MD1171Z - Method for treating the decellularized liver matrix to enhance cell adhesion - Google Patents

Method for treating the decellularized liver matrix to enhance cell adhesion Download PDF

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Publication number
MD1171Z
MD1171Z MDS20160156A MDS20160156A MD1171Z MD 1171 Z MD1171 Z MD 1171Z MD S20160156 A MDS20160156 A MD S20160156A MD S20160156 A MDS20160156 A MD S20160156A MD 1171 Z MD1171 Z MD 1171Z
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Moldova
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liver
matrix
decellularized
decellularized liver
amount
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MDS20160156A
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Romanian (ro)
Russian (ru)
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Марианна ЖИАН
Виорел НАКУ
Виталие КОБЗАК
Константин ПАЛАДИЙ
Валериана ПАНТЯ
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Государственный Медицинский И Фармацевтический Университет "Nicolae Testemitanu" Республики Молдова
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Abstract

The invention relates to regenerative medicine and tissue engineering and can be used for treating the decellularized liver matrix to enhance cell adhesion.Summary of the invention consists in that is washed the decellularized liver matrix by injecting into the portal vein a mixture of phosphate buffered saline, streptomycin 100 µg/ml, penicillin, 100 U/ml and amphotericin B 0.25 µg/ml, in an amount of 20 ml/10 g of decellularized liver, then is injected a solution of type I collagen, obtained from bovine Achilles tendon in a concentration of 0.01%, dissolved in acetic acid 0.1 M, in an amount of 160…240 ml/10 g of decellularized liver, after which the hepatic matrix is perfused with phosphate buffered saline with a pH 7.4 in an amount of 300 ml/10 g of liver at a rate of 5 ml/min.

Description

Invenţia se referă la medicina regenerativă şi ingineria tisulară şi poate fi utilizată pentru prelucrarea matricei decelularizate a ficatului pentru sporirea adeziunii celulare. The invention relates to regenerative medicine and tissue engineering and can be used for processing decellularized liver matrix to enhance cell adhesion.

Cu aceeaşi destinaţie, în condiţii de laborator matricea decelularizată a unui lob al ficatului este recelularizată cu câte 1,5x107 hepatocite la fiecare 10 min până se ajunge la 2x108 celule [1]. În timpul procesului de recelularizare nu se utilizează suplimentar nici o substanţă pentru mărirea aderării celulelor la matricea extracelulară a ficatului decelularizat. For the same purpose, under laboratory conditions the decellularized matrix of a liver lobe is recellularized with 1.5x107 hepatocytes every 10 min until 2x108 cells are reached [1]. During the recellularization process, no additional substance is used to increase cell adhesion to the extracellular matrix of the decellularized liver.

Dezavantajul constă în aceea că în timpul procesului de recelularizare are loc pierderea excesivă de hepatocite din cauza adeziunii insuficiente a hepatocitelor injectate de matricea hepatică decelularizată. The disadvantage is that during the recellularization process, excessive hepatocyte loss occurs due to insufficient adhesion of the injected hepatocytes to the decellularized hepatic matrix.

Problema pe care o rezolvă invenţia constă în utilizarea unei metode noi, ce ar permite amplificarea adeziunii hepatocitelor de matricea decelularizată a ficatului şi ar împiedica pierderea lor excesivă după injectare. The problem solved by the invention consists in using a new method, which would allow the amplification of hepatocyte adhesion to the decellularized liver matrix and would prevent their excessive loss after injection.

Esenţa invenţiei constă în aceea că se spală matricea decelularizată a ficatului prin injectarea în vena portă a unui amestec de soluţie tampon fosfat salin, streptomicină 100 µg/ml, penicilină 100 U/ml şi amfotericină B 0,25 µg/ml, în cantitate de 20 ml/10 g de ficat decelularizat, apoi se injectează soluţie de colagen tip I, obţinut din tendonul Achile de bovină în concentraţie de 0,01%, dizolvat în 0,1M de acid acetic, în cantitate de 160...240 ml/10 g de ficat decelularizat, după care matricea hepatică se perfuzează cu soluţie tampon fosfat salin cu un pH 7,4 în cantitate de 300 ml/10 g de ficat cu o viteză de 5 ml/min. The essence of the invention consists in washing the decellularized liver matrix by injecting into the portal vein a mixture of phosphate buffer saline, streptomycin 100 µg/ml, penicillin 100 U/ml and amphotericin B 0.25 µg/ml, in an amount of 20 ml/10 g of decellularized liver, then injecting a solution of type I collagen, obtained from bovine Achilles tendon in a concentration of 0.01%, dissolved in 0.1M acetic acid, in an amount of 160...240 ml/10 g of decellularized liver, after which the liver matrix is perfused with phosphate buffer saline with a pH of 7.4 in an amount of 300 ml/10 g of liver at a rate of 5 ml/min.

