same conditions of transmitted light intensity and the white balance.

The images of blue-stained mucus were quantitatively analyzed in the HSV color model using ImageJ software. Only the blue areas more than the threshold value of 0.5 in the 8-bit images of H (hue) were extracted. For these areas, the inverse of the V values (brightness) in the 8-bit image with 255 levels were calculated so that higher mucus production would result in a higher value, because abundant mucus was significantly stained to give lower light transmission and brightness. The amount of mucus was quantified as the sum of the number of pixels of each brightness multiplied by the inverted value of brightness in each level as shown in the formula (2). For comparison, each value of the mucus production for the four different culture conditions was normalized with respect to the highest value.

$$\begin{align}& {\text{Amount of the mucus production}}\nonumber\\ & \quad = \mathop \sum \limits_{i = 0}^{254} {\text{Inverted value of brightness }}\nonumber\\ & \qquad \times {\text{ Number of pixels }}\end{align} \tag{ 2 }$$

Immunofluorescent staining of mucin 2 (MUC2) for CLSM observation was performed using anti-MUC2 antibody [996/1] (Abcam, Cambridge, UK) and Alexa Fluor 488 goat anti-mouse IgG1 (Thermo Fisher).

All experiments were conducted at least three times. In the statistical analyses, a multiple-group test was performed with one-way analysis of variance (ANOVA) using GraphPad Prism 5J software (GraphPad, MA, USA), and then the Bonferroni method was used to analyze difference between the groups.

The gelatin microfibers were uniformly electrospun and tightly adhered to the paper towel material without any adhesive molecule probably by the physical interaction. The microfiber layer and the paper towel stably maintained the dual-layer structure even after immersion in water for one day. On the other hand, the dual-layer substrates made of other paper materials (Kimwipe paper, copy paper, and qualitative filter paper) were easily separated into a paper layer and a microfiber layer, either automatically or by lightly peeling them off with tweezers (supplementary figure 1). Moreover, among the paper materials examined, the paper towel exhibited high water absorption ratio (supplementary table 1), suggesting it can provide the culture medium to the fiber layer and the cells. Therefore, hereafter, the paper towel was focused as the paper material for the dual-layer scaffold. The SEM image showed that the average diameter of the gelatin microfibers was 0.76 ± 0.13 µm. The average pore size and porosity of the fiber layer were calculated as 4.59 µm² and 56.6%, respectively (figure 3(a)). The microfibers were crosslinked with GA and their morphologies were maintained even after 14 d of immersion in the medium (figure 3(a)) [20]. The paper layer also stably retained its structure and mechanical strength in the medium (data not shown) [21, 22], suggesting that the dual-layer scaffold could be stable during long-term cell culture experiments. The SEM image showed that the pore size and porosity of Transwell with 0.4 µm pore polyester membrane were apparently smaller and lower than the dual-layer scaffold (figure 3(b)), respectively. The average pore size and porosity were calculated as about 0.13 µm² and 0.46%, respectively. Thus, the dual-layer scaffold with higher porosity was expected to more efficiently supply the medium to the cells cultured on the gelatin microfiber layer.

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The SEM images showed that Caco-2 cells were successfully cultured on the dual-layer scaffold because the cells adhered well and stably remained on the microfiber layer without penetrating into the pores between the fibers (supplementary figure 2). In the ALI culture using the dual-layer scaffold (dual-layer scaffold/ALI culture), three-dimensional (3D) convex structures like villous architectures of intestine began to form from day 7 and continuously grew until day 12 (figure 4). The morphology was retained at least 21 d in culture. The high magnification SEM image showed dense microvilli approximately 0.5 µm long on the apical side of the cells (figure 5).

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The cross-sectional images by the CLSM Z-stack observation showed that the villous architectures were about 100 µm in height and diameter in the dual-layer scaffold/ALI culture (figure 5). Furthermore, the histological images also showed 3D convex structures like villous architectures (figures 6(a) and (b)). As in the CLSM image, the height of these villous architectures was about 100 µm and some luminal structures were also observed. On the other hand, the villous architectures were found to be partially composed of multilayers of cells, which could not be seen in the CLSM image due to the low resolution. The microvilli were confirmed by the TEM observation (figure 6(c)). The TEM images also showed tight junction structures characteristic of epithelial tissue as well as desmosomal junctions (figures 6(c) and (d)). Tight junction formation was also suggested by the immunofluorescent staining of ZO-1 (supplementary figure 3). These results indicated that the paper-based dual-layer scaffold in the ALI culture was superior for fabricating 3D intestinal tissue from Caco-2 cells.

