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Method Article

Establishing a Three-Dimensional Coculture Module of Epithelial Cells Using Nanofibrous Membranes

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DOI:

10.3791/67780

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December 27th, 2024

 ,  ,  , 
1Department of Pharmacology, School of Medicine, Ajou University, 2Department of Medical Sciences, The Graduate School, Ajou University, 33D Immune System Imaging Core Center, Ajou University

In This Article

Summary

Here, we demonstrate how epithelial cells cultured with fibroblasts in the nanofibrous membrane-based two-layer system can stably adhere to and grow on the membrane.

Abstract

Technical hurdles in a culture of epithelial cells include dedifferentiation and loss of function. Biomimetic three-dimensional (3D) cell culture methods can enhance cell culture efficiency. This study introduces an advanced two-layered culture system intended to cultivate epithelial cells as tissue-like layers with the culture of fibroblasts within a 3D environment. Polyvinyl alcohol (PVA) and poly(ε-caprolactone) (PCL) nanofibrous membranes (NMs) were fabricated via electrospinning and utilized as a physiologically relevant extracellular matrix for the culture of epithelial cells and fibroblasts, respectively. In the upper insert wells, lung epithelial cells were cultivated on the PVA NM, and in the lower chambers, fibroblasts were cultured on the PCL NM. This configuration eliminates direct cell-cell contact and facilitates the examination of paracrine signaling mediated by soluble factors. Confocal microscopy was employed to analyze the distribution, growth pattern, and expression of intracellular proteins, including zona occludens in epithelial cells. Z-stacking techniques enabled detailed 3D reconstructions, providing precise insights into the integrity of tight junctions and spatial organization within the epithelial layer. Scanning electron microscopy (SEM) assessed the morphological characteristics of cell types on the nanofibrous membranes. SEM imaging revealed intricate cell surface structures and interactions with the nanofibers, offering a comprehensive perspective on cellular architecture and cell interaction with nanofibrous structure. The Cell Counting Kit-8 (CCK-8) assay is a simple method for measuring epithelial cell and fibroblast growth rates over time. It provides the proliferative behaviors and potential synergistic effects of coculturing these cells. These findings highlight the effectiveness of a simple insert co-culture system for simultaneous culture of fibroblasts and epithelial cells, which is crucial in various physiological and pharmacological contexts, including epithelial tissue regeneration, tumor microenvironment with endothelial, immune, and other stroma cells, toxicity assay, and drug activity test.

Introduction

Over the decades, the classic two-dimensional (2D) monolayer culture has been essential for understanding infections, pharmacology, and toxicology1,2. However, it is important to consider the complexity, dynamic interactions between multi-compositions, and three-dimensional (3D) architecture of the tissue microenvironment. Thus, the 2D cell culture has limitations in mimicking tissue-like structures3,4. For example, there is a lack of cell-to-cell and cell-to-extracellular matrix (ECM) signaling, which is essential in cell differentiation, proliferation, and cellular functions in the vivo microenvironment. For these reasons, the systems of 3D cell cultures have been developed5. The cells in 3D culture grow and interact with the surrounding extracellular matrix in three dimensions. Furthermore, a 3D coculture approach, composed of more than one cell type, has been developed to bridge the gap between simplistic single-cell type in vitro models and the dynamic cellular interactions that occur in vivo6. 3D coculture systems have gained popularity in the study of cell-cell communication and cell-extracellular matrix (ECM) interactions7,8,9,10. Current 3D coculture systems have several forms and can be separated into direct and indirect coculture8,9. In direct coculture, various cell functions in cultured cells can be mainly affected by direct contact rather than paracrine effects. In comparison, indirect coculture has a greater advantage in investigating paracrine interaction by using barriers or layers to separate different cell types.

Various materials and methods are utilized to fabricate biomimetic scaffolds mimicking the ECM using biocompatible and biodegradable substances10. The nanofibrous scaffold structure mimics the configuration of native ECM in biological tissues, providing a 3D architecture for cell culture11,12. Electrospun nanofibrous membranes (NMs) have been used in direct and indirect coculture of different types of cells13,14,15.