Avantajele metodei revendicate constau în aceea că este simplă, economă şi eficientă. Metoda propusă ameliorează şi majorează viteza procesului de recelularizare a ficatului prin sporirea adeziunii matricei extracelulare hepatice. The advantages of the claimed method are that it is simple, economical and efficient. The proposed method improves and increases the speed of the liver recellularization process by increasing the adhesion of the hepatic extracellular matrix.

Rezultatul constă în aceea că respectiva metodă - eficientă, sigură, economă şi uşor de implementat, permite o adeziune mai rapidă, evitându-se pierderi excesive de hepatocite injectate în matricea hepatică decelularizată în timpul procesului de recelularizare. The result is that the method - efficient, safe, economical and easy to implement - allows for faster adhesion, avoiding excessive losses of hepatocytes injected into the decellularized hepatic matrix during the recellularization process.

În scopul creşterii adeziunii hepatocitelor de matricea extracelulară a ficatului decelularizat şi evitării pierderii lor excesive, s-a utilizat soluţie de colagen tip I obţinut din tendon Achile de bovină cu o concentraţie de 0,01% dizolvat în 0,1 M de acid acetic, care se injectează foarte încet în matricea hepatică decelularizată prin vena portă. In order to increase the adhesion of hepatocytes to the extracellular matrix of the decellularized liver and avoid their excessive loss, a solution of type I collagen obtained from bovine Achilles tendon with a concentration of 0.01% dissolved in 0.1 M acetic acid was used, which was injected very slowly into the decellularized hepatic matrix through the portal vein.

Modalitatea de creştere a adeziunii hepatocitelor de matricea extracelulară a ficatului decelularizat constă în injectarea soluţiei colagen tip I obţinut din tendon Achile de bovină, cu concentraţia de 0,01% dizolvat în 0,1 M de acid acetic, după o spălare prealabilă cu soluţie PBS şi un compus antibiotic-antimicotic (streptomicină 100 µg/ml, penicilină 100 U/ml, amfotericină B 0,25 µg/ml). Cantitatea de soluţie de colagen injectată este direct proporţională cu masa ficatului. La 10 g de masă umedă de ficat se utilizează 200 ml ±40 ml de soluţie de colagen, acesta se injectează foarte lent pentru a permite ataşarea fibrelor de colagen de matricea hepatică decelularizată evitând formarea conglomeratelor şi obturarea vaselor intrahepatice. Pentru a verifica dacă vasele matricei decelularizate nu sunt obturate de fibrele de colagen şi a aduce pH-ul la unul normal de 7,2…7,4 pentru a asigura viabilitatea celulelor, matricea hepatică se perfuzează cu PBS în volum de 300 ml/10 g ficat decelularizat, cu o viteză de 5 ml/min şi un pH egal cu 7,4. Totodată se urmăreşte umplerea şi extravazarea uniformă a lichidului din matricea hepatică decelularizată. The method of increasing the adhesion of hepatocytes to the extracellular matrix of the decellularized liver consists of injecting a solution of type I collagen obtained from bovine Achilles tendon, with a concentration of 0.01% dissolved in 0.1 M acetic acid, after a prior wash with PBS solution and an antibiotic-antimycotic compound (streptomycin 100 µg/ml, penicillin 100 U/ml, amphotericin B 0.25 µg/ml). The amount of collagen solution injected is directly proportional to the mass of the liver. For 10 g of wet mass of liver, 200 ml ± 40 ml of collagen solution is used, it is injected very slowly to allow the attachment of collagen fibers to the decellularized hepatic matrix, avoiding the formation of conglomerates and the obstruction of intrahepatic vessels. To verify that the vessels of the decellularized matrix are not clogged by collagen fibers and to bring the pH to a normal one of 7.2…7.4 to ensure cell viability, the liver matrix is perfused with PBS in a volume of 300 ml/10 g of decellularized liver, at a rate of 5 ml/min and a pH equal to 7.4. At the same time, the uniform filling and extravasation of the fluid from the decellularized liver matrix is monitored.

Exemplu Example

Prin vena portă matricea extraxelulară a 10 g de ficat decelularizat se spală cu 20 ml soluţie PBS şi antibiotic antimicotic. Apoi cu viteza de 1 ml/min se injectează 200 ml de soluţie de colagen tip I de 0,01%, obţinut din tendon Achile de bovină dizolvat în acid acetic de 0,1 M. Ulterior, matricea decelularizată se spală cu o viteză de 5 ml/min cu 300 ml soluţie PBS cu pH-ul de 7,4. Through the portal vein, the extracellular matrix of 10 g of decellularized liver is washed with 20 ml of PBS solution and antifungal antibiotic. Then, at a rate of 1 ml/min, 200 ml of 0.01% type I collagen solution, obtained from bovine Achilles tendon dissolved in 0.1 M acetic acid, is injected. Subsequently, the decellularized matrix is washed at a rate of 5 ml/min with 300 ml of PBS solution with pH 7.4.

Metoda corespunde cerinţelor şi normelor sanitare, necesită utilizarea soluţiei de colagen de 0,01% şi a matricei hepatice decelularizate şi se efectuează în condiţii sterile. The method complies with sanitary requirements and norms, requires the use of 0.01% collagen solution and decellularized liver matrix and is performed under sterile conditions.

