Culturing and Applications of Rotating Wall Vessel Bioreactor Derived 3D Epithelial Cell Models

Summary

A rotating cell culture system that allows epithelial cells to grow under physiological conditions resulting in 3-D cellular aggregate formation is described. The aggregates generated display in vivo-like characteristics not observed in conventional culture models and serve as a more accurate organotypic model system for a multitude of scientific investigations.

Abstract

Cells and tissues in the body experience environmental conditions that influence their architecture, intercellular communications, and overall functions. For in vitro cell culture models to accurately mimic the tissue of interest, the growth environment of the culture is a critical aspect to consider. Commonly used conventional cell culture systems propagate epithelial cells on flat two-dimensional (2-D) impermeable surfaces. Although much has been learned from conventional cell culture systems, many findings are not reproducible in human clinical trials or tissue explants, potentially as a result of the lack of a physiologically relevant microenvironment.

Here, we describe a culture system that overcomes many of the culture condition boundaries of 2-D cell cultures, by using the innovative rotating wall vessel (RWV) bioreactor technology. We and others have shown that organotypic RWV-derived models can recapitulate structure, function, and authentic human responses to external stimuli similarly to human explant tissues ^1-6. The RWV bioreactor is a suspension culture system that allows for the growth of epithelial cells under low physiological fluid shear conditions. The bioreactors come in two different formats, a high-aspect rotating vessel (HARV) or a slow-turning lateral vessel (STLV), in which they differ by their aeration source. Epithelial cells are added to the bioreactor of choice in combination with porous, collagen-coated microcarrier beads (Figure 1A). The cells utilize the beads as a growth scaffold during the constant free fall in the bioreactor (Figure 1B). The microenvironment provided by the bioreactor allows the cells to form three-dimensional (3-D) aggregates displaying in vivo-like characteristics often not observed under standard 2-D culture conditions (Figure 1D). These characteristics include tight junctions, mucus production, apical/basal orientation, in vivo protein localization, and additional epithelial cell-type specific properties.

The progression from a monolayer of epithelial cells to a fully differentiated 3-D aggregate varies based on cell type^{1, 7-13}. Periodic sampling from the bioreactor allows for monitoring of epithelial aggregate formation, cellular differentiation markers and viability (Figure 1D). Once cellular differentiation and aggregate formation is established, the cells are harvested from the bioreactor, and similar assays performed on 2-D cells can be applied to the 3-D aggregates with a few considerations (Figure 1E-G). In this work, we describe detailed steps of how to culture 3-D epithelial cell aggregates in the RWV bioreactor system and a variety of potential assays and analyses that can be executed with the 3-D aggregates. These analyses include, but are not limited to, structural/morphological analysis (confocal, scanning and transmission electron microscopy), cytokine/chemokine secretion and cell signaling (cytometric bead array and Western blot analysis), gene expression analysis (real-time PCR), toxicological/drug analysis and host-pathogen interactions. The utilization of these assays set the foundation for more in-depth and expansive studies such as metabolomics, transcriptomics, proteomics and other array-based applications. Our goal is to present a non-conventional means of culturing human epithelial cells to produce organotypic 3-D models that recapitulate the human in vivo tissue, in a facile and robust system to be used by researchers with diverse scientific interests.

Protocol

All steps should be performed under BSL-2 conditions in a Laminar flow hood.

1. Preparing the STLV Bioreactor

1. Assemble the STLV bioreactor according to manufacturer's protocol and perform detoxification protocol to ensure sterility of the bioreactor. Cover open ports with luer caps and fill the STLV with 95% ethanol for 24 h.
2. Remove ethanol and fill the STLV with sterilized distilled water for 24 h.
3. Repeat step 1.2 with sterilized distilled water only.
4. With the tool supplied by vendor, loosen all screws, caps and center plug from the STLV, and autoclave at 110 °C for 20 min in a sterilization pouch.....

Discussion

Utilization of the RWV bioreactor technology presented here may provide researchers with the capability to advance their current cell culture system to a more physiologically relevant organotypic cell culture model. The RWV bioreactor cell culture system provides a low shear microenvironment that enables cells to form 3-D cellular aggregates with in vivo-like characteristics, including tight junctions, mucin production, extracellular processes (i.e. microvilli), and cellular polarity. The majority of the data an.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Alexa Fluor 488	Invitrogen	A21131	Used at 1:500 dilution
FACSDiva	BD Biosciences		Flow cytometer
β-tublin antibody	Calbiochem	654162	Used at 1:5000 dilution
Bio-Plex 2000	Bio-Rad	171-000205	v5 software
Bioreactor and components	Synthecon	RCCS-4
Cell strainer	BD Biosciences	352340	40μm pore size
Conical tube (50mL)	Corning	5-538-60
Coverslips	VWR international	48366067
Cytokine bead array kits	Bio-Rad	Custom human kit
Cytodex beads	Sigma-Aldrich	C3275
DPBS	GIBCO, by Life Technologies	14190
EDTA	Sigma-Aldrich	ED-500G	Ethylenediaminetetraacetic acid
Epithelial specific antibody (ESA)	Chemicon International	CBL251	Used at 1:50 dilution
Fetal Bovine Serum (FBS)	GIBCO, by Life Technologies	10438	Heat inactivated
HARV (Disposable)	Synthecon	D-405
Hydrochloric acid	Sigma-Aldrich	258148	37%
Involucrin antibody	Sigma-Aldrich	I 9018
Microscope slides	VWR international	16004-368
MTT reagent	MP Biomedicals	194592	3-(4,5-Dimethylthiazolyl 1-2)-2,5-Diphenyl Tetrazolium Bromide
MUC1 antibody (microscopy)	Santa Cruz Biotechnology, Inc.	Sc-7313	Used at 1:50 dilution
MUC1 antibody (flow cytometry)	BD Biosciences	559774	Also called CD227, use 20μL per test
Paraformaldehyde	Electron Microscopy Sciences	15710	Diluted to 4% in DPBS
Petri dish (small)	BD Biosciences	353002
Polystyrene tube with filter	BD Biosciences	352235
Polystyrene flow tube	BD Biosciences	352058
PR antibody	Dako	M3569	Used at 1:100 dilution
ProLong Gold	Invitrogen	P36931	Mounting media with DAPI
RNeasy Mini Kit	Qiagen	74903
Sodium dodecyl sulfate	Sigma-Aldrich	71725
Sterilization pouch	VWR international	11213-035
Stopcocks (one-way)	MedexSupply	MX5061L
Syringe (10mL)	BD Biosciences	309604	Luer-lock tip
Syringe (5mL)	BD Biosciences	309603	Luer-lock tip
Trypan Blue	Invitrogen	T10282
Vp5 antibody	Santa Cruz Biotechnology, Inc.	sc-13525	HSV-2 antibody Clone 6F10; used at 1:5000 dilution