Культивирование и применение вращающихся стен биореактора полученных 3D эпителиальных клеточных моделях
Summary
Вращающейся системе культуры клеток эпителия, который позволяет клеткам расти в физиологических условиях в результате 3-D сотовой формировании совокупного описано. Агрегаты порожденных дисплей<em> В естественных условиях</em>-Подобных характеристик не наблюдается в обычных моделях культуры и служить более точным органотипической модельной системой для множества научных исследований.
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 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.
Protocol
Discussion
Использование технологии RWV биореактор, представленные здесь может дать исследователям возможность для продвижения своей нынешней системе культуры клеток на более физиологически соответствующих органотипической модели культуры клеток. Ячейка RWV биореактор культуры система обеспе?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Авторы хотели бы поблагодарить Брук Хельм ее техническую экспертизу и Андрей Ларсена за его анализа белков. Эта работа была частично финансируется Альтернативы развития исследований Фонда (MMHK) грант NIH и NIAID передаваемых половым путем и противомикробных средств совместной научно-исследовательский центр IU19 AI062150-01 (MMHK). Мы выражаем глубокую признательность биологии размножения для повторного использования цифр.
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| Alexa Fluor 488 | Invitrogen | A21131 | Used at 1:500 dilution |
| FACSDiva | BD | Flow cytometer | |
| β-tublin antibody | Calbiochem | 654162 | Used at 1:5000 dilution |
| Bio-Plex 2000 | BioRad | 171-000205 | v5 software |
| Bioreactor and components | Synthecon | RCCS-4 | |
| Cell strainer | BD Falcon | 352340 | 40μm pore size |
| Conical tube (50mL) | Corning | 5-538-60 | |
| Coverslips | VWR | 48366067 | |
| Cytokine bead array kits | BioRad | Custom human kit | |
| Cytodex beads | Sigma | C3275 | |
| DPBS | Gibco | 14190 | |
| EDTA | Sigma | ED-500G | Ethylenediaminetetraacetic acid |
| Epithelial specific antibody (ESA) | Chemicon | CBL251 | Used at 1:50 dilution |
| Fetal Bovine Serum (FBS) | Gibco | 10438 | Heat inactivated |
| HARV (Disposable) | Synthecon | D-405 | |
| Hydrochloric acid | Sigma | 258148 | 37% |
| Involucrin antibody | Sigma | I 9018 | |
| Microscope slides | VWR | 16004-368 | |
| MTT reagent | MP Biomedicals, LLC | 194592 | 3-(4,5-Dimethylthiazolyl 1-2)-2,5-Diphenyl Tetrazolium Bromide |
| MUC1 antibody (microscopy) | Santa Cruz | Sc-7313 | Used at 1:50 dilution |
| MUC1 antibody (flow cytometry) | BD Pharmingen | 559774 | Also called CD227, use 20μL per test |
| Paraformaldehyde | Electron Microscopy Sciences | 15710 | Diluted to 4% in DPBS |
| Petri dish (small) | BD Falcon | 353002 | |
| Polystyrene tube with filter | BD Falcon | 352235 | |
| Polystyrene flow tube | BD Falcon | 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 | 71725 | |
| Sterilization pouch | VWR | 11213-035 | |
| Stopcocks (one-way) | Medex | MX5061L | |
| Syringe (10mL) | BD | 309604 | Luer-lock tip |
| Syringe (5mL) | BD | 309603 | Luer-lock tip |
| Trypan Blue | Invitrogen | T10282 | |
| Vp5 antibody | Santa Cruz | sc-13525 | HSV-2 antibody Clone 6F10; used at 1:5000 dilution |
References
- Herbst-Kralovetz, M. M., et al. Quantification and comparison of toll-like receptor expression and responsiveness in primary and immortalized human female lower genital tract epithelia. Am. J. Reprod. Immunol. 59 (3), 212-224 (2008).
- Hjelm, B. E., Berta, A. N., Nickerson, C. A., Arntzen, C. J., Herbst-Kralovetz, M. M. Development and characterization of a three-dimensional organotypic human vaginal epithelial cell model. Biol. Reprod. 82, 617-627 (2009).
- Khaoustov, V. I., et al. Induction of three-dimensional assembly of human liver cells by simulated microgravity. In Vitro Cell Dev. Biol. Anim. 35, 501-509 (1999).
