several cell lines were isolated during independent experiments from dissected annulus fibrosus ( af ) tissue of mature bovine intervertebral discs ( ivd ) via a reproducible non - enzyme driven protocol .
the cell lines were frozen at low passage number and they recovered well after freeze - thawing ( see fig .
preliminary characterization of the af cells was carried out with bovine specific rna probes derived from bovine genomic dna using plate rna in situ hybridisation ( pish )   for col1a1 and col2a1 expression , two structural proteins found in the mature ivd .
less type - ii collagen fibers were described for the outer af in rabbit   correlating with a common notion that type ii collagen is higher in the np than the af , .
the dissected outer af of mature bovine ivds was the source for our af cell lines and we did not detect col2a1 expression by either rna in situ hybridization ( sish ) , , ,   on sections of the outer af tissue or by pish on the cells derived from the outer af , while col2a1 expression was very prominent in cells of the np as shown by sish on the same section ( fig .
the discrepancy between our findings and that of increased col2a1 expression in the bovine af over the np reported by minoque et al .   might reflect differences in defining the af .
we see col1a1 expression in af and np cells by sish in vivo and by pish in vitro ( fig .
skinned bovine tails were collected fresh from local abattoirs , remained chilled and were processed within 2  h. tail pieces were immersed in 10% povidone - iodine solution , rinsed with tap water , followed by immersion in 70% etoh prior to removing all fat and muscle tissue .
ivds were dissected away from adjacent vertebrae endplates , briefly dipped in 70% etoh and rinsed with 1   pbs/10% gentamicin prior to separating the outer af from the remaining ivd tissue .
outer af tissue was cut into smaller pieces using sterile procedures and placed in uncoated as well as 0.1% gelatin coated 35  mm culture dishes ( falcon ) .
sterile filtered fbs - hi with 10% gentamicin and 5  g / ml amphotericin b ( all gibco ) was added prior to the incubation at 37c , 5% co2 and atmospheric o2 .
after 24  h the fbs mix was diluted 1:1 with standard dmem based growth medium containing 1   dmem with 4.5  g / l glucose , 1   pyruvate , 1   glutamax , 1   nonessential amino acids , 10% v / v hi - fbs , 0.48% v / v gentamicin ( all gibco ) , 0.12  mm beta - mercapthoethanol ( sigma ) and additional 5  g / ml amphotericin b and the tissue .
following 48  h of incubation cells had attached to the bottom of the wells and were expanded in fresh standard dmem based growth medium ( see above ) .
cell lines derived in such manner from af tissue could be passaged with 0.05% trypsin / edta ( gibco ) at 1:10 dilutions for more than 10 passages without slowing down in population growth or dramatic changes in morphology ( fig .
early and late passages were subjected to plate rna in situ hybridization ( pish )   for preliminary gene expression analysis ( fig .
during embryogenesis , the af part of the ivd is believed to be of sclerotomal origin , .
cultured cells derived from the outer af of mature bovine caudal ivds with our procedure were assayed for the expression of two major collagen genes col1a1 and col2a1 .
the observed in vitro expression of these two genes mirrored the in vivo expression in cells of the mature the af : presence of col1a1 expression and absence of col2a1 expression in cells of the outer af ( fig .
 the adult bovine ( bos taurus ) intervertebral disc is primarily comprised of two major tissue types : the outer annulus fibrosus ( af ) and the central nucleus pulposus ( np ) . 
 we isolated several primary cell lineages of passage ( p ) 0 cells from the af tissue omitting typically used enzymatic tissue digestion protocols . 
 the cells grow past p10 without signs of senescence in dmem  +  10% fcs on 0.1% gelatin coated / uncoated surfaces of standard cell culture plates and survive freeze - thawing . 
 preliminary analysis of the af derived cells for expression of the two structural genes col1a1 and col2a1 was performed by pish recapitulating the expression observed in vivo . 
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