Ciliogenesis in cryopreserved mammalian tracheal epithelial cells cultured at the air-liquid interface.
To determine air-liquid interface (ALI) culture derived from cryopreserved mammalian tracheal ciliated cells is a viable ciliated cell model for the investigations of regulatory mechanisms of ciliary beat frequency (CBF), two studies were performed using ovine and porcine tracheae obtained from local slaughterhouses. The protease-digested tracheal ciliated cells were harvested and cultured at the ALI using collagen-coated, porous membrane inserts. In study 1, the ALI culturing protocols were established using non-cryopreserved ovine tracheal ciliated cells. Ciliogenesis was documented with immuno-histology and electron micrographs. Vigorous beating cilia were video-recorded. CBF was measured by laser light scattering. The functional integrity of the autonomic receptors of the ciliated cells was confirmed with the stimulatory responses of CBF using luminal methacholine and basolateral terbutaline. In study 2, porcine tracheal ciliated cells stored in liquid nitrogen for a minimum of 4 weeks were used. The cryopreserved cells were thawed and cultured using the ALI protocol established in study 1. After two months, cilia outgrowths were confirmed using video microscopy and scanning electron micrograph (SEM). The trans-epithelial resistances were 28.5 kOmega (n=4). Luminal applications of 1 microM and 10 microM methacholine stimulated CBF from a baseline of 7.4+/-0.2 Hz to 8.4+/-0.8 Hz and 7.7+/-0.4 Hz, respectively (n=5). Basolateral applications of 1 microM and 10 microM terbutaline stimulated CBF from a baseline of 7.5+/-0.3 Hz to 8.2+/-0.4 Hz and 8.0+/-0.4 Hz, respectively (n=5). These data demonstrated that a ciliated cell bank can be established using cryopreserved ciliated cells for pulmonary drug discovery and toxicological screening.
Mao
Hua
H
BioTechPlex Corporation, 1205 Linda Vista Dr., San Marcos, CA 92078, USA. hmao@biotechplex.com
Wang
Yuchi
Y
Yuan
Weihua
W
Wong
Lid B
LB
D000388:Air; D000818:Animals; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D002923:Cilia / Q000502:physiology*; D015925:Cryopreservation; D004847:Epithelial Cells / Q000502:physiology* / Q000648:ultrastructure; D008855:Microscopy, Electron, Scanning; D046529:Microscopy, Electron, Transmission; D018715:Microscopy, Video; D009024:Morphogenesis; D012756:Sheep; D013552:Swine; D014132:Trachea / Q000166:cytology*
In Vitro Cell Dev Biol. 1988 May;24(5):420-8
3372447
Am J Respir Cell Mol Biol. 1994 Feb;10(2):192-201
7509163
Am J Respir Cell Mol Biol. 1994 Mar;10(3):271-7
8117445
J Appl Physiol (1985). 1988 Oct;65(4):1895-901
2846499
Biochem Biophys Res Commun. 1995 Oct 24;215(3):1014-21
7488025
J Cell Sci. 1999 Dec;112 ( Pt 23):4357-66
10564653
Cell Biol Toxicol. 1992 Jul-Sep;8(3):141-50
1446252
Genome Biol. 2001;2(7):RESEARCH0026
11516339
Lab Invest. 1971 Jan;24(1):55-66
4322702
Virchows Arch B Cell Pathol Incl Mol Pathol. 1984;45(2):221-40
6143448
J Physiol. 1999 Nov 1;520 Pt 3:851-65
10545149
Am J Respir Cell Mol Biol. 1997 Jun;16(6):724-31
9191474
Am J Respir Cell Mol Biol. 1996 Jan;14(1):19-26
8534482
Am J Respir Cell Mol Biol. 1995 Mar;12(3):329-38
7873199
Biol Cell. 1994;82(2-3):211-6
7606217
J Appl Physiol (1985). 1988 Aug;65(2):971-81
3049516
Ann Biomed Eng. 1998 Jul-Aug;26(4):666-78
9662158
Exp Lung Res. 1995 Nov-Dec;21(6):957-70
8591796
Am J Physiol. 1998 Oct;275(4):L827-35
9755116
Cryobiology. 1990 Dec;27(6):591-5
1962747
Lab Invest. 1975 Oct;33(4):451-60
1186125
In Vitro Cell Dev Biol Anim. 1993 Jun;29A(6):481-92
7687243
Am J Physiol. 1998 Aug;275(2):C440-8
9688598
Lab Invest. 1987 Jun;56(6):654-64
2439773
Cryobiology. 1985 Apr;22(2):105-10
3979080
Cryobiology. 1989 Aug;26(4):328-32
2766780
Cancer Chemother Rep 3. 1973 Mar;4(2):43-7
4729337