title: An improved 3D tetraculture system mimicking the cellular organisation at the alveolar barrier to study the potential toxic effects of particles on the lung.
abstract: BACKGROUND
  Exposure to fine and ultra-fine ambient particles is still a problem of concern in many industrialised parts of the world and the intensified use of nanotechnology may further increase exposure to small particles. Complex in vitro coculture systems may be valuable tools to study particle-induced processes and to extrapolate effects of particles on the lung. A system consisting of four different human cell lines which mimics the cell response of the alveolar surface in vitro was developed to study native aerosol exposure (Vitrocell™ chamber). The system is composed of an alveolar type-II cell line (A549), differentiated macrophage-like cells (THP-1), mast cells (HMC-1) and endothelial cells (EA.hy 926), seeded in a 3D-orientation on a microporous membrane.
  
  
  RESULTS
  The spatial distribution of the cells in the tetraculture was analysed by confocal laser scanning microscopy (CLSM), showing a confluent layer of endothelial and epithelial cells on both sides of the transwell. Macrophage-like cells and mast cells can be found on top of the epithelial cells. The cells formed colonies under submerged conditions, which disappeared at the ALI. To evaluate the response to oxidative stress, the dichlorodihydrofluorescein diacetate (DCFH-DA) assay was used together with 2,2'-azobis-2-methyl-propanimidamide-dihydrochloride (AAPH) as inducer of oxidative stress. The tetraculture showed less induction of reactive oxygen species (ROS) production after being treated with a positive control compared to the monocultures of EA.hy 926, THP-1 and HMC-1. Submerged cultures showed elevated ROS and IL-8 levels compared to ALI cultures. The Vitrocell™ aerosol exposure system was not significantly influencing the viability. Using this system, cells were exposed to an aerosol of 50 nm SiO2-Rhodamine NPs in PBS. The distribution of the NPs in the tetraculture after exposure was evaluated by CLSM. Fluorescence from internalized particles was detected in CD11b-positive THP-1 cells only.
  
  
  CONCLUSIONS
  The system can be used in conjunction with a native aerosol exposure system and may finally lead to a more realistic judgement regarding the hazard of new compounds and/or new nano-scaled materials in the future. The results for the ROS production and IL-8 secretion suggest that submerged exposure may lead to an overestimation of observed effects.
authors.forename: Sebastian G
authors.initials: SG
authors.lastname: Klein
authors.forename: Tommaso
authors.initials: T
authors.lastname: Serchi
authors.forename: Lucien
authors.initials: L
authors.lastname: Hoffmann
authors.forename: Brunhilde
authors.initials: B
authors.lastname: Blömeke
authors.forename: Arno C
authors.initials: AC
authors.lastname: Gutleb
mesh_terms: D000336:Aerosols; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D018920:Coculture Techniques; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism; D019570:Inhalation Exposure; D016209:Interleukin-8 / Q000378:metabolism; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism; D008407:Mast Cells / Q000187:drug effects* / Q000378:metabolism; D053758:Nanoparticles; D018384:Oxidative Stress / Q000187:drug effects; D052638:Particulate Matter / Q000633:toxicity*; D011650:Pulmonary Alveoli / Q000098:blood supply* / Q000187:drug effects* / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D018570:Risk Assessment; D012822:Silicon Dioxide / Q000633:toxicity*
chemical_list: D000336:Aerosols; C508606:CXCL8 protein, human; D018836:Inflammation Mediators; D016209:Interleukin-8; D052638:Particulate Matter; D017382:Reactive Oxygen Species; D012822:Silicon Dioxide
references.citation: Part Fibre Toxicol. 2007 Sep 25;4:9
references.pmid: 17894871
references.citation: Toxicol Appl Pharmacol. 2003 Aug 1;190(3):278-85
references.pmid: 12902199
references.citation: Exp Lung Res. 1984;6(3-4):197-213
references.pmid: 6092046
references.citation: J Leukoc Biol. 1996 Apr;59(4):555-61
references.pmid: 8613704
references.citation: Adv Colloid Interface Sci. 2007 Oct 31;134-135:167-74
references.pmid: 17574200
references.citation: Cell Biol Toxicol. 2010 Oct;26(5):481-98
references.pmid: 20340042
references.citation: Exp Toxicol Pathol. 2008 Jun;60(2-3):163-80
references.pmid: 18479892