Epithelial cells form a physical barrier of the major organs, whereas fibroblasts are predominant stroma cells that mediate ECM remodeling and regulate the neighboring epithelium6,16,17. Epithelial cells and fibroblasts interact with laminin in the basement membrane and fibronectin in the interstitial matrix, respectively, through integrin receptors on the cell surface18. When epithelial cells are cultured on the poly(vinyl alcohol) (PVA) NMs with diameters of 150 - 250 nm and micropores, they form multi-layers instead of cell aggregates and spheroids, and their growth patterns are similar to those of the cells in epithelial tissue19. In comparison, the poly(caprolactone) (PCL) NMs with 400-1,500 nm diameters and 10-50 µm micropores provide a similar spatial dimensionality to the interstitial matrix for the growth of cultured fibroblasts13,14. Due to their biocompatibility, PVA and PCL membranes are commonly used polymers in tissue engineering20,21.

This study introduces an advanced two-layer culture system using porous electrospun NMs to cultivate epithelial cells as tissue-like layers and fibroblasts within a 3D structure. The protocol can be divided into five key stages: nanofiber fabrication, fiber post-crosslink and quality assessment, preparation of membrane-attached culture well, cell seeding and 3D coculture, and assays of 3D cultured cells.

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Protocol

1. Fabrication of water-stable PVA nanofibers

  1. Weigh 0.02 g of poly(acrylic acid) (PAA) and 1 g of PVA, then add them into a clean glass bottle containing a magnetic bar. Add 7.8 mL of distilled water to the bottle, place the bottle on a plate stirrer, and set the temperature to 84 °C, the speed to 500 rpm, and the time to 12 h.
  2. Let the bottle cool to room temperature before adding 0.2 mL of glutaraldehyde (GA). Place the bottle on the plate stirrer, and set the speed to 500 rpm, time to 30 min, and temperature to room temperature.
  3. Use a 5 mL syringe for electrospinning, separate the plungers from the barrels, and clean small plastic debris inside the barrels and the tip of plungers.
  4. Use needles to block the liquid flow, then pour PVA solution into each barrel (volume: 2.5-3 mL each). Assemble the plungers, then remove the needles.
    NOTE: There might be a bubble in the solution, so wait until the bubble disappears. This takes 10-30 min.
  5. Assemble a new 27 G metal needle, then place it into the electrospinning machine.
  6. Wrap the collector well with foil.
  7. Let the injector panel return to the original position. Use the following settings for machine: (each pump) injection volume: 2 mL, injection rate: 6 µL/min, roller speed: 100 rpm, tip to collector distance: 14 cm. Check the liquid flow for each pump.
  8. Run the machine with an electrical potential of approximately 12 kV.
  9. Ensure that the humidity is 40 - 50% in the beginning and 25 - 30% at the end of the process. Use a humidifier when the humidity decreases and place dishes containing distilled water inside the machine the day before electrospinning to increase humidity.
    NOTE: This is a critical factor in the successful fabrication of PVA nanofibers.
  10. Check the flow regularly. If the needle is blocked, temporarily stop the machine. Use a laboratory wipe to remove the drop and wait until the new drop appears. If not, change the needle, then continue running the machine.
  11. The process of PVA nanofiber fabrication takes ~6 h to complete. After the electrospinning process finishes, detach the foil containing the fiber mat from the collector and store the nanofiber mat in desiccant under vacuum.

2. Treatment of a PVA nanofibrous membrane with hydrochloric acid (HCl) vapor and assessment of fiber quality

NOTE: The water stability of PVA NMs is well preserved after the post-crosslinking reaction by HCl vapor. This process is optimized for suitable thickness, pore size, and roughness of the NMs. PVA is a very hydrophilic polymer, and electrospun PVA nanofibers are unstable in water contact and humid conditions. PVA NMs should be treated with HCl vapor just after the fabrication of the PVA polymer.

  1. Cut a nanofiber mat into pieces of the desired size for the trans-well insert.
  2. Prepare a small dish containing 1.5 mL of pure HCl. Place the HCl-containing dish and nanofiber pieces inside a vacuum chamber and close the lid.
    NOTE: HCl vapor is used as a postcrosslink agent to make the nanofiber water-stable.
  3. Open the valve connected to the vacuum pump, wait 10 s until the HCl fume rises, and then close the valve. Treat the membranes with HCl fume for 2 min.
  4. After treatment with HCl vapor, add some drops of distilled water (DW) to the membranes, put them on a glass slide, and remove the membrane from the foil. After air drying for 20 min, the membranes naturally stick to the slide.
  5. Coat the membranes on carbon tape with a thin layer of gold using an autosputter coater. Obtain scanning electron microscope (SEM) images.
  6. After crosslinking PVA NMs with HCl, wash them 3x with 1x PBS, and check the pH after soaking them in culture media to confirm the absence of residual HCl.

