Rotary Culture System Research Articles

Morphological and biochemical integrity of human liver slices in long-term culture: effects of oxygen tension.

We tested the effects of low (20% O2) and high (70% O2) oxygen tension on the morphological and biochemical integrity of human liver slices incubated for up to 72 h in supplemented Williams' E medium in a dynamic rotating culture system. High oxygen tension was more effective than low oxygen tension for preserving morphological integrity in long-term culture (48-72 h). After 72 h of culture with 70% O2, the lobular pattern was well preserved, and the survival of hepatocytes (approximately 80%) and other cell types was good. Immunohistochemical studies showed good preservation of the region-specific expression of CYP2EI and CYP3A4 isoenzymes for up to 72 h of incubation in 70% O2. As compared to 20% O2, the oxidized glutathione content and reactive oxygen species production were slightly increased in 70% O2, suggesting that minimal oxidative stress occurred with the high oxygen tension. In conclusion, despite slight oxidative stress associated with high oxygen tension, 70% O2 appeared more appropriate than 20% O2 for preserving the morphological and biochemical integrity of human liver slices cultured in a dynamic organ culture system for up to 72 h.

AN IN VITRO STUDY FOR METABOLIC CHARACTERIZATION OF HEPATOCYTE (HP) MULTIAGREGATES

414 The aim of our current study was to culture rat HP as multiagregate spheroids to provide an in vitro model for metabolic characterization prior to establish transplantation in in vivo studies. HP were isolated from Sprague Dawley rats using two-step enzyme dispersion method with collagenase and were later cultured in a continuous rotating culture system. The total number of cells isolated per animal was 2.5 − 3.0 × 103 cells and the viability was 78% (69-88 %). After 48 hours, 100% of the cultures were viables with a maximal duration of 144 hours in culture. Total DNA measurement was utilized to evaluate cell number, showing a slight decrease throughout the culture time, but differences were not statistically significant (n=4; p<0.005). HP viability in culture was evaluated by LDH leakage, results showed less than 2% release after 5 days in culture (n=5). Total protein/total DNA ratio increased 2 fold throughout the culture time (day 0: 18.8 ± 25.68 mg protein/mg DNA; day 5: 36.7 ± 21.22 mg protein/mg DNA; n=4) suggesting an active synthesis of protein by the HP in culture. Samples were incubated in media supplemented with NH4Cl and urea synthesis and amonium clereance was determined at different time-points, results showed a marked decrease in the rate of urea synthesis after one day in culture when comparing with freshly isolated HP and only a slight decrease thereafter. Moreover, amonium clearence was observed throughout the different time-points. Under electron microscopy, the isolated cells were spherical with 17−30 ∓ in diameter finding ocasional binucleated cells. HP were seeded at 1×106 cells/ml and after 24 hours in culture formed multiagregates with 150-600u in diameter. By 72 hours the multiagregates showed a compact structure where it was difficult to differenciate individual HP. By day 5 HP re-established cell to cell communication and resembled the architectural structure of the liver. Over 90% of HP were associated as spheroids. Enzyme activities were measured spectrophotometrically: Hexoquinase activity was very low (n=16; 1.6+0.35 umol/min/mg protein) corresponding to an in vivo conditions, whereas Glucoquinase activity was 4 fold higher (n=16; 6.24+0.29 umol/min/mg protein) than that observed for Hexoquinase. The induced Glucoquinase activity might be as a result of the aviability of insulin in the culture media. These findings suggest that the culture contains a highly pure and functional parenchymal cell preparation (HP). These results show an adequate way for in vitro metabolic evaluation of HP spheroids prior to in vivo studies in animal models with FHF.

多孔質担体への苗条原基の捕捉と種苗大量増殖への応用

Development of an efficient system for shoot mass propagation through the entrapment of shoot primordia was investigated on Stevia rebaudiana. Shoot primordia of Stevia rebaudiana could be trapped into cubes of reticulate form matrix of loofah, sponge of Luffa aegyptica, when the primordia were cultured in a rotary culture system (flask scale) or in the drum type jar fermentor system in the presence of the cubic matrices. The shoot primordia grew to form uniform clumps staying in loofah matrices and the growth was stimulated by the entrapment compared to the control culture without the matrices. Shoots were efficiently induced from the entrapped clumps of primordia under subsequent static culture condition and were acclimatized successfully without removing the matrices.

Ａ／Ｊ系マウス全はい培養法を用いたエストラドオールの口唇裂発生抑制作用の検討

I studied the effect of estradiol on prevention of cleft lip in A/J mice using whole embryo culture system. Embryos of the 10.5th day of gestation (plug day=0) were harvested from untreated pregnant mice, pregnant mice injected subcutaneously with 25μg/kg of estradiol or the solvent of hormone on the 9. 5th day of gestation. As a control of untreated A/J mouse embryos, C 57 BL/6 mouse embryos of same developmental stage were used. These embryos were cultured for 48 hours in pure rat serum containing 2mg/ml of glucose and antibiotics with continuous supply of 95% 02 and 5% CO2 mixture gas by a rotating culture system at 25 to 10 rpm. Both A/J and C 57 BL/6 mouse embryos showed manifest growth and development in vitro. The incidence of cleft lip in vivo in A/J and C 57 BL/6 mice at the 18th day of gestation is 6.2% and 0% respectively. No cleft lip was observed in cultured C 57 BL/6 mouse embryos. Cleft lip was observed in 51.2% of untreated and 52.2% of solvent treated A/J mouse embryos in vitro. By contrast, in estradiol treated group, the incidence of cleft lip was decreased significantly to 21.3% (p<0.001). These results indicate that the effects of estradiol treatment act to the embryos before primary palate formation, and that the genetical predisposition of cleft lip in A/J mouse embryos become resistant to cleft lip.