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.
5. Once the STLV is cooled, tighten screws and repeat steps 1.1-1.4 to ensure sterility and complete detoxification.
6. Tighten screws of cooled the STLV and screw on pre-sterilized one-way stopcocks to each port.
7. Open stopcock by positioning the knobs vertically.
8. Remove the plungers from a 10 mL and 5 mL luer-lock syringe and re-sleeve plungers back in wrappers to maintain sterility. Screw syringes onto stopcocks (luer-lock tip syringes are required for this step).
9. Add Dulbecco's Phosphate-Buffered Saline (DPBS) to the 10 mL syringe until the STLV is full and begins to fill the 5 mL syringe.
10. Replace the sterile plungers into each syringe and remove residual bubbles occurring in bioreactor by alternating between driving plungers of the syringes.
11. Leave the syringes attached to bioreactor after removing all bubbles. Close stopcocks and attach the STLV to the rotating vertical platform and rotate for a minimum of 24 h to monitor for leaks.

2. Preparing Microcarrier Beads

1. Add 15 mL DPBS to ~250 mg cytodex microcarrier beads in a 50 mL conical tube and autoclave under same conditions as the STLV (1.4).
2. After cooling, remove DPBS, add media used to grow epithelial cell of interest, and swirl tube to resuspend beads.
3. Repeat wash steps with media two more times. After final wash add 15 mL growth media to beads.

3. Seeding Epithelial Cells and Beads into the STLV Bioreactor

1. Grow epithelial cells of interest as monolayers in tissue culture flasks until you obtain ≥1x10⁷ cells. Remove cells from the flask (i.e. trypinsization, EDTA), count cells to establish cellular concentration, and determine cellular viability by trypan blue exclusion staining¹.
2. To the conical tube containing prepared beads (see 2.3), add desired concentration of cells (2x10⁵-1x10⁷ epithelial cells)^{2, 8}, depending on your epithelial cell type of intent (Figure 1A). Ensure all cells are transferred by rinsing the conical tube.
3. Remove DPBS and syringes from the STLV.
4. Remove plug from center port and add the epithelial cell/bead suspension into the STLV using 10 mL serological pipette. Rinse conical tube to ensure all cells/beads are transferred to the STLV and replace center port plug.
5. Remove plungers from a 5 and 10 mL syringes and re-sleeve plungers back in wrappers to maintain sterility. Screw syringes into ports with open stopcocks. Fill the STLV with media, replace plungers, and close stopcocks.
6. Place the newly seeded STLV in a 37 °C incubator for 30 min with no rotation.
7. Open stopcocks, remove bubbles using plungers, and then close stopcocks.
8. Place the STLV in a 37 °C, 5% CO₂ humidified incubator rotating at 20 rpm (Figure 1B). Cultures must be rotated continuously 24 h a day except for when sampling, changing media, or harvesting aggregates (see next section).

4. Culturing, Sampling, and Photo Documentation of Cultures

1. After an initial 96-120 h of culturing cells in the bioreactor, change media by tilting the STLV and allowing cells/beads to settle. Remove syringes, open both stopcocks, and pour off ~75% of the media from side port (Figure 1C). Based on cellular metabolism of media, change media every day or every other day after initial seeding and 96-120 h culture period.
2. Screw on 5 and 10 mL syringes void of plungers and follow refeeding protocol as described above (1.7-1.11), then screw closed the STLV back in place on rotating platform.
3. Monitor growth and viability of aggregates every 5-7 days after seeding into the STLV by carefully removing a small amount of aggregates (~200 μL) from the center port into two 1.5 mL tubes. To avoid aggregate shearing, for all aggregate transfers use a 1000 μL pipette tip that has been cut ~2 cm from the point and sterilized.
4. Replace center plug, exchange with new syringes, add fresh media to the STLV as described above (1.7-1.11), and place the STLV back in the incubator with rotation (see 3.8).
5. For monitoring cellular viability, use one of the two 1.5 mL tube aggregates aliquoted in step 4.3, and remove cells from beads in the same manner the epithelial cells are removed from tissue culture flasks (i.e. trypsinization). Monitor viability by trypan blue exclusion staining¹.
6. For imaging cellular aggregates, add 0.5-1 mL of media to the second 1.5 mL aggregate aliquot and transfer to a small Petri dish using a cut-off 1000 μL pipette tip. Image aggregates using an inverted light microscope (Figure 1D).

