EXPRESSION OF THE MICROFOLD CELLS IN THREE-DIMENSIONAL COCULTURE SYSTEM FOR IN VITRO CULTIVATION OF HUMAN NOROVIRUS
DOI:
https://doi.org/10.22159/ijap.2019.v11s5.T0052Keywords:
Human Norovirus, Microfold cells, Three-dimensional culture, Alginate hydrogelAbstract
Introduction: Human Norovirus (HuNoV), a food-borne virus is the leading cause for acute gastroenteritis. However, its inability to propagate in vitro
persists as major challenge in understanding HuNoV biology.
Objective: This study aims to determine an effective culture system for HuNoV.
Methods: The Caco-2 cells were cocultured with Raji B cells on alginate hydrogel beads. Scanning electron microscopy (SEM) was performed to confirm
the three-dimensional (3D) cells morphology. Western blot (WB) analysis was performed to detect protein markers expressed by Microfold (M) cells.
Results: Optimization of Caco-2 cells monoculture in the alginate hydrogel beads showed optimum number of cells of 1 × 106 cells/ml, indicated
by the intact structure of the beads. Result of SEM showed clear structure of monoculture in the alginate hydrogel beads indicated by the presence
of smooth and regular apical surface while the coculture showed reduced apical surface of M cells. The result of WB showed downregulation of
Ulex europaeus antibody expression.
Conclusion: It is evident that the expression of M cells grown in 3D alginate hydrogel beads was successful, indicated by the structural morphology
seen under SEM as well as expression of protein marker by M cells. This established in vitro system is highly potential for cultivation of HuNoV.
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References
organization of Norwalk virus. Virology 1993;195:51-61.
2. Matthews JE, Dickey BW, Miller RD, Felzer JR, Dawson BP, Lee AS,
et al. The epidemiology of published Norovirus outbreaks: A review of
risk factors associated with attack rate and genogroup. Epidemiol Infect
2012;140:1161-72.
3. Almand EA, Moore MD, Jaykus LA. Norovirus binding to ligands
beyond histo-blood group antigens. Front Microbiol 2017;8:2549.
4. Singh BK, Leuthold MM, Hansman GS. Structural constraints on
human Norovirus binding to histo-blood group antigens. mSphere
2016;1:e49-16.
5. Straub TM, Hutchison JR, Bartholomew RA, Valdez CO, Valentine NB,
Dohnalkova A, et al. Defining cell culture conditions to improve human
Norovirus infectivity assays. Water Sci Technol 2013;67:863-8.
6. Mabbott NA, Donaldson DS, Ohno H, Williams IR, Mahajan A.
Microfold (M) cells: Important immunosurveillance posts in the
intestinal epithelium. Mucosal Immunol 2013;6:666-77.
7. Karst SM, Wobus CE. A working model of how noroviruses infect the
intestine. PLoS Pathog 2015;11:e1004626.
8. Jones MK, Watanabe M, Zhu S, Graves CL, Keyes LR, Grau KR, et al.
Enteric bacteria promote human and mouse Norovirus infection of B
cells. Science 2014;346:755-9.
9. Gonzalez-Hernandez MB, Liu T, Payne HC, Stencel-Baerenwald JE,
Ikizler M, Yagita H, et al. Efficient Norovirus and reovirus replication in
the mouse intestine requires microfold (M) cells. J Virol 2014;88:6934-43.
10. Kernéis S, Bogdanova A, Kraehenbuhl JP, Pringault E. Conversion
by Peyer’s patch lymphocytes of human enterocytes into M cells that
transport bacteria. Science 1997;277:949-52.
11. Beloqui A, Brayden DJ, Artursson P, Préat V, des Rieux A. A human
intestinal M-cell-like model for investigating particle, antigen and
microorganism translocation. Nat Protoc 2017;12:1387-99.
12. Voo WP, Ooi CW, Islam A, Tey BT, Chan ES. Calcium alginate
hydrogel beads with high stiffness and extended dissolution behavior.
Euro Polym J 2016;75:343-53.
13. Lee KY, Mooney DJ. Alginate: Properties and biomedical applications.
Prog Polym Sci 2012;37:106-26.
14. Ab-Rahim S, Selvaratnam L, Raghavendran HR, Kamarul T.
Chondrocyte-alginate constructs with or without TGF-?1 produces
superior extracellular matrix expression than monolayer cultures. Mol
Cell Biochem 2013;376:11-20.
15. Kleiveland CR. Co-culture Caco-2/Immune Cells. In: Verhoeckx K,
Cotter P, López-Expósito I, Kleiveland C, editors. The Impact of Food
Bioactives on Health. Cham, Switzerland: Springer; 2015. p. 197-205.
16. Wang X, Hao T, Qu J, Wang C, Chen H. Synthesis of thermal
polymerizable alginate-GMA hydrogel for cell encapsulation.
J Nanomater 2015;2015:1-8.
17. Schimpel C, Werzer O, Fröhlich E, Leitinger G,
Absenger-Novak M, Teubl B, et al. Atomic force microscopy as
analytical tool to study physico-mechanical properties of intestinal
cells. Beilstein J Nanotechnol 2015;6:1457-66.
18. Casteleyn C, Van den Broeck W, Gebert A, Tambuyzer BR, Van
Cruchten S, Van Ginneken C, et al. M cell specific markers in man
and domestic animals: Valuable tools in vaccine development. Comp
Immunol Microbiol Infect Dis 2013;36:353-64.
19. Takanashi S, Saif LJ, Hughes JH, Meulia T, Jung K, Scheuer KA,
et al. Failure of propagation of human Norovirus in intestinal epithelial
cells with microvilli grown in three-dimensional cultures. Arch Virol
2014;159:257-66.
20. Renfeng L, Xiangqin T, Songlin Q, Yanyan Y, Enmin Z, Gaiping Z.
Morphological and immunohistochemical identification of villous
M cells in the small intestine of newborn piglets. Int J Morphol
2015;33:1261-8.
21. Masuda K, Kajikawa A, Igimi S. Establishment and evaluation of
an in vitro M cell model using C2BBe1 cells and Raji cells. Biosci
Microflora 2011;30:37-44.
22. Jang MH, Kweon MN, Iwatani K, Yamamoto M,
Terahara K, Sasakawa C, et al. Intestinal villous M cells: An antigen
entry site in the mucosal epithelium. Proc Natl Acad Sci U S A
2004;101:6110-5.
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