Basic Medical Sciences, University of Arizona College of Medicine - Phoenix
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Radtke, A. L., Herbst-Kralovetz, M. M. Culturing and Applications of Rotating Wall Vessel Bioreactor Derived 3D Epithelial Cell Models. J. Vis. Exp. (62), e3868, doi:10.3791/3868 (2012).
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 type1, 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.
All steps should be performed under BSL-2 conditions in a Laminar flow hood.
1. Preparing the STLV Bioreactor
2. Preparing Microcarrier Beads
3. Seeding Epithelial Cells and Beads into the STLV Bioreactor
4. Culturing, Sampling, and Photo Documentation of Cultures
5. Transferring and Harvesting Aggregates
6. Potential Analysis, Applications and Assays Conducted with Aggregates
7. Representative Results
An example of a differentiated human epithelial aggregate grown in the STLV bioreactor system can be seen in Figure 2. The SEM and TEM images are collected from human vaginal cells grown in the STLV for 39 days, where microridges, invaginations, intracellular secretory vesicles, and microvilli on the apical surface can be observed. Further demonstration of in vivo-like characteristics of epithelial cells grown in the bioreactor can be observed by confocal immunofluorescence microscopy images (Figure 3) of 3-D vaginal cells expressing mucin (MUC1) and markers specific for epithelial cells (ESA) and terminal differentiation (Involucrin; INV).
As shown, 3-D aggregates display ultrastructural features similar to tissues, however their more physiologically relevant phenotypes also serve as an excellent platform for evaluating toxicity of compounds. The MTT assay, a commonly used assay to measure cell viability, metabolism or proliferation, can be used to measure toxicity to various chemicals and compounds (Figure 4). Triton X-100, a detergent that destroys cellular membranes, was used as a positive control for validating the MTT assay. An additional method to measure toxicity following treatment with test compounds is trypan blue exclusion (Figure 5). Following treatment with the nonoxynol-9 (N-9), the vaginal aggregates demonstrated a toxicity response similar to that of cervical explant models, but a different profile compared to monolayers 2, 16.
Additional studies that demonstrate the ability of epithelial cells, grown in the bioreactor, to function and signal similarly to tissues in vivo, can be observed in Figure 6. Upon stimulation with molecules derived from microbes that are recognized by the Toll-like Receptors (TLR), the aggregates respond and secrete pro-inflammatory cytokines, including IL-61. Expression of the progesterone receptor (PR), a receptor that responds to hormone stimulation, can also be observed in the vaginal cells both at the RNA and protein levels (Figure 7 and Figure 8, respectively). The 3-D aggregates not only have the capability to respond to pathogen and hormone molecules, but are also able to functionally support a herpes simplex virus type 2 (HSV-2) infection. A confocal microscopy image of HSV-2 infected 3-D vaginal aggregates (Figure 9) demonstrates infection. Parallel 2-D vaginal epithelial cell cultures are not shown as a result of the severe cellular destruction caused by HSV-2 infection. RWV-derived 3-D aggregates have also been used to quantify bacterial and viral replication through intracellular growth curves and plaque assays, respectively8, 9. Lastly, the physical and functional properties of individual cells within the aggregates can also be quantified by flow cytometry. For example, we employed a flow cytometry assay to measure MUC1 surface expression on individual vaginal cells (Figure 10). Vaginal 3-D aggregates expressed a lower percentage of MUC1 (27.7%) compared to monolayers (67.2%). The lower percentage of MUC1 on the 3-D aggregates surface compared to monolayers may be a result of the majority of MUC1 being secreted from the cells as observed in the vaginal tract 17, 18.
Figure 1. Schematic for culturing human epithelial cells in RWV and potential applications using RWV-derived aggregates. A) Epithelial cells are initially grown as monolayers in a tissue culture flask. Once confluent, cells are removed from flask and combined with microcarrier beads in the STLV bioreactor. Scale bar: 80 μm. B) The STLV rotates on a platform to create a constant free fall environment of low fluid shear, allowing the cells to attach and grow on beads thereby forming visible cellular aggregates. C) After 96 h, the media in the STLV must be changed to accommodate for cellular metabolism. The media is poured out of a side port, fresh media is replaced through an attached 10 mL syringe (plunger removed), and syringe plungers are replaced and used to extrude bubbles. D) After ~5 days (d) the aggregates begin to form. The aggregates are sampled every 7 days and imaged by light microscopy to monitor their developmental progression (20x magnification of 3-D human epithelial vaginal cells). E) Once complete aggregate formation and cellular differentiation has occurred, the aggregates are harvested from the bioreactor and transferred into a 50 mL conical tube. F) Aggregates can be seeded into a 1.5 mL tube or a multi-well plate format to carry out experimental analysis and assays. G) Outline of potential assays and analyses that can be conducted on the aggregates. This representative list of analyses is not meant to be an exhaustive catalog of all potential downstream applications.