3. Fabrication of PCL nanofibers and nanofiber quality assessment

  1. Weigh 1.5 g of PCL and add it to a clean glass bottle. Add 8.5 mL of chloroform to the bottle, place the bottle on a plate stirrer, and dissolve PCL in chloroform for 12 h at room temperature.
  2. Repeat steps 1.3 to 1.9, except for steps 1.7 and 1.8, set the injection rate at 8 µL/min and the voltage at 17.5 kV.
    NOTE: PCL fabrication takes ~3.5 h to finish.
  3. Cut a small piece of the membrane and coat the pieces by auto-sputter process for 5 min. Obtain SEM images.

4. Assembly of two layers of membranes in a trans-well

NOTE: In this protocol, we use a 24-well plate and its respective insert well. The PVA NM is attached to the insert well, while the PCL NM is attached to the bottom well.

  1. Cut a nanofiber mat into round pieces, with a diameter of 10 mm and 13 mm for PVA and PCL NMs, respectively.
  2. Use polydimethylsiloxane (PDMS) Sylgard 184 as an adhesive agent to attach the membranes to the insert and chamber well. Before using, mix Sylgard 184 Base (part A) and Curing Agent (part B) in a plastic cup in a 10:1 mass ratio. Use a clean glass slide to mix the PDMS solution vigorously for 2-3 min until the entire mixture is filled with bubbles. Let the bubble form and disappear for 10 min before using; meanwhile, let the slide warm to 100 °C.
  3. Spread the PDMS on a glass slide, and dip the bottom edge of the insert well slightly in the PDMS. Keep the insert well upside down on the slide warmer for the PDMS to harden (~10-15 min). Carefully check with a 200 µL tip to make sure the PDMS is not sticky.
  4. Treat the PVA NM pieces as described in steps 2.2 to 2.4.
  5. Gently press the insert well onto the PVA NM piece so that the bottom of the insert well faces the nanofiber side of the membrane piece. Add a drop of distilled water to the center of the well and use forceps to remove the foil. Put the insert well in a new 24-well plate, and let the membrane dry.
  6. When attaching the PCL NM piece to the bottom well, be sure to dip the 200 µL tip in the PDMS mixture and dot it precisely at the four corners and in the center.
  7. Place the plate on the slide warmer and allow the PDMS to harden for 5-7 min to ensure the quality of the assembly.
  8. Put the PCL NM piece into the PDMS-containing well, with the nanofiber side of the membrane attached to the PDMS. Use a small clean brush to press the foil side of the membrane piece slightly for complete attachment. Add 0.2 mL of 70% ethanol to the well to easily remove the foil.
  9. Sterilize the insert and the bottom well in the trans-well setup under UV light overnight.
    NOTE: The mixed PDMS can be stored at -20 °C for further use. CAUTION: The PCL nanofibrous membrane-attached plate can be stored dry but must be wet with 70% ethanol before use.
  10. The next day, wash the insert and bottom well by adding 200 µL and 500 µL of 1x phosphate-buffered saline (PBS), respectively, for 3 x 10 min. The insert and bottom well are ready to use.

5. Cell seeding and assembly of two layers for the coculture model

NOTE: MLE-12 cells were cultured in 1x Dulbecco's Modified Eagle Medium (DMEM)/F12 with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin antibiotic. NIH3T3 cells were cultured in 1x DMEM high glucose with 10% FBS and 1% penicillin/streptomycin antibiotic.

  1. Maintain the cells in a 150 mm culture dish. When they are 70-80% confluent, wash the cells with 10 mL of 1x PBS and trypsinize them with 2 mL of prewarmed 0.05% trypsin/EDTA solution for 3 min at 37 °C.
  2. Gently tap the dish to dislodge the cells from the bottom, and then add 8 mL of complete culture media to each type of cell to neutralize the trypsin enzyme. Transfer the cell suspension to a 50 mL centrifuge tube, centrifuge at 400 × g for 5 min, and discard the supernatant. Stain the cells with Cell-Tracker to observe the distribution of seeded cells on NM (see section 6 for more details).
  3. Resuspend the cell pellet in 5 mL of complete DMEM/F12 media. Determine the cell number and adjust to 6 × 106 MLE-12 cells/mL and 8 × 105 NIH3T3 cells/mL.
    NOTE: Complete DMEM/F12 media is used for coculture with MLE-12 and NIH3T3 cells.
  4. Fill the bottom well with 500 µL of complete DMEM/F12 media (the well without PCL). Transfer the PVA nanofibrous membrane-attached insert to the trans-well, and add 50 µL of MLE-12 cell suspension. Incubate the cells at 37 °C and 5% CO2.
  5. Simultaneously, fill the PCL nanofibrous membrane-attached bottom well with 500 µL of NIH3T3 cell suspension and incubate the cells at 37 °C and 5% CO2.
  6. After 2 h, assemble the MLE-12 cell-containing insert and the NIH3T3 cell-containing bottom well in the trans-well setup. This is the coculture model with MLE-12 cells as the upper layer and NIH3T3 cells as the lower layer. Coculture the cells in the two-layered membranes for the desired time.

