Procedure for the preparation of unilamellar vesicles for...

A - Human Necessities – 01 – N

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A01N 1/02 (2006.01) C12N 5/073 (2010.01) C12N 5/075 (2010.01) C12N 5/076 (2010.01)

Patent

CA 2653905

In order to prepare unilamellar vesicles of low density lipids, lipids of plant origin are first predissolved; a suitable quantity of lipids, from 4 to 12 mg/ml is mixed with hot pure water and stirred for some 30 minutes, the suspension obtained is held at 50-60°C for one hour and stirred for 2 minutes every 20 minutes. High molecular weight hyalurone is added to the lipid suspension up to a final concentration of 1 mg/ml and the suspension of lipids and hyalurone is emulsified using ultrasound, pumped through a homogenizer valve and immediately passed through a polycarbonate membrane, obtaining a final suspension with 70% of the particles in suspension having a size from 5 to 15 nM, which means that HA and LDL single layer vesicles can be used for cryoconservation and the culture of various mammal cells, especially stem cells.

Pour la préparation des vésicules unilamellaires de lipides de basse densité, on obtient d'abord une présolution de lipides d'origine végétale. Une quantité appropriée de lipides, de 4 à 12 mg/ml, est mélangée avec de l'eau pure chauffée et agitée pendant environ 30 minutes, et la suspension ainsi obtenue est maintenue à 50-60°C pendant une heure et agitée pendant 2 minutes à chaque 20 minutes. On ajoute une hyalurone de poids moléculaire élevé à la suspension lipidique jusqu'à une concentration finale de 1 mg/ml. La suspension de lipides et d'hyalurone est alors émulsionnée par sonification, pompée au moyen d'une valve d'homogénéisation, puis immédiatement amenée à traverser une membrane de polycarbonate, d'où l'obtention d'une suspension finale dont environ 70% des particules en suspension présentent une taille comprise entre 5 et 15 nM, ce qui permet d'utiliser les vésicules monocouche de HA et LDL pour la cryoconservation et la culture de différentes cellules de mammifères, et notamment de cellules germinales.

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