Research Report New Chamber System for Maintaining Differentiated Guinea Pig Respiratory Epithelial Cells Between Air and Liquid Phases INTRODUCTION cells isolated from human airways. do KB Adler, JE Schwarz differentiated function M,. Whitcutt' and R_ Wu" erpless Excess secrelion nearly as well as cells from guinex pig mucUS University of Vermont; 'Lake respiralory airways the pathogenetic when   cultured this This system; Placid Cell Science Center and spcctrum basis for development o will emphasize [echnical repon University of California at of pulmonary disordcrs, ranging Irom procedure and the use of ot inc Aspects Davis chronic bronchitis and emphysema t0 cells derivcd [rvm guinea pig airways; Ihe common cold: Thus. definition involvcd mcchansms noma METHODS AND MATERIALS stimulatcd secretion Of mucus by goblet cells and glands lining the airways has Uch gonl Ol pulmonary reseurchers Isolation of Guinea Pig Tart of thc problcm has Tracheobronchial Epithelial Cells Tor decudes been the Iack of an appropriatc modcl Male;   Hantley (250- with which study guinea pigs Lhc Un sysicm 300 g) maintained week post-receipf sectelion: drinking Inthis repon, describc (cch- with   tetrucyeline   adted control for respiratory infec icque which allows tor toration of waler differentiated ciliated ticn; anesthetized by injection ABSTRACT and mucin - sccrering epithelium in an in vitr o SYS- Irapenloneally ot sodiumpentobarbutol. ICm).   Proteolytically-dissociated   cells The trachea and mainslemn bronchi are Tracheobronchial cpaheliat cells from dissected free and opened lorigitudinal- from guinea pig trachea Iilneren Jitociane nrmeolsucaln suinea pig are Iy. They are thenrinsed withcold  Ca bronchi collagcn ullugen Mainained {lalfclenaed Hea frec   Hanks balanced gcl wnich sits atop transprtng, Cruss- salts solurion Tratonareni Crons-Minacd ninoceuiose (HBSS linked   nitrocellulose  membrane. The and placed in 4 Pelri dish con- Ine cells ure Jed Jra mcuinrt(e' Ccu taining XIV; pronase cell cultutes are "fed" from below only. 0S (Type Jefined mediwm nite ICrurt-rtt Coz, Sigma Chemical Sc Louis. MO) humidified air inler- cxposcu naled Irt and incubated at 37" C in 959 air/5% Mainiemanceof the cellcul- Accoove Manleonta COz The epitheliumt for polarized (umcsintnismanncr aows (cm polarieed  eeenation cels Uunps gently teased off with fine forcens anda differentiation isolated cells hall afer cight rul (Le/ue dus alterabout $ davs the nseudo stcnle #I0 scalpel blade , and thc cell pScudoswralfitd ciliated columuutue TC stralifed columnar cpithelium formed sneet genty dspersed by trituration formed viritelly identicol #Hucre with sterile Pasteur pipette The cell is virlually dentical morphologically thar seed 6cuctro JcEnT and functionally lo mucosal epitbel- suspension is then centriluged at [25 g intact Otinial 4uur Suitem com for I0 the pellet iUMI In (nc intac( AIJITIAI, Dires Ie advantages 0} txull (Run F-12 meeliur resusperderel ( ulrures The becn Ham ` culture systcm {roh- e{uunoles niuiar containing described primary rescarch [0R fetal calf Tic sys - pant Icmm cut6 Wnrlenr TCcuaua (FCS). The   suspended previous manuscript (151. cells ideally 0 airnoy paper Wfed aenan centrifuged and the cell peller again bas full characterization Lhe cele cpirhelial function. SNOR Secrtry lular growth kinetics analysis of suspendedinF.[2 medium IOSFCS_ sponses Irritunts miedulors centrifuged The cells are third time #ell cellular secrenons- As pointed OuI in studlles Ini Olng haeurre{laset that paper. cclls from other rodent air- unider idemical contditicns, Ard Ihe cell cell diffcreriation Wnc occrmi hamsier ad rabbit; and pellet suspended cold, -fice cuch Ways cuacancncs Ler (1987) Hiu -chnicices 50578728
