Author/Editor		Jagrič, Tomaž; Marš, Tomaž; Grubič, Zoran; Jagrič, Timotej; Geršak, Borut
Title		Kulture človeških endotelnih celic, izoliranih iz arterije mamarije interne
Translated title		Human endothelial cell cultures isolated from internal mammary artery
Type		članek
Source		Zdrav Vestn
Vol. and No.		Letnik 75, št. 10
Publication year		2006
Volume		str. 621-8
Language		slo
Abstract		Background The aim of ourstudy was to prepare homogenic and long tern cultures of endothelial cells (EC) from a human internal mammary artery (IMA) obtained during a cardiac by pass graft operation (CABG). In this way we would be able to assess the conditions suitable for the preparation of sufficient number of autologous arterial ECs and their use in a population mostly affected with cardiovascular diseases. Methods Seven 52-76 years old male patients diagnosed with ischemic heart disease were included in ourstudy. Samples of internal mammary artery were collected during CABG performed on these patients. EC cells were prepared from these samples using two approaches. in situ digestion with proteolytical enzymes and proteolytic digestion from the previously resected IMA vessels. Cell morphology and growth kinetics were studied in 3 weeks old cultures. EC cell specificy was determined by immunohystochemical staining for von Willebrand's factor. Results Cell growth could be observed only in cultures prepared with the in situ proteolytic digestion. Cells sowed the typical EC morphology and were positive for von Willebrand's factor. The maximal cell number in colonies (11 in average, 51 at the most) was reached after 6 days in culture. The average cell doubling time of the five cultures was 82 ± 43 hours. Conclusions The study proved that our method of isolation is specific for ECs. Using a small IMA sample we were able to collect a number of colonies and sustain them in long term cultures, but the end numbers of cells is still too small for their application in tissue engineering (TE). Our further efforts will be focused on the optimization of the conditions which would allow sufficient proliferation of EC so that small IMA sample would be enough for the TE.
Summary		Izhodišča Cilj raziskave je pripraviti homogene kulture endotelnih celic (EC), izoliranih iz človeške arterije mamarije interne (AMI), dobljene med srčno obvodno operacijo, in jih vzdrževati v dolgoročnih kulturah. Na ta način bi skušali ugotovili pogoje, pri katerih bi lahko v nadaljnjem delu s celičnimi kulturami prišli do zadostnega števila avtolognih arterijskih EC celic za uporabo pri populaciji, ki jo kardiovaskularne bolezni najbolj prizadenejo. Metode V raziskavo je bilo vključenih 7 moških, starih 52-76 let, z diagnosticirano ishemično srčno boleznijo. Pri vseh je bila opravljena obvodna operacija. Iz žilnih vzorcev, ki smo jih pridobili med operacijo, smo osamili celice na dva načina: s proteolitično razgradnjo tkiva in situ ali pa s proteolitično razgradnjo iz predhodno razkosanih žilnih preparatov. Spremljali smo morfologijo in kinetiko rasti EC v 3 tedne starih kulturah. Celično specifičnost smo dokazali z imunohistokemičnim določanjem von Willebrandovega faktorja. Rezultati Rast celic je bile prisotna le v kulturah, ki smo jih pripravili s prvo metodo. Celice so kazale tipične morfološke značilnosti EC in izražale von Willebrandov faktor. Največje število celic v kolonijah (v povprečju 11, maksimalno pa 51) so kulture dosegle v 6 dneh. Podvojitven čas celic je bil 82 ± 43 ur. Zaključki Z raziskavo smo pokazali, da je predstavljena metoda osamitve EC iz majhnega vzorca AMI primerna za pridobivanje vitalnih celic. Metoda je selektivna EC in omogoča njihovo rast v dolgoročnih kulturah. Končno število celic v kolonijah je za zdaj premajhno za uporabo v tkivnem inženirstvu, vendar bi se z optimizacijo metode v prihodnjih raziskavah lahko bolj približali temu cilju.
Descriptors		ENDOTHELIUM, VASCULAR CELLS, CULTURED MAMMARY ARTERIES VON WILLEBRAND FACTOR MYOCARDIAL ISCHEMIA CORONARY ARTERY BYPASS MICROSCOPY, PHASE-CONTRAST IMMUNOHISTOCHEMISTRY