- Papadaki, M., et al. Tissue engineering of functional cardiac muscle: molecular, structural, and electrophysiological studies. Am. J. Physiol. Heart Circ. Physiol. 280, 168-178 (2001).
- Ishikawa, M., et al. Reconstitution of hepatic tissue architectures from fetal liver cells obtained from a three-dimensional culture with a rotating wall vessel bioreactor. J. Biosci. Bioeng. 111, 711-718 (2011).
- Carvalho, H. M., Teel, L. D., Goping, G., O’Brien, A. D. A three-dimensional tissue culture model for the study of attach and efface lesion formation by enteropathogenic and enterohaemorrhagic Escherichia coli. Cell Microbiol. 7, 1771-1781 (2005).
- Nickerson, C. A., et al. Three-dimensional tissue assemblies: novel models for the study of Salmonella enterica serovar Typhimurium pathogenesis. Infection and Immunity. 69, 7106-7120 (2001).
- Honer zu Bentrup, K., et al. Three-dimensional organotypic models of human colonic epithelium to study the early stages of enteric salmonellosis. Microbes Infect. 8, 1813-1825 (2006).
- Carterson, A. J., et al. A549 lung epithelial cells grown as three-dimensional aggregates: alternative tissue culture model for Pseudomonas aeruginosa pathogenesis. Infection and Immunity. 73, 1129-1140 (2005).
- Smith, Y. C., Grande, K. K., Rasmussen, S. B., O’Brien, A. D. Novel three-dimensional organoid model for evaluation of the interaction of uropathogenic Escherichia coli with terminally differentiated human urothelial cells. Infection and Immunity. 74, 750-757 (2006).
- Sainz, B., TenCate, V., Uprichard, S. L. Three-dimensional Huh7 cell culture system for the study of Hepatitis C virus infection. Virol J. 6, 103 (2009).
- Duray, P. H., et al. Invasion of human tissue ex vivo by Borrelia burgdorferi. J. Infect Dis. 191, 1747-1754 (2005).
- Margolis, L. B., et al. Lymphocyte trafficking and HIV infection of human lymphoid tissue in a rotating wall vessel bioreactor. AIDS Res. Hum. Retroviruses. 13, 1411-1420 (1997).
- Reed, L. J., Muench, H. A simple method of estimating fifty percent endpoints. Am. J. Hyg. 27, 493-497 (1938).
- Beer, B. E., et al. In vitro preclinical testing of nonoxynol-9 as potential anti-human immunodeficiency virus microbicide: a retrospective analysis of results from five laboratories. Antimicrob Agents Chemother. 50, 713-723 (2006).
- Hickey, D. K., Patel, M. V., Fahey, J. V., Wira, C. R. Innate and adaptive immunity at mucosal surfaces of the female reproductive tract: stratification and integration of immune protection against the transmission of sexually transmitted infections. J. Reprod. Immunol. 88, 185-194 (2011).
- Andersch-Bjorkman, Y., Thomsson, K. A., Holmen Larsson, J. M., Ekerhovd, E., Hansson, G. C. Large scale identification of proteins, mucins, and their O-glycosylation in the endocervical mucus during the menstrual cycle. Mol. Cell Proteomics. 6, 708-716 (2007).
- Barrila, J., et al. 3D cell culture models: Innovative platforms for studying host-pathogen interactions. Nature Reviews Microbiology. 8, 791-801 (2010).
- Vamvakidou, A. P., et al. Heterogeneous breast tumoroids: An in vitro assay for investigating cellular heterogeneity and drug delivery. J. Biomol Screen. 12, 13-20 (2007).
- Jin, F., et al. Establishment of three-dimensional tissue-engineered bone constructs under microgravity-simulated conditions. Artif Organs. 34, 118-125 (2010).
- Vertrees, R. A., et al. Development of a three-dimensional model of lung cancer using cultured transformed lung cells. Cancer Biol Ther. 8, 356-365 (2009).
- Hwang, Y. S., et al. The use of murine embryonic stem cells, alginate encapsulation, and rotary microgravity bioreactor in bone tissue engineering. Biomaterials. 30, 499-507 (2009).
- Pei, M., He, F., Kish, V. L., Vunjak-Novakovic, G. Engineering of functional cartilage tissue using stem cells from synovial lining: a preliminary study. Clin. Orthop Relat. Res. 466, 1880-1889 (2008).