Această metodă se utilizează pentru mărirea adeziunii hepatocitelor în timpul recelularizării ficatului decelularizat în cadrul Laboratorului de Inginerie Tisulară şi Culturi Celulare. This method is used to increase hepatocyte adhesion during recellularization of decellularized liver within the Tissue Engineering and Cell Culture Laboratory.

1. Uygun B., Price G., Saeidi N., Izamis M., Berendsen T., Yarmush M., Uygun K. Decellularization and Recellularization of Whole Livers. JoVE, 48, 2011. http://www.jove.com/details.php?id=2394 1. Uygun B., Price G., Saeidi N., Izamis M., Berendsen T., Yarmush M., Uygun K. Decellularization and Recellularization of Whole Livers. JoVE, 48, 2011. http://www.jove.com/details.php?id=2394

Claims (1)

Metodă de prelucrare a matricei decelularizate a ficatului pentru sporirea adeziunii celulare, care constă în aceea că se spală matricea decelularizată a ficatului prin injectarea în vena portă a unui amestec de soluţie tampon fosfat salin, streptomicină 100 µg/ml, penicilină 100 U/ml şi amfotericină B 0,25 µg/ml, în cantitate de 20 ml/10 g de ficat decelularizat, apoi se injectează soluţie de colagen tip I, obţinut din tendonul Achile de bovină în concentraţie de 0,01%, dizolvat în 0,1M de acid acetic, în cantitate de 160...240 ml/10 g de ficat decelularizat, după care matricea hepatică se perfuzează cu soluţie tampon fosfat salin cu un pH 7,4 în cantitate de 300 ml/10 g de ficat cu o viteză de 5 ml/min.Method of processing decellularized liver matrix to enhance cell adhesion, which consists in washing the decellularized liver matrix by injecting into the portal vein a mixture of phosphate buffer saline, streptomycin 100 µg/ml, penicillin 100 U/ml and amphotericin B 0.25 µg/ml, in an amount of 20 ml/10 g of decellularized liver, then injecting a solution of type I collagen, obtained from bovine Achilles tendon in a concentration of 0.01%, dissolved in 0.1M acetic acid, in an amount of 160...240 ml/10 g of decellularized liver, after which the liver matrix is perfused with phosphate buffer saline with a pH of 7.4 in an amount of 300 ml/10 g of liver at a rate of 5 ml/min.
MDS20160156A 2016-12-23 2016-12-23 Method for treating the decellularized liver matrix to enhance cell adhesion MD1171Z (en)

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SU1053803A1 (en) * 1981-11-05 1983-11-15 2-Й Московский Ордена Ленина Государственный Медицинский Институт Им.Н.И.Пирогова Method of preserving isolated liver cells
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SU1463757A1 (en) * 1987-04-02 1989-03-07 Институт проблем криобиологии и криомедицины АН УССР Method of producing isolated cells of liver
SU1513390A1 (en) * 1987-10-28 1989-10-07 Тюменский государственный медицинский институт Method of investigating liver cells
SU1564550A1 (en) * 1986-02-06 1990-05-15 Харьковский научно-исследовательский институт терапии Method of isolating cells from liver and myocardium
SU1578192A1 (en) * 1988-04-26 1990-07-15 Институт проблем онкологии им.Р.Е.Кавецкого Method of growing cells of liver
SU1673969A1 (en) * 1986-04-08 1991-08-30 Научно-исследовательский институт физико-химической медицины Method for production of suspension for reconstruction of liver membranes and cells
MD1015Y (en) * 2015-05-12 2016-03-31 Ip Universitatea De Stat De Medicină Şi Farmacie "Nicolae Testemiţanu" Din Republica Moldova Method for decellularization of liver in experimental animals

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SU952958A1 (en) * 1980-08-01 1982-08-23 Институт проблем онкологии им.Р.Е.Кавецкого Method for preparing isolated liver cells
SU1053803A1 (en) * 1981-11-05 1983-11-15 2-Й Московский Ордена Ленина Государственный Медицинский Институт Им.Н.И.Пирогова Method of preserving isolated liver cells
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SU1564550A1 (en) * 1986-02-06 1990-05-15 Харьковский научно-исследовательский институт терапии Method of isolating cells from liver and myocardium
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SU1463757A1 (en) * 1987-04-02 1989-03-07 Институт проблем криобиологии и криомедицины АН УССР Method of producing isolated cells of liver
SU1513390A1 (en) * 1987-10-28 1989-10-07 Тюменский государственный медицинский институт Method of investigating liver cells
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MD1015Y (en) * 2015-05-12 2016-03-31 Ip Universitatea De Stat De Medicină Şi Farmacie "Nicolae Testemiţanu" Din Republica Moldova Method for decellularization of liver in experimental animals

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Title
Uygun B., Price G., Saeidi N., Izamis M., Berendsen T., Yarmush M., Uygun K. Decellularization and Recellularization of Whole Livers. JoVE, 48, 2011. http://www.jove.com/details.php?id=2394 *

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