Figure 2. Examining morphological and structural characteristics of 3-D epithelial cell aggregates using electron microscopy. (A) Scanning electron microscopy (SEM) image of a 3-D human epithelial vaginal cell aggregate. Scale bars: 200 μm (100x), 100 μm (200x), 50 μm (500x). Transmission electron microscopy (TEM) image of a 3-D human epithelial vaginal cell aggregate with arrows pointing to (B) intracellular secretory vesicles and microvilli or (C) "cytoplasmic processes". Scale bars: 2 μm (modified from Hjelm et al. 2010)2.
Figure 3. Confocal immunofluorescence microscopy used to identify junctional differentiation and protein markers in 3-D epithelial cell aggregates. Human 3-D vaginal cells harvested from bioreactor after 32 days, fixed, and stained with (A) mucin antibody MUC1, (B) antibody specific for epithelial cells, ESA, or (C) anti-Involucrin (INV) antibody that recognizes terminally differentiated epithelial cells. Aggregates were indirectly labeled with Alexa 488 secondary antibody (green). Scale bar: 60 μm (modified from Hjelm et al. 2010)2.
Figure 4. MTT assay is used to measure cellular viability. Human 3-D vaginal aggregates were seeded in a 24 well plate and treated with media alone (untreated) or 0.01%, 0.1% or 1.0% Triton X-100 detergent for 1 h at 37 °C. Following treatment, media was removed and the MTT assay was performed to measure cellular viability. ** represents p<0.001; one-tailed Student's t-test comparing Triton X-100 treated cells to untreated control.
Figure 5. Utilizing 3-D epithelial cell aggregates to screen compound toxicity. (A) Nonoxynol-9 (N-9) dose-dependent viability curve 24 h post-treatment of 3-D human vaginal epithelial cell model (red line/bars) compared to same cell type grown as confluent monolayers (black line/bars). (B) N-9 TC50 levels of vaginal epithelial cell cultures in (A) at 1.5 h, 4 h, 8 h, and 24 h post treatment. * represents p<0.05; one-tailed Student's t-test comparing monolayers to 3-D cells at each exposure time. (Modified from Hjelm et al. 2010)2.
Figure 6. Analysis of cytokine production in 3-D epithelial cells in response to toll-like receptor (TLR) agonist stimulation. Three-D human vaginal cells were stimulated with FSL-1 (TLR2/6), PIC (TLR3), FLAG (TLR5), and CL097 (TLR7/8) for 24 h and secreted cytokines were measured by cytometric bead array. IL-6 is shown as a representative proinflammatory cytokine produced and measured. * represents p<0.05; one-tailed Student's t-test comparing stimulated samples to PBS group. (Modified from Hjelm et al. 2010)2.
Figure 7. Monitoring gene expression in 3-D epithelial cells. Semi-quantitative RT-PCR analysis of progesterone receptor (PR) expression in monolayer (ML) or 3-D human vaginal epithelial cells. GAPDH is shown as a loading control. Amplification products shown are a result of transcript expression occurring after 30 PCR cycles. Arrow heads point to PR bands only occurring in the 3-D sample.
Figure 8. Protein expression analysis of 3-D epithelial cells. Western blot analysis of monolayer and 3-D human vaginal epithelial cell whole cell lysates (30μg) probed with an anti-progesterone receptor (PR) antibody. β-tubulin was used as a probe for loading control.
Figure 9. Three-D human epithelial cell model supports a productive viral infection. Confocal immunofluorescence microscopy image of 3-D human vaginal epithelial cell aggregate infected with HSV-2. Cells were infected at an MOI of 1.0 for two hours, washed, fixed 24 h post infection (4% PFA), and stained with a HSV-2 specific VP5 capsid antibody (green) and the nuclear stain DAPI (blue). Scale bar: 50 μm.
