Morphological and functional integrity of precision-cut rat liver slices in rotating organ culture and multiwell plate culture: effects of oxygen tension.
We examined the maintenance of functional and morphological integrity of precision-cut rat liver slices cultured in various incubation systems and conditions for 72 h. Slices were incubated (37 degrees C) for 6, 24, 48, and 72 h in supplemented Williams E medium in 6-well plastic culture plates on a gyratory shaking platform (WPCS) or in a rotating organ culture system (ROCS) using 5% CO2--95% air (WPCS/air or ROCS/air) or 5% CO2--70% O2--25% N2 (WPCS/O2 or ROCS/O2). Biochemical and functional parameters of slices maintained in WPCS/air or WPCS/O2 were almost totally inhibited after 24 h, in keeping with the extensive and diffuse coalescing coagulative necrosis typical of post-ischemic injury affecting almost all the slice surface after 48 h. As compared to freshly isolated slices, slices maintained in ROCS/air for 72 h showed stable ATP and GSH content, increased protein synthesis, and a slight steady decrease in GST activity, while ATP and GST activity remained stable and protein synthesis and GSH content increased in slices incubated in ROCS/O2 for 72 h. The extent of coagulative necrosis was markedly lower in longitudinal sections from slices incubated for 72 h in ROCS/O2 than in ROCS/air. Transversal sections from slices kept in ROCS/air for 72 h showed a thick central band of necrotic cells edged by two peripheral layers of viable hepatocytes, whereas most of the slice was composed of viable hepatocytes lined by two thin layers of necrotic cells after 72 h in ROCS/O2. ROCS/O2 emerged as the system best preserving the histological and functional integrity of rat liver slices in long-term culture.
Départment Sécurité du Médicament, Centre de Recherche de Vitry-Alfortville, Rhône-Poulenc Rorer S.A., Vitry sur Seine, France.
H J
HJ
Toutain
V
V
Moronvalle-Halley
J P
JP
Sarsat
C
C
Chelin
D
D
Hoet
D
D
Leroy
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D046508:Culture Techniques; D019803:Glutathione Disulfide / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D008099:Liver / Q000033:anatomy & histology / Q000502:physiology*; D008297:Male; D009924:Organ Culture Techniques; D010100:Oxygen / Q000378:metabolism*; D014176:Protein Biosynthesis; D011506:Proteins / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors
D011506:Proteins; D000255:Adenosine Triphosphate; D005982:Glutathione Transferase; D010100:Oxygen; D019803:Glutathione Disulfide