6. Cell distribution assay

  1. Prepare 5 µM of Cell-Tracker red and green solution in a total volume of 2 mL of complete DMEM/F12 medium.
  2. After trypsinization, mix MLE-12 cells with previously prepared Cell-Tracker red solution and NIH3T3 with Cell-Tracker green solution. Incubate the cells at 37 °C and 5% CO2 for 30 min to ensure optimal cell labeling. Add 8 mL of complete DMEM/F12 medium to the samples and centrifuge at 400 × g for 5 min.
    NOTE: Cell Tracker cannot affect cell viability and proliferation. Hence Cell-Tracker-stained cells can be cultured until confluence. The fluorescence intensity in the stained cells might reduce 4-5 days after culture.
  3. Repeat steps 5.3 to 5.6.
  4. While observing under a confocal microscope, transfer the insert well from the 24-well plate to a 12-well plate.
    NOTE: This minimizes the distance between the objective lens and the cells, allowing for the observation of cell morphology.

7. Confocal observations of 3D cocultured cells

NOTE: Epithelial cell attachment to a PVA NM is weak compared to that of fibroblasts to a PCL NM. Discard old media manually by pipette aspiration and add new media or solution gently to the wall of wells. During the staining process, try not to let the membranes dry.

NOTE: Cell attachment can be improved by coating laminin or incorporating peptides of integrin-binding motifs in PVA nanofibers19.

  1. Transfer the insert to a new 24-well plate. Discard the old media and add 150 µL of a 4% paraformaldehyde (PFA) solution. Incubate for 15 min at room temperature.
  2. Remove the 4% PFA solution and wash the adhered cells with 150 µL of 1x PBS.
  3. For blocking and permeabilization, add 150 µL of 5% normal goat serum and 0.2% Triton X-100 diluted in 1x PBS and incubate for 30 min at room temperature.
  4. Prepare the Alexa Fluor 594-conjugated anti-zona occludens Ab (1:400) and Phalloidin-iFlour 488 (1:1,000) by diluting them in antibody diluent reagent. Remove the blocking solution, add 150 µL of the fluorophore solution per well, and incubate for 1 h at room temperature.
    NOTE: Prepare the antibody solutions with utmost precision, as it will directly impact the quality of the results. Avoid exposure to light.
  5. Discard the antibody solution and wash the cells with 150 µL of 1x PBS for 10 min.
  6. Add 150 µL of Hoechst 33342 diluted in 1x PBS (1:1,000) and incubate for 10 min; avoid exposure to light.
  7. Remove the Hoechst 33342 solution and wash the adhered cells with 150 µL of 1x PBS for 10 min.
  8. Prepare a clean glass slide and add a precise amount of aqueous mounting gel. Use forceps with pointed tips to delicately detach the membranes from the surrounding edge. Place the membranes on the mounting gel, ensuring the side containing the cells is facing upward. Add a precise amount of mounting gel and cover with a cover slide. Seal the slides with nail polish, ensuring a precise and complete seal.
  9. Observe cells under a confocal microscope using the following imaging settings: Laser Power control: 405 Intensity 10; Laser Power control: 488 Intensity 10; Laser Power control: 561 Intensity 10; Detector sensitivity: 420LP (600 value); Detector sensitivity: 525/50 (700 value); Detector sensitivity: 561LP (750 value).

8. Assay of cell attachment to nanofibers

  1. Discard the old media and wash the cells with 150 µL of 1x PBS.
  2. Fix the cells with 150 µL of 3% GA diluted in 1x PBS for 1 h at room temperature.
  3. Wash the cells with 150 µL of 1x PBS in 10 min.
  4. Fix the cells secondarily by adding 150 µL of 1% osmium tetroxide (OsO4) diluted in 1x PBS for 1 h.
    CAUTION: OsO4 and its vapor are harmful. It must be used in a fume hood.
  5. Discard the solution and wash the cells with 300 µL of 1x PBS for 2 x 10 min.
  6. Dehydrate the cells with graded ethanol from 50%, 70%, 90%, and 95% to 100%, for 10 min in each step. Perform the last dehydration step by adding fresh 100% ethanol for 10 min.
  7. Air dry the cells and membranes completely. Use forceps with the pointed tips to detach the membranes, and place the membranes on the specimen holder using a two-side tap. Perform ion-sputtering for 5 min before observing the cells under SEM at 2,000x magnification.