Figure 10. Individual cell analysis in 3-D human epithelial cell model by flow cytometry. (A) Monolayer or (B) 3-D human vaginal epithelial aggregates were dissociated with EDTA, labeled with a FITC conjugated MUC1 antibody (blue), an isotype control (green), or PBS (red), and FITC expression was quantified by flow cytometry. Numbers represent the percentage of cells that stained positive for mucin antibody that remained after gating out background fluorescence from the isotype control stained cells. Non-uniform peaks are a result of the multitude of glycosolation patterns and varying levels of MUC1 on the surface of these cells.
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 and examples presented here are using vaginal epithelial cells grown in the RWV system, however the RWV system also has been used to culture other epithelial cell types including small and large intestine, lung, bladder, liver, and tonsil epithelial cells to form 3-D organotypic models 1, 7-13, 18. Additionally, this system has been used to culture cell types other than epithelial cells, and currently the development of complex, multicellular organotypic culture models are underway 2, 18.
The protocols outlined here are meant to serve as a guide, and may need to be modified based upon the cell type of interest. The most common modifications to the protocol for culturing epithelial cells in the RWV system include the length of time the cells are cultured in the STLV, decision and timing of aggregate transfer from the STLV to the HARV, and the cell/bead ratio for the initial seeding in the STLV. A more specialized modification that may be considered for successful aggregate formation are the properties of the microcarrier bead or scaffold, such as surface area, diameter, shape, surface coatings, density, charge, core material, and porosity. In addition, adjustments to the base culture medium, formulation, or supplements, may be necessary to optimize the culture conditions and maximize aggregate formation. Adjustments can also be made to the bioreactor system and its components, including the size of bioreactor vessel and the rotational speed. For an enhanced assessment of the microenvironment within the bioreactor, a perfused culture system is also available that allows for continuous monitoring of pH, oxygen, and glucose levels within the culture vessel ensuring a prolific environment for aggregate formation. The potentially lengthy time to optimize culture conditions, aggregate formation, cellular differentiation and to characterize each new model system, as well as the initial expense of the bioreactor system, are noteworthy drawbacks to this system. However, any new culture system implemented in a laboratory will have similar disadvantages.
We and others have shown that RWV-derived models utilizing human cells may be a valuable tool for predicting the efficacy, toxicity and pharmacokinetics of vaccines, microbicides, biologics and pharmaceuticals in a fashion that is relevant to humans 1, 2, 18. With the success of this bioreactor culture system to produce authentic human responses, combined with its flexibility, the applications of the RWV bioreactor system is currently being expanded to such fields as tumor modeling, regenerative medicine, and tissue engineering 2, 18-23. To advance our RWV-generated mucosal models we are working to create a more complex multicellular system that replicates both the structure and function of the human mucosal tissue. However, the authors acknowledge that it may be necessary to utilize or integrate multiple model systems to reproduce the complex interactions that drugs or vaccines have with human physiological systems. Overall, our goal of using the RWV bioreactor and its experimental applications is to better understand mucosal tissue biology and the cellular responses to environmental insults in human organotypic models.
The authors have nothing to disclose.
The authors would like to thank Brooke Hjelm for her technical expertise and Andrew Larsen for his protein analysis. This work was funded in part by the Alternatives Research Development Foundation (MMHK) Grant and the NIH NIAID Sexually Transmitted Infections and Topical Microbicides Cooperative Research Center IU19 AI062150-01(MMHK). We gratefully acknowledge Biology of Reproduction for reuse of figures.
|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|
|Cytokine bead array kits||Bio-Rad||Custom human kit|
|DPBS||GIBCO, by Life Technologies||14190|
|Epithelial specific antibody (ESA)||Chemicon International||CBL251||Used at 1:50 dilution|
|Fetal Bovine Serum (FBS)||GIBCO, by Life Technologies||10438||Heat inactivated|
|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|
|Syringe (10mL)||BD Biosciences||309604||Luer-lock tip|
|Syringe (5mL)||BD Biosciences||309603||Luer-lock tip|
|Vp5 antibody||Santa Cruz Biotechnology, Inc.||sc-13525||HSV-2 antibody Clone 6F10; used at 1:5000 dilution|