AJEBAK 56 (Pt. 3) 3Ti--i77 (1978)
Brief Communicatioji;
IN VITRO GROWTH OF OVINE SQUAMOUS CELL CARCINOMA hy M. H. Jl'N. IV H. JOHNSON, D. J. MACUIRK AM. M. S. P R . \ T T '
(From the Department of Tropical N'etfrinary Science, JanU'S Cook University of North Queensland, Townsville. Qu'^^"=*l'i"d 4811. Australia and
(Accepted for publication January 26, 1978.)
Summar>'. Cultured In fragiiR'nt explantation following a selrctivc trypsinisation procedure. 22/.Jt (71'/) specimens of o\iiie sqnamous cell carciiKiina yielded pure strains of epithelial cells which to (late have passaged vinoii)iisly to the 29th level of passage. In e M . S . I'R.VIT
procedures
Iiiitiall}, standard trxpsinisalion procedures «t 37° were used for cell dispi-rsal (Johnson p (J h ami Smith. 19fi2). crlls licinn seeded hitn jjlaNs iir plastii- dishes at 5 x 10'' pi-r ml of \ iabitcells (as dfteniiiiu'd Iiy ti\paii blue t'xtliisidii tet liiiifitu'). The iiiwliuiii was a mixture of aiiial parts MSLS (JohiiMin and Smith, U)(i2) and 199 .supplemented with -Hyji heatinactivated foetal calf •icriim ( F C S ) , rethiced tu 10'.' for maiiitenanie. Subseiiuentlv. tr\p!iinisation was omilUnl, and OSCX: was cultored directly from chopped fruKments usinH the sanw medium. Approximiitel) 6 fraKineiibi were seedes;d disease ( M D ) vinis, and porcine parvoxirus ( P P \ ' ) « e r e adsorbwl to partlv fonned mfinolayers of OSCC and O F E C cells at an estimated concentration of 1 infictions unit jicr 10 eells. Co\erslip preparations were stained witb H & E dnriim a 21-day period of ohscrMition. Demonstration of passenger or other \ li uses in OSCC cnltures was attempted by c'ocu]ti\ati()n of OSCC cells with O F E C dtiriiiK an H5-day observation period entiiiliiit; 10 passages. Coverslip monolayers were examined at each pa.ssage level.
RESULTS. Of 12 attempts to obtain OSCC moiiolayeni by routuie trypsinisalion, none were successful. In eacb case, tr>psinised cells multiplied sIowK in suspension tiuHnu a .l-wt-ek period, after wbich rapid deKeneration occurred, hi contrast, skin from all of 4 o\iiie f- both trypsinisation and explant techniijues from normal aclntt and foetal o\ine skin. All 31 monolayer strains of OSCC initially contained variable proportions of cells showing epithelial and fibroblastic morphology. By use of a selective detachment procedure (Owen.s et al. 1976) using trypsin-versene, it was po.ssible to achieve 71% success (22/31) in establishing OSCC strains of apparently pure epitheli(}id morphology. This technique is simple and appears more practical than other selection teehniques such as those described by McAllister and Ket>d (1968) and Aaronson et al (1970).
Acknowledgements. This work was supported by financial grants from the World Health Organization. The authors wish to thank Dr. Peter Hopkins (Toorak Sheep Field Researeh Station. Department of Primar\' Indnstries) for providing tumor specimens throughout this projeet, and Messrs. R. Jones. J. Marriott and J. Darley for valuable teehnieal assistance.
377
OVINE SQUAMOUS CELL CARCINOMA REFERENCES. AAROXSON,
S.
A.. TODARO,
G.
]..
and
FREEMAN, A. E, (1970): Kxp. Cell. Rt-s..
61, 1.
Pcdiat. Rci.. 2, 356. MOOHE, G . K.. MEHHICK.
C. F. A. (1974): " Handbook of histopalhological and lii,st(ithL'niital technique.s". Third edition. Biitterworth & Co. Ltd., London, p. 341 and p. 489. GiARD, D. J.. AAIIONSON, S. A.. TODABO, G. J., AHNSTEIN, P.. KEHSEV, j . H.. Dosnt.
H.. and PARKS, W . F. (1973): / . Nat.
Cancer Inst., 51. 1417. Bull. Epizoot. Dis. Afr.. 10, 417. P.
W,,
and
ENTWISTLK,
(1977): Br, /, Cancer. 35, 110.
S. R., WOODS
L. K., and ARAUASZ. \ .
M. ( 1975):
Cancer Res.. 35, 2684. OWENS. R. R., SMITH, H . S.. NELSON-REES,
W. A,, and Si'HiNCEH. E. L. (1976); /. Nat. Cancer Imt., 56. 843. SEMAN. G.. HUNTEH, S. J., MILLEH, R. C .
and DMOCHOWSKI, L . (1976): Cancer. 37, 1814. THDNEPSON. S. W . . and HUNT, R. D . (1966):
JOHNSON. R. H., and SMITH, V. W. (1962):
LAUDS.
MCALLISTEH. R. M., and RKKD. G . (196H):
K. \V.
"Selt'cted histochemical and histopatholngical method.s". Charlt'S C, Thomas, Puhiislicr. Illinois, p. 790. ToMKiNN. G. A., and PYE, D . (1968): } . Nat. Cancer
Imt.. 4 0 ( 1 ) , 9 1 .