9. Cell growth assay

NOTE: In this protocol, the CCK-8 assay is used to quantify cell growth rate following the manufacturer's instructions.

  1. Prepare the CCK-8 mixture by mixing 9 parts of prewarmed DMEM/F12 (by volume) with 1 part of the CCK-8 reagent. After removing the old culture media, add 150 µL of the CCK-8 mixture to the cell-containing well.
  2. Transfer the insert to the new 24-well plate to separate it from the bottom well containing cells. Incubate the cells for 45 min at 37 °C and 5% CO2.
  3. Collect the supernatant and transfer it to a 1.5 mL tube. Centrifuge at 1,800 × g for 3 min.
  4. Add 100 µL of the supernatant from the microcentrifuge tube to the 96-well plate. Read optical density (OD) at 450 nm.

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Results

This protocol outlines the critical steps for coculturing MLE-12 lung epithelial cells and NIH3T3 fibroblasts by constructing a nanofibrous membrane-based two-layer model (Figure 1). This model suits live cell imaging, immunohistochemistry, and endpoint quantitative analysis of cocultured cells. Other cell types may also be utilized by optimizing cell density and growth conditions.

SEM shows the uniform distribution of fabricated PVA nanofibers without any bead fo...

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Discussion

The transition from 2D culture to 3D culture models represents a significant advancement in developing in vitro coculture models. A 3D culture model must mimic the tissue microenvironment in which cells can proliferate, aggregate, and differentiate22. The use of scaffold-based 3D coculture models enhances the replication of tissue architecture. In this study, an NM-based trans-well system provides indirect 3D coculture of two types of cells. The PCL NM with large pores in the lower chambe...

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Disclosures

The authors declare no conflicts of interest.

Acknowledgements

This work was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HR16C0001); the Basic Science Research Capacity Enhancement Project through the Korea Basic Science Institute (National Research Facilities and Equipment Center) grant funded by the Ministry of Education (2019R1A6C1010003); and National Research Foundation of Korea (NRF) grants funded by the Korean Government (MSIT) (2022R1A4A5032702).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
0.05% Trypsin/EDTAWelgeneLS015-01
100x Penicilin/StreptomycinGibco10378016
20x PBSLPS solutionCBP007A
4% ParaformaldehydeBiosesangPC2031-050-00
Alexa Fluor-594 conjugated ZO-1 antibodyInvitrogen339194
Antibody diluent OP QuantoEpredia10129-576
CCK-8 assay kitDonginbioCCK-3000
Cell culture dish (150x20)SPL20151
CellTracker Green CMFDAInvitrogenC7025
CellTracker Red CMTPXInvitrogenC34552
ChloroformSamchunC0584
Conical tubeSPL50050
Cover glassCorningCLS2980245
DMEM high glucoseWelgeneLM001-05
DMEM/F12WelgeneLM 002-05
DURAN Desiccator bases with plane flange, screw threadDWK Life Sciences 7.022 260
DURAN Glass Desiccator Lid & StopcockDaihan ScienceSM.2444061
DURAN Stopcock, with PTFE SpindleDWK Life Sciences 10322671
Electrospinning machineNanoNCESR200RD
Ethyl alcohol, PureSigma Aldrich459844
FBSSigma AldrichTMS-013-BKR 
Fluorescence Laser Confocal Scanner Module K1-fluoNanoscope Systems
Gel/mountBiomeda Corp.M01
Glutaraldehyde solutionSigma Aldrich340855
Hoechst 33342InvitrogenH1399
Metal nozzle 27GNanoNC
Nail polishNature republic
Normal goat serumVector LaboratoriesS-1000
Osmium tetroxideSigma Aldrich201030
Phalloidin-iFlour 488abcamab176753
Plastic Syringe_Lure Lock (10 mL)HENKE SASS WOLFAL10
Poly(acrylic acid)Sigma Aldrich323667
Poly(vinyl alcohol)Sigma Aldrich341584
PolycaprolactoneSigma Aldrich440744
Pure HCLDuksan1129
Scanning Electron MicroscopySECSNE-4500M
Slide glassMarienfeld SuperiorK15663717
Sylgard 184 setomniscienceOMNI.05255
Synergy H1 Multimode ReaderBiotek
Triton X-100Sigma AldrichX100

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