Be-Doubleflow Application Notes: Gut-on-chip 1

This application note focused on work developed by Ainia on the development of a gut-on-chip model using Be-Doubleflow device. It is the part one of two application notes.

Introduction

This guide compares the Be-Doubleflow device to an insert platform as control as in vitro platforms for gut models. Cell viability, layer formation and cell differentiation into intestinal cell phenotypes were evaluated in both systems.

Materials

1. Be-Doubleflow device

Figure 1 Be-Doubleflow device and cell culture scheme in the device channels.

Be-Doubleflow design (Figure 1) consists of two perfusable channels connected via a PET porous membrane. This device is optimal for epithelium/endothelium barrier models when flux plays a role in both sides of the co-culture. In this work, the upper/apical channel is destined for intestine lumen and the lower/basal channel as endothelial vessel.

* The volumes presented in the table are theoretical values calculated for the standard products. Changes in the device features of custom chips may modify the exact channel volume.

2. Rocker

Typically, the medium volume of the channels is limited. To promote optimal cell growth, it is essential to refresh the supply of nutrients and oxygen. Therefore, to replenish the cell culture medium (after cell attachment but before connecting any perfusion system) the rocker (orbital shaker) (Figure 2) constitutes a practical tool (video).

Several microfluidic devices can be placed simultaneously, ensuring the renewal of the medium within the channel by tilting the devices back and forth. The rocker also permits control over the degree of inclination, speed and working time.

3. Cell types

For the development of an intestinal monolayer, researchers consider two specific cell types:

First, researchers use the Caco-2 cell line (ATCC® HTB-37TM, USA). This cell line has been a standard in in vitro intestinal models since the 1990s. They culture these cells in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% fetal bovine serum (FBS). Additionally, the medium includes antibiotics, such as penicillin (100 U/mL) and streptomycin (30 µg/mL), as well as 1% non-essential amino acids (NEAA). Researchers then incubate the cells at 37 °C in a humidified atmosphere containing 5% CO₂.

In addition, researchers use the HT-29 cell line (ATCC® HTB-38TM, USA). This cell line is modified to a mucus-secreting cell type with methotrexate (MTX). They culture these cells in McCoy’s 5a Medium Modified, supplemented with 10% FBS and antibiotics. The antibiotics include penicillin (100 U/mL) and streptomycin (30 µg/mL). Afterward, they place the cells in a CO₂ incubator set at 37 °C, maintaining a humidified 5% CO₂ atmosphere.

By maintaining these two cell types under precise conditions, researchers ensure robust and reproducible intestinal monolayer models for further study.

4. Traditional culture insert

Traditional culture inserts (0.4 µm porous size) adapted for 6-well cell culture plates are used as the control system.

Methods

1.    Be-Doubleflow cell culture

Before seeding, prewarm the Be-Doubleflow device in the incubator overnight to minimise the formation of air bubbles.

1. Fill the top channel of the device with 100 µL of 0.1 mg/mL of collagen or 0.1% gelatin (diluted in PBS) and incubate at 37°C for 30 minutes. Wash the channel by smoothly adding 100 µL of PBS into the inlet and removing it at the outlet well with the pipette. Repeat the washing step three times.
2. Aspire gently the dilution buffer completely before seeding.
3. Pipette through the pinhole of the apical channel 50 µL of culture medium with 10⁶ Caco-2/HT-29/MTX (9:1) cells resuspended. Cover the inlets and incubate the cells for 5-6 h).
4. After cell attachment, add 300 µL of culture medium to the medium reservoirs. Add PBS/water to the evaporation reservoirs, cover and keep the device in the incubator in the rocker for up to 7 or 21 days. Change cell culture medium every 2-3 days.

 2.    Insert culture

1. Fill the bottom of the apical side of the insert with 100 µL of 0.1 mg/mL of collagen or gelatin (diluted in PBS) and incubate at 37 °C for 30 minutes. Wash the insert with PBS three times.
2. Pipette the cell solution containing 6×10⁵ cells/cm² (same as the Be-Doubleflow).

 3.    Viability and proliferation assays

To assess the viability and proliferation of the cells during culture, researchers can perform assays such as alamarBlue (DAL1025, ThermoFisher Scientific, USA) cell viability. The active ingredient, resazurin, enters the cell membrane. Then, live cells reduce it to resorufin, which produces pink fluorescence. Researchers detect this fluorescence to evaluate cell viability.

Furthermore, the assay quantitatively evaluates the number of cells and viable cells. Researchers use an absorbance/fluorescence-based microplate reader for this assessment.