references.citation: Int J Cancer. 1976 Jan 15;17(1):62-70
references.pmid: 175022
references.citation: Exp Toxicol Pathol. 2005 Jul;57 Suppl 1:213-26
references.pmid: 16092729
references.citation: Free Radic Biol Med. 1995 Dec;19(6):805-12
references.pmid: 8582653
references.citation: Toxicol In Vitro. 2011 Dec;25(8):1953-65
references.pmid: 21756993
references.citation: Toxicol Lett. 2004 Apr 1;149(1-3):243-53
references.pmid: 15093270
references.citation: Blood. 1986 Oct;68(4):825-9
references.pmid: 3092887
references.citation: Am J Respir Cell Mol Biol. 1994 Jun;10(6):594-603
references.pmid: 8003338
references.citation: Part Fibre Toxicol. 2009 Oct 08;6:26
references.pmid: 19814802
references.citation: Chemosphere. 2006 Dec;65(10):1784-90
references.pmid: 16762398
references.citation: Free Radic Biol Med. 2008 May 1;44(9):1689-99
references.pmid: 18313407
references.citation: Toxicol Appl Pharmacol. 2007 Jul 15;222(2):141-51
references.pmid: 17599375
references.citation: Am J Respir Cell Mol Biol. 2005 Apr;32(4):281-9
references.pmid: 15640437
references.citation: Cancer Res. 1982 Apr;42(4):1530-6
references.pmid: 6949641
references.citation: Toxicol In Vitro. 2011 Oct;25(7):1251-8
references.pmid: 21570460
references.citation: Am Rev Respir Dis. 1988 Mar;137(3):507-9
references.pmid: 3345032
references.citation: Toxicol In Vitro. 2013 Mar;27(2):774-81
references.pmid: 23261642
references.citation: Cell Tissue Res. 2009 Apr;336(1):91-105
references.pmid: 19238447
references.citation: Int J Cancer. 1980 Aug;26(2):171-6
references.pmid: 6970727
references.citation: Small. 2009 Sep;5(18):2067-76
references.pmid: 19642089
references.citation: Toxicol In Vitro. 2013 Feb;27(1):409-17
references.pmid: 22906573
references.citation: J Leukoc Biol. 1994 Feb;55(2):253-8
references.pmid: 8301222
references.citation: Lancet. 1953 Feb 14;1(6755):336-8
references.pmid: 13012086
references.citation: Altern Lab Anim. 2009 Feb;37(1):89-141
references.pmid: 19292579
references.citation: Paediatr Respir Rev. 2006;7 Suppl 1:S73-5
references.pmid: 16798602
references.citation: Biointerphases. 2007 Dec;2(4):MR17-71
references.pmid: 20419892
references.citation: Environ Health Perspect. 2006 Mar;114(3):412-9
references.pmid: 16507465
references.citation: Part Fibre Toxicol. 2006 Aug 14;3:11
references.pmid: 16907977
references.citation: Environ Health Perspect. 2002 Feb;110 Suppl 1:103-12
references.pmid: 11834468
references.citation: Arch Toxicol. 2012 Apr;86(4):517-8
references.pmid: 22322267
references.citation: Curr Opin Allergy Clin Immunol. 2008 Apr;8(2):103-9
references.pmid: 18317016
references.citation: J Nanobiotechnology. 2004 Dec 08;2(1):12
references.pmid: 15588280
references.citation: Leuk Res. 1988;12(4):345-55
references.pmid: 3131594
references.citation: Environ Health Perspect. 2005 Jul;113(7):823-39
references.pmid: 16002369
references.citation: J Appl Toxicol. 2008 Apr;28(3):249-53
references.pmid: 18300327
references.citation: J Allergy Clin Immunol. 2002 Sep;110(3):349-56
references.pmid: 12209079
references.citation: J Aerosol Med. 2005 Spring;18(1):1-22
references.pmid: 15741770
references.citation: Am J Respir Crit Care Med. 2008 Feb 15;177(4):426-32
references.pmid: 17932382
references.citation: J R Soc Interface. 2010 Feb 6;7 Suppl 1:S27-40
references.pmid: 19586954
references.citation: Nanotoxicology. 2011 Jun;5(2):195-207
references.pmid: 20804439
references.citation: Eur Respir J. 2008 Nov;32(5):1184-94
references.pmid: 18653652
references.citation: J Allergy Clin Immunol. 1998 Oct;102(4 Pt 1):539-54
references.pmid: 9802360
references.citation: Hum Exp Toxicol. 2011 Jul;30(7):579-90
references.pmid: 20554636
references.citation: J Pharm Pharmacol. 2001 Nov;53(11):1447-56
references.pmid: 11732747
references.citation: Toxicology. 2009 Jun 16;260(1-3):142-9
references.pmid: 19464580
references.citation: Toxicol Lett. 2006 Jan 25;160(3):218-26
references.pmid: 16137845
references.citation: Toxicol In Vitro. 2011 Dec;25(8):1516-34
references.pmid: 21963807
references.citation: Toxicol Lett. 2003 Apr 11;140-141:205-11
references.pmid: 12676467
references.citation: Exp Toxicol Pathol. 2005 Jul;57 Suppl 1:189-204
references.pmid: 16092727
references.citation: Microsc Res Tech. 1997 Sep 1;38(5):488-99
references.pmid: 9376652
references.citation: Curr Opin Pulm Med. 2001 Jan;7(1):20-6
references.pmid: 11140402
references.citation: Part Fibre Toxicol. 2005 Oct 21;2:10
references.pmid: 16242040