5. Transferring and Harvesting Aggregates

1. For some epithelial cell models it is necessary to transfer the aggregates to a disposable HARV to increase culture aeration². Approximately one week prior to harvesting aggregates for analysis, remove syringes and stopcocks from the STLV and pour off ~50% of the media from side port.
2. Remove the STLV's center plug and carefully pour all contents into a 50 mL conical tube from central port (Figure 1E). Rinse the STLV two times with 5 mL media and combine rinse with rest of aggregates.
3. Continue with transferring the aggregates as described in 5.2, but transfer aggregates from the 50 mL conical tube to the HARV.
4. Carefully harvest aggregates from the HARV after ~1 week or from the STLV (based on cell type) as was performed above (5.2). See below for potential assay formats, assays and analysis following aggregate harvest.

6. Potential Analysis, Applications and Assays Conducted with Aggregates

1. Seeding aggregates for various experimental formats. Transfer desired amount of aggregates from the 50 mL conical tube to a 1.5 mL tube, 24 well plate, or 96 well plate using a sterilized cut-off 1000 μL pipette tip (Figure 1F).
2. Washing and preparing aggregates for analysis. Remove media after aggregates have settled. Dispense DPBS directly to center of tube or well so all aggregates are stirred up. Once aggregates have settled to bottom, remove DPBS and repeat as many times as needed.
3. Shipping aggregates for off-site experimentation and analysis. Add desired amount of aggregates to a 50 mL tube and wash aggregates as in 6.2. Completely fill 50 mL tube with media, cap, and parafilm around cap to avoid leakage. Securely ship aggregates to desired location overnight at ambient temperature.
4. Fixing aggregates for electron microscopy (Figure 2). Scanning, transmission or immuno-electron microscopy can be conducted by transferring 150-300 μL aggregates to a 1.5 mL tube and washing aggregates 3 times with DPBS (6.2). Add 200 μL of application-specific (SEM, TEM, immuno-EM, etc.) electron microscopy fixative for the desired incubation period. Remove fix, wash aggregates 2 times with DPBS, and proceed as outlined in Hjelm et al. 2010 for scanning and transmission electron microscopy².
5. Immunofluorescence microscopy imaging (Figure 3). Transfer ~100 μL aggregates to a 1.5 mL tube and wash aggregates (6.2). Fix and label aggregates with antibody under similar conditions as with monolayers, except in a 1.5 mL tube. To mount, place 1 drop of mounting media on microscope slide, transfer labeled aggregates on top of mounting media with a cut-off 1000 μL pipette tip, place coverslip over aggregates, seal coverslip with nail polish, and dry slide overnight².
6. Measuring cellular viability/proliferation using MTT assay (Figure 4). Transfer aggregates to a 24 well plate and replace media with 625 μL phenol red free media. Add 62.5 μL of 5 mg/mL Thiazolyl Blue Tetrazolium Bromide (MTT) to each well and incubate for 4 h at 37 °C. Add 625 μL of 100 mg/mL SDS-0.01 M HCl to each well and incubate overnight. Read absorbance at 570 nm and obtain % cell viability using equation:

7. Toxicology studies. Transfer aggregates to 24 well plate and perform trypan blue exclusion on ≥2 wells for initial cell viability/concentration. Add test compound at ranging concentrations to duplicate wells. For cell viability (Figure 5A), wash aggregates and perform trypan blue exclusion after treatment. For TC₅₀ (Figure 5B), take cellular viability of duplicate wells for each concentration over time and use Reed Muench method to determine toxic concentration 50%^{2, 15}.
8. Cytometric bead array (CBA) or ELISA. Cellular supernatants can be taken after seeding cells in any experimental format diagrammed. Stimulate seeded aggregates as with cells grown as monolayers, collect supernatants (≥120 μL), and store at -80 °C for analysis by ELISA or CBA (Figure 6).
9. RNA analysis. Transfer ≥500 μL of aggregates to 1.5 mL tube and wash aggregates with DPBS. Continue extraction using Qiagen RNAeasy kit and protocol for animal cells with homogenization of lysate using 20-gauge needle. Make sure to let beads settle at the bottom of the tube, prior to transferring supernatant, and DO NOT transfer empty beads to the spin column (beads will clog column membrane resulting in low RNA yield) (Figure 7).
10. Protein analysis. Harvest aggregates under the same methods as with monolayers using a 1.5 mL tube or larger experimental format. However, after lysis of the aggregates, allow the beads to settle and transfer lysate to a separate tube before running a protein gel or other form of protein analysis (Figure 8).
11. Infection studies. Seed aggregates into desired experimental format. Use at least two wells/samples for trypsinizing cells from beads to enumerate cell number and quantify cell viability. Infect aggregates with pathogen of interest at selected multiplicity of infection as performed with cells grown as monolayers (Figure 9). Wash steps must be performed as in 6.2.
12. Flow cytometry: Seed aggregates in 50 mL tube, wash aggregates (6.2), and add 2mM EDTA for 5-10 min at 37 °C. Add 5-10 mL media to cells and pass cells through a cell strainer. Aliquot 1x10⁶

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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