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In contrast, even though the microvilli were formed, flat and single layer tissue structures were observed in other culture conditions: submerged culture using the dual-layer scaffold and ALI and submerged culture using Transwell (figure 5). Among them, the cells in the submerged culture using the dual-layer scaffold took vertically long shape to make the tissue thick. Regardless of the culture methods, the cells cultured using the Transwell in this study showed flat and thin shape. Even when the ALI culture was performed using Transwell membranes of 0.4 µm pores with high density or of larger pore size (1.0 and 3.0 µm), the 3D tissue was not formed and only the flat tissues were produced (supplementary figure 4). Therefore, Transwell of 0.4 µm pores with normal density was focused as a control sample (figures 3, 5, 7–9, supplementary figures 3 and 5).

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Time course analyses of ANPEP and CYP3A4 activities were performed for the Caco-2-derived engineered intestinal tissues fabricated under four different conditions (ALI and submerged culture using the dual-layer scaffold and Transwell, respectively).

In the analysis of ANPEP activity (figure 7), the averaged values for the dual-layer scaffold/ALI culture increased from day 5 (10 µmol min⁻¹ l⁻¹) to day 10 (20 µmol min⁻¹ l⁻¹) and they were slightly larger than the submerged culture. However, from day 10 to day 21, the ANPEP activity in the ALI culture was almost constant and therefore became slightly smaller than in the submerged culture. The similar profiles were also obtained for Transwell; the activity of the ALI culture was larger until day 10, but the submerged culture became comparable on day 21. In the comparison between the scaffolds, the ANPEP activity of the dual-layer scaffold was larger than Transwell up to day 10 in both culture methods. But the ANPEP activity became almost the same in all culture systems on day 21 without statistical differences at P < 0.05.

In the analysis of CYP3A4 activity (figure 8(a)), the values in each culture method increased almost two-fold from day 5 to day 10, reaching a maximum on day 10, and decreased on day 21. The averaged values for the dual-layer scaffold were higher than those of Transwell in the both culture methods. In each culture scaffold, the value was higher in the submerged culture than in the ALI culture. Thus, the CYP3A4 activity was the highest in the submerged culture using the paper-based dual-layer scaffold and it showed the maximum value of about 4000 RLU on day 10 of culture.

The statistical analysis on the CYP3A4 activity values was performed (figure 8(b)). There was no statistical difference between the ALI culture using the dual-layer scaffold on day 12, when the 3D villous architectures were formed, and the submerged culture using Transwell on day 21, which previous studies have shown to be necessary for cell differentiation and maturation [9, 12, 13]. On day 12 of culture, a significant difference was found between the submerged culture using the dual-layer scaffold and the ALI culture using Transwell at P < 0.05.

Mucus productions of the engineered intestinal tissues were analyzed using alcian blue staining, which stains acidic mucopolysaccharides blue (figure 9, supplementary figure 4). The microscopic images in figure 9(a) apparently showed that in both ALI and submerged cultures, the tissues fabricated by the paper-based dual-layer scaffold were more deeply stained blue throughout all incubation periods than those of Transwell. In particular, the engineered intestinal tissue fabricated by the dual-layer scaffold/ALI culture showed more remarkable staining images from day 10 of culture.

The quantitative analysis clearly confirmed that the amount of mucus production of the engineered tissues in the paper-based dual-layer scaffold/ALI culture was the largest throughout all incubation periods among the four different culture conditions (figures 9(b) and (c)). It gradually increased from day 5 and reached the maximum value on day 12, coinciding with the timing of the villous architecture formation. Similarly, in the submerged culture with the dual-layer scaffold, the mucus quantity increased until day 12 and remained almost constant to day 21. These mucus productions of the tissues fabricated using the paper-based dual-layer scaffold were significantly larger than those of Transwell, even though they continuously increased until day 21. There were statistically significant differences between the dual-layer scaffold/ALI culture on day 12 and the Transwell/ALI culture on day 12 or the Transwell/submerged culture on day 21 at P < 0.01 (figure 9(c)). When compared between the experiments using Transwell, the ALI culture showed larger values than the submerged culture, suggesting that the ALI culture worked to promote mucus production. The conventional submerged culture using Transwell was the lowest.