Brief Communicatioji;
IN VITRO GROWTH OF OVINE SQUAMOUS CELL CARCINOMA hy M. H. Jl'N. IV H. JOHNSON, D. J. MACUIRK AM. M. S. P R . \ T T '
(From the Department of Tropical N'etfrinary Science, JanU'S Cook University of North Queensland, Townsville. Qu'^^"=*l'i"d 4811. Australia and
(Accepted for publication January 26, 1978.)
Summar>'. Cultured In fragiiR'nt explantation following a selrctivc trypsinisation procedure. 22/.Jt (71'/) specimens of o\iiie sqnamous cell carciiKiina yielded pure strains of epithelial cells which to (late have passaged vinoii)iisly to the 29th level of passage. In e M . S . I'R.VIT
procedures
Iiiitiall}, standard trxpsinisalion procedures «t 37° were used for cell dispi-rsal (Johnson p (J h ami Smith. 19fi2). crlls licinn seeded hitn jjlaNs iir plastii- dishes at 5 x 10'' pi-r ml of \ iabitcells (as dfteniiiiu'd Iiy ti\paii blue t'xtliisidii tet liiiifitu'). The iiiwliuiii was a mixture of aiiial parts MSLS (JohiiMin and Smith, U)(i2) and 199 .supplemented with -Hyji heatinactivated foetal calf •icriim ( F C S ) , rethiced tu 10'.' for maiiitenanie. Subseiiuentlv. tr\p!iinisation was omilUnl, and OSCX: was cultored directly from chopped fruKments usinH the sanw medium. Approximiitel) 6 fraKineiibi were seedes;d disease ( M D ) vinis, and porcine parvoxirus ( P P \ ' ) « e r e adsorbwl to partlv fonned mfinolayers of OSCC and O F E C cells at an estimated concentration of 1 infictions unit jicr 10 eells. Co\erslip preparations were stained witb H & E dnriim a 21-day period of ohscrMition. Demonstration of passenger or other \ li uses in OSCC cnltures was attempted by c'ocu]ti\ati()n of OSCC cells with O F E C dtiriiiK an H5-day observation period entiiiliiit; 10 passages. Coverslip monolayers were examined at each pa.ssage level.
RESULTS. Of 12 attempts to obtain OSCC moiiolayeni by routuie trypsinisalion, none were successful. In eacb case, tr>psinised cells multiplied sIowK in suspension tiuHnu a .l-wt-ek period, after wbich rapid deKeneration occurred, hi contrast, skin from all of 4 o\iiie f- both trypsinisation and explant techniijues from normal aclntt and foetal o\ine skin. All 31 monolayer strains of OSCC initially contained variable proportions of cells showing epithelial and fibroblastic morphology. By use of a selective detachment procedure (Owen.s et al. 1976) using trypsin-versene, it was po.ssible to achieve 71% success (22/31) in establishing OSCC strains of apparently pure epitheli(}id morphology. This technique is simple and appears more practical than other selection teehniques such as those described by McAllister and Ket>d (1968) and Aaronson et al (1970).
Acknowledgements. This work was supported by financial grants from the World Health Organization. The authors wish to thank Dr. Peter Hopkins (Toorak Sheep Field Researeh Station. Department of Primar\' Indnstries) for providing tumor specimens throughout this projeet, and Messrs. R. Jones. J. Marriott and J. Darley for valuable teehnieal assistance.
377
OVINE SQUAMOUS CELL CARCINOMA REFERENCES. AAROXSON,
S.
A.. TODARO,
G.
]..
and
FREEMAN, A. E, (1970): Kxp. Cell. Rt-s..
61, 1.
Pcdiat. Rci.. 2, 356. MOOHE, G . K.. MEHHICK.
C. F. A. (1974): " Handbook of histopalhological and lii,st(ithL'niital technique.s". Third edition. Biitterworth & Co. Ltd., London, p. 341 and p. 489. GiARD, D. J.. AAIIONSON, S. A.. TODABO, G. J., AHNSTEIN, P.. KEHSEV, j . H.. Dosnt.
H.. and PARKS, W . F. (1973): / . Nat.
Cancer Inst., 51. 1417. Bull. Epizoot. Dis. Afr.. 10, 417. P.
W,,
and
ENTWISTLK,
(1977): Br, /, Cancer. 35, 110.
S. R., WOODS
L. K., and ARAUASZ. \ .
M. ( 1975):
Cancer Res.. 35, 2684. OWENS. R. R., SMITH, H . S.. NELSON-REES,
W. A,, and Si'HiNCEH. E. L. (1976); /. Nat. Cancer Imt., 56. 843. SEMAN. G.. HUNTEH, S. J., MILLEH, R. C .
and DMOCHOWSKI, L . (1976): Cancer. 37, 1814. THDNEPSON. S. W . . and HUNT, R. D . (1966):
JOHNSON. R. H., and SMITH, V. W. (1962):
LAUDS.
MCALLISTEH. R. M., and RKKD. G . (196H):
K. \V.
"Selt'cted histochemical and histopatholngical method.s". Charlt'S C, Thomas, Puhiislicr. Illinois, p. 790. ToMKiNN. G. A., and PYE, D . (1968): } . Nat. Cancer
Imt.. 4 0 ( 1 ) , 9 1 .
