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

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 toxicology^1,2. 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 structures^3,4. For example, there is a lack of cell-to-cell and cell-to-extracellular matrix (ECM) signaling, which is essential in cell differentiation, prolife....

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.......

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.......

Materials

Name	Company	Catalog Number	Comments
0.05% Trypsin/EDTA	Welgene	LS015-01
100x Penicilin/Streptomycin	Gibco	10378016
20x PBS	LPS solution	CBP007A
4% Paraformaldehyde	Biosesang	PC2031-050-00
Alexa Fluor-594 conjugated ZO-1 antibody	Invitrogen	339194
Antibody diluent OP Quanto	Epredia	10129-576
CCK-8 assay kit	Donginbio	CCK-3000
Cell culture dish (150x20)	SPL	20151
CellTracker Green CMFDA	Invitrogen	C7025
CellTracker Red CMTPX	Invitrogen	C34552
Chloroform	Samchun	C0584
Conical tube	SPL	50050
Cover glass	Corning	CLS2980245
DMEM high glucose	Welgene	LM001-05
DMEM/F12	Welgene	LM 002-05
DURAN Desiccator bases with plane flange, screw thread	DWK Life Sciences	7.022 260
DURAN Glass Desiccator Lid & Stopcock	Daihan Science	SM.2444061
DURAN Stopcock, with PTFE Spindle	DWK Life Sciences	10322671
Electrospinning machine	NanoNC	ESR200RD
Ethyl alcohol, Pure	Sigma Aldrich	459844
FBS	Sigma Aldrich	TMS-013-BKR
Fluorescence Laser Confocal Scanner Module K1-fluo	Nanoscope Systems
Gel/mount	Biomeda Corp.	M01
Glutaraldehyde solution	Sigma Aldrich	340855
Hoechst 33342	Invitrogen	H1399
Metal nozzle 27G	NanoNC
Nail polish	Nature republic
Normal goat serum	Vector Laboratories	S-1000
Osmium tetroxide	Sigma Aldrich	201030
Phalloidin-iFlour 488	abcam	ab176753
Plastic Syringe_Lure Lock (10 mL)	HENKE SASS WOLF	AL10
Poly(acrylic acid)	Sigma Aldrich	323667
Poly(vinyl alcohol)	Sigma Aldrich	341584
Polycaprolactone	Sigma Aldrich	440744
Pure HCL	Duksan	1129
Scanning Electron Microscopy	SEC	SNE-4500M
Slide glass	Marienfeld Superior	K15663717
Sylgard 184 set	omniscience	OMNI.05255
Synergy H1 Multimode Reader	Biotek
Triton X-100	Sigma Aldrich	X100