Towards the development of a simplified long-term organ culture method for human scalp skin and its appendages under serum-free conditions

A long-term culture method of beating heart explants of adult newt has been developed recently in our laboratory. This method has enabled us to study the ultrastructure and contractile activities of beating adult heart explants at selected intervals. Adult newt heart ventricle was excised and washed with amphibian balanced salt solution and cut into several pieces of approximately 0. 5-1. 0 mm. Heart pieces were cultured for 2 months at 25°C in a medium containing 90% modified Leibovitz medium (L-15), 10% fetal calf serum and 1% penicillin-Streptomycin. In first week of culture, more than 37% of heart explants were attached to the substrate, and more than 33% established pulsation rates. Some explants had a rate of 3-12 beat/min., while others had a rate of 28-67 beat/min. A few cells migrated from the explants and were attached to the substrate around the explants. These migrated cells did not show pulsation.

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Abstract 312: Mechanisms Underlying Impaired KCl-induced Contractility After Long-term Serum-free Organ-culture of Rat Isolated Mesenteric Artery

Hypertension ◽

10.1161/hyp.62.suppl_1.a312 ◽

2013 ◽

Vol 62 (suppl_1) ◽

Author(s):

Tomoka Morita ◽

Muneyoshi Okada ◽

Hideyuki Yamawaki

Keyword(s):

Smooth Muscle ◽

Organ Culture ◽

Mesenteric Artery ◽

Cell Function ◽

Mesenteric Arteries ◽

Muscle Contractility ◽

Smooth Muscle Contractility ◽

Serum Free ◽

Functional Changes

(Background and aim) Organ culture of blood vessels is a useful technique to investigate the long-term effects of drugs. Organ culture in a serum-free condition is so far the best way to maintain differentiated cell function. However some functional changes may occur from freshly isolated blood vessel (fresh) such as decreased contractility. Mammalian/mechanical target of rapamycin (mTOR) complex 1 is atypical serine/threonine kinase which integrates various signals induced by growth factors, stress, energy status, oxygen, and amino acids. Here, we investigated the mechanism of decrease in smooth muscle contractility after long-term serum-free organ culture specifically focusing on mTOR. (Methods and results) Rat isolated mesenteric arteries were cultured for 5 days without (0% serum) or with rapamycin (Rap). In 0% serum, absolute contraction by KCl significantly decreased from fresh (n=21 for fresh, n=7 for 0% serum, p<0.01 ). In Rap, the decreased contraction was significantly normalized (n=7, p<0.05 vs. 0% serum). In both 0% serum and Rap, sensitivity to KCl significantly increased from fresh ( p<0.01 vs. fresh). In 0% serum, mTOR expression significantly increased from fresh (n=8, p<0.01 ) which was significantly normalized by rapamycin (n=8, p<0.01 ). Morphological examinations showed degenerative changes in smooth muscle layer of 0% serum which was improved by rapamycin. In 0% serum, expression of myocardin, a master regulator of smooth muscle gene expression significantly decreased from fresh (n=9, p<0.01 ), which was significantly normalized by rapamycin (n=9, p<0.01 ). (Conclusion) Addition of rapamycin prevented the decreased contractility in serum-free organ cultured mesenteric artery perhaps by normalizing the mTOR and downstream myocardin expression as well as arterial degenerative morphological damage. Further studies are needed to clarify how mTOR controls myocardin expression and also how decreased myocardin expression leads to the decreased smooth muscle contractility. This experimental system may be useful for the hypertension research.

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