1. At the chosen timepoint, remove the device from the incubator and place it in the cell culture hood.
2. Remove the medium from the inlet and outlet reservoirs without emptying the cell channel.
3. Wash the channel with PBS smoothly adding 100 µL of PBS into the inlet and removing it at the outlet well with the pipette. Repeat the washing step three times.
4. Place 100 µL of the alamarBlue reagent in the inlet and wait for it to reach the outlet. If needed, slowly aspirate the liquid through the pinhole of the outlet.
5. Place the device in the incubator for 1 h at 37 °C.
6. Place the device in a plate reader or fluorescence spectrophotometer with 530/590 nm (excitation/emission) filter settings.
7. To calculate the cell viability relative to the values obtained at 7 days, use the following equation:

4.    Microscopy monitoring

The monolayer formation can be tracked through a phase contrast microscopy on specific timepoints (days 4 and 7 for the results presented).

5.    RNA isolation and Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

Differentiation markers can be studied through qRT-PCR on the intended culture days (in case of the presented results it was at days 7 and 21).

1. At the chosen timepoint, remove the device from the incubator and place it in the cell culture hood.
2. Remove the medium from the inlet and outlet reservoirs without emptying the cell channel.
3. Wash the channel with PBS smoothly adding 100 µL of PBS into the inlet and removing it at the outlet well with the pipette. Repeat the washing step three times.
4. Pipette 100 µL of extraction buffer from Maxwell kit to detach the cultured cells.
5. Place the sample in MAXWELL equipment (Promega, Madison, WI, USA) for cellular RNA isolation and purification.
6. Use Nanodrop (Thermo Fisher Scientific) for quantification and quality evaluation of the extracted RNA.
7. Collect 1 μg of extracted RNA and use the High-Capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA) to obtain cDNA.
8. Place the cDNA obtained with the commercial primers selected in TAQMAN™ fast advanced master mix (Thermo Fisher Scientific) for real-time PCR.

Table 1: Genes evaluated in the results presented and their reference from Thermo Fisher Scientific.

9. Quantify the gene expression relative to a reference/housekeeping gene, demonstrating the magnitude of the physiological changes in                the genes of interest. Use the formula 2-ΔΔCT.

Results obtained using the guide protocol

Cell viability and proliferation with two different coatings

Researchers used gelatin and collagen as coating solutions and assessed cell viability and proliferation after 7, 14, and 21 days of culture. The results showed that both gelatin and collagen coatings maintained high cell viability, similar to the control system after 7 days of culture. After this time point, the cell viability increased in both coatings, matching the control (Figure 3). Viability values above 100% indicated cell proliferation, which researchers observed in both coatings.

Between gelatin and collagen coatings, they found no significant differences. However, when comparing the results at day 21, the gelatin coating in the Be-Doubleflow device showed a significant difference compared to the control. As a result, researchers chose the gelatin coating for the next experiments.

Figure 3 Cell viability (and proliferation) in the culture on the Be-Doubleflow device and control system for 7, 14 and 21 days. (A) Cell viability with collagen coating and (B) cell viability with gelatin coating. Data are presented as the mean ± standard error of the mean. Unpaired t-test with Welch’s correction statistical analysis performed: *** = p < 0.001.

Epithelial monolayer evaluation

We observed the morphology of the cells in both systems with a gelatin coating on days 4 and 7. The epithelial monolayer formed in the Be-Doubleflow system showed no gaps, which validated its integrity. This result was similar to the monolayer formed in the control system (Figure 4).

Figure 4: Phase contrast images of the cell (Caco-2: HT-29/MTX) monolayer on the control system (Transwell) and Be-Doubleflow at day 4 and day 7 of culture.

Cell differentiation in the model

The cell differentiation was evaluated on days 7 and 21 in Be-Doubleflow and in the control system. Results showed that the cells cultured in Be-Doubleflow devices had a significant increase in Goblet and Paneth cell biomarkers after 7 and 21 days, compared with control, which points to a more heterogeneous cell population, more reminiscent of the native tissue (Figure 5). Between days 7 and 21, there are no significant differences between biomarket expression levels, which can indicate that these biomarkers are expressed as early as the first time point.

Figure 5 Gene expression of cells cultured in control system and Be-Doubleflow device at days 7 (A) and 21 (B). Data are presented as the mean ± standard error of the mean. Unpaired t-test with Welch’s correction statistical analysis performed: * = p < 0.05; ** = p < 0.01; *** = p < 0.005; **** = p < 0.001.

Conclusions

The Be-Doubleflow device provides a suitable platform for intestine modeling as a gut-on-a-chip. In this model, researchers observed high cell viability and proliferation rates, similar to the control system. Additionally, the Be-Doubleflow device improved cell differentiation. Specifically, they saw an increase in mucin- and defensin-secreting cells in the gut-on-chip model. Moreover, gene expression in these cells increased as early as day 7. This early gene expression suggests quicker differentiation in the microfluidic system, compared to the standard 21 days of culture.

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References

Ashammakhi, N., Nasiri, R., Barros, N. R. de, Tebon, P., Thakor, J., Goudie, M., Shamloo, A., Martin, M. G., & Khademhosseini, A. (2020). Gut-on-a-chip: Current progress and future opportunities. Biomaterials, 255, 120196.