Research Report New Chamber System for Maintaining Differentiated Guinea Pig Respiratory Epithelial Cells Between Air and Liquid Phases INTRODUCTION cells isolated from human airways. do erpless differentiated function nearly as well as cells from guinex pig when   cultured this system; This repon will emphasize [echnical Aspects ot inc procedure and the use of cells derivcd [rvm guinea pig airways; KB Adler, JE Schwarz M,. Whitcutt' and R_ Wu" University of Vermont; 'Lake Placid Cell Science Center and University of California at Davis Excess secrelion mucUS respiralory airways the pathogenetic basis for development o spcctrum of pulmonary disordcrs, ranging Irom chronic bronchitis and emphysema t0 Ihe common cold: Thus. definition mcchansms involvcd noma stimulatcd secretion Of mucus by goblet cells and glands lining the airways has Uch gonl Ol pulmonary reseurchers Tor decudes Tart of thc problcm has been the Iack of an appropriatc modcl sysicm with which study Lhc Un sectelion: Inthis repon, describc (cch- icque which allows tor toration of differentiated ciliated and mucin - sccrering epithelium in an in vitr o SYS- ICm).   Proteolytically-dissociated   cells from guinea pig trachea Iilneren bronchi Mainained collagcn gcl wnich sits atop transprtng, Cruss- linked   nitrocellulose  membrane. The cell cultutes are "fed" from below only. cxposcu humidified air inler- Accoove Mainiemanceof the cellcul- (umcsintnismanncr aows for polarized differentiation isolated cells hall alterabout $ davs TC the nseudo stralifed columnar cpithelium formed is virlually dentical morphologically and functionally lo mucosal epitbel- iUMI In (nc intac( AIJITIAI, The culture systcm becn described pant primary rescarch paper previous manuscript (151. bas full characterization Lhe cele lular growth kinetics analysis of cellular secrenons- As pointed OuI in that paper. cclls from other rodent air- Ways cuch hamsier ad rabbit; and METHODS AND MATERIALS Isolation of Guinea Pig Tracheobronchial Epithelial Cells Male;   Hantley guinea pigs (250- 300 g) maintained week post-receipf with   tetrucyeline   adted drinking waler control for respiratory infec ticn; anesthetized by injection Irapenloneally ot sodiumpentobarbutol. The trachea and mainslemn bronchi are dissected free and opened lorigitudinal- Iy. They are thenrinsed withcold  Ca frec   Hanks balanced salts solurion (HBSS and placed in 4 Pelri dish con- taining 0S pronase (Type XIV; Sigma Chemical Coz, Sc Louis. MO) and incubated at 37" C in 959 air/5% COz The epitheliumt (cm gently teased off with fine forcens anda stcnle #I0 scalpel blade , and thc cell sneet genty dspersed by trituration with sterile Pasteur pipette The cell suspension is then centriluged at [25 g for I0 the pellet resusperderel Ham ` F-12 meeliur containing [0R fetal calf Icmm (FCS). The   suspended cells centrifuged and the cell peller again suspendedinF.[2 medium IOSFCS_ The cells are centrifuged third time unider idemical contditicns, Ard Ihe cell pellet suspended cold, Ler -fice ABSTRACT Tracheobronchial cpaheliat cells from suinea pig are Jitociane nrmeolsucaln {lalfclenaed ullugen Hea Tratonareni Crons-Minacd ninoceuiose mcuinrt(e' Ine cells ure Jed Jra Ccu nite ICrurt-rtt  Jefined mediwm naled Irt Manleonta polarieed  eeenation Uunps cels (Le/ue dus rul afer cight pScudoswralfitd ciliated columuutue #Hucre formed viritelly identicol 6cuctro JcEnT thar seed intact Otinial 4uur Suitem com Dires Ie advantages 0} txull (Run ( ulrures e{uunoles niuiar {roh- Wnrlenr cut6 TCcuaua Tic sys - Wfed ideally aenan 0 airnoy cpirhelial function. SNOR Secrtry sponses Irritunts miedulors haeurre{laset #ell studlles Ini Olng cell diffcreriation Wnc occrmi cuacancncs (1987) Hiu -chnicices 50578728