Hepatocyte Isolation, Culture, and Its Clinical Applications

Lee, Doo-Hoon; Lee, Kwang‐Woong

doi:10.7599/hmr.2014.34.4.165

Cited by 9 publications

(7 citation statements)

References 48 publications

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“…Jak dotąd stosowane były: hodowle przestrzenne, mające na celu odtworzenie warunków in vivo, opłaszczanie podłoży hodowlanych polimerami wspomagającymi adhezję (laminina, fibronektyna, kolagen), wyspecjalizowane media hodowlane czy też kokultury, czyli współhodowle z innymi komórkami (np. fibroblastami) [21]. Najnowsze doniesienia literaturowe wskazują, iż najbardziej aktywne komórki można pozyskać izolując hepatocyty z wątrób uzyskanych od młodych mężczyzn.…”

Section: Przeszczep Hepatocytówunclassified

Kliniczne metody wspomagania niewydolnej wątroby

Ciezkowska

Pluta

2019

Postepy Biochem

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Choroby wątroby prowadzące do jej niewydolności są jedną z najczęstszych przyczyn zgonów na całym świecie. Jak dotąd jedynym skutecznym sposobem leczeniem ostrej niewydolności wątroby jest jej przeszczep. Niestety niedobór odpowiednich dawców stanowi główne ograniczenie tej terapii. Z tego powodu naukowcy stale szukają alternatywnych rozwiązań dla transplantacji tego organu. Najbardziej obiecujące są systemy wspomagania wątroby i przeszczep komórkowy. Bez wątpienia hepatocyty są najlepszym źródłem komórek do zastosowania w terapiach wątrobowozastępczych. Jednakże izolowane ludzkie hepatocyty bardzo szybko odróżnicowują się i tracą swoje funkcje. Dlatego stale poszukuje się nowych źródeł komórek, które mogłyby zastąpić hepatocyty. Obecnie panuje pogląd, że aby pomóc pacjentom w walce z chorobami wątroby, potrzeba interdyscyplinarnego podejścia do rozwiązania tego problemu.

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Section: Przeszczep Hepatocytówunclassified

Kliniczne metody wspomagania niewydolnej wątroby

Ciezkowska

Pluta

2019

Postepy Biochem

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“…Several techniques, including slow speed centrifugation steps followed after partial collagenase digestion, and Ficoll fractionation, sorted based on the markers expression, have been used to culture neonatal murine LDSCs [ 11 , 14 , 19 – 21 ]. The enzyme digestion step for dissociating cells from liver tissue is too critical which determines the success of isolating viable neonatal liver stem cells [ 22 ]. Although the repetitive digestion with various enzyme combinations gives good yield, long-term enzyme digestion leads to toxicity which limits the success of higher yield in isolating viable cell [ 23 ].…”

Section: Introductionmentioning

confidence: 99%

An Efficient Protocol for Deriving Liver Stem Cells from Neonatal Mice: Validating Its Differentiation Potential

Dhanasekaran

Sithambaram

Govarthanan

et al. 2015

Analytical Cellular Pathology

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The success of liver regeneration depends on the availability of suitable cell types and their potential to differentiate into functional hepatocytes. To identify the stem cells which have the ability to differentiate into hepatocytes, we used neonatal liver as source. However, the current protocol for isolating stem cells from liver involves enzymes like collagenase, hyaluronidase exposed for longer duration which limits the success. This results in the keen interest to develop an easy single step enzyme digestion protocol for isolating stem cells from liver for tissue engineering approaches. Thus, the unlimited availability of cell type favors setting up the functional recovery of the damaged liver, ensuring ahead success towards treating liver diseases. We attempted to isolate liver stem derived cells (LDSCs) from mouse neonatal liver using single step minimal exposure to enzyme followed by in vitro culturing. The cells isolated were characterized for stem cell markers and subjected to lineage differentiation. Further, LDSCs were induced to hepatocyte differentiation and validated with hepatocyte markers. Finally, we developed a reproducible, efficient protocol for isolation of LDSCs with functional hepatocytes differentiation potential, which further can be used as in vitro model system for assessing drug toxicity assays in various preclinical trials.

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“…Що вищий рівень організації досліджуваного об'єкта, то важчим є проведення експериментів, але більш достовірним трактування результатів. Для дослідження впливу медичних препаратів і патогенезу хвороб печінки часто використовують ізольовані гепатоцити, які зберігають функціональні властивості, характерні для цілісного органа [8,17]. Такі дослідження здійснюють на гепатоцитах, які виділили неперфузійним методом, методом перфузії печінки in vitro та in situ або на культурі гепатоцитів.…”

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“…Оскільки життєздатність і функціональний стан клітин може залежати від способу їхнього виділення (у т.ч. від складу розчинів), дослідники часто модифікують ці методи виділення, емпірично підбираючи, на їхню думку, оптимальні умови [1,5,8]. Причому в багатьох випадках залишається незрозумілим, за рахунок якої модифікації методів виділення збільшується життєздатність клітин.…”

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Dependence of the adaptive capacity of liver mitochondria on preparation method

Mazur¹,

Merlavsky²,

Manko³

et al. 2020

VLUBS

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When conducting studies on isolated hepatocytes, it is important to obtain cells that retain the functional properties that are characteristic of the whole organ. Increased blood viscosity during liver perfusion, decreased perfusion pressure in blood vessels, and hence hypoxia, are among the factors that may affect the functional state of isolated hepatocytes. The functional state of cells can be estimated by the adaptive capacity of mitochondria, by inducing maximal respiration rate by uncoupling respiration and oxidative phosphorylation due to the addition of FCCP. The research aimed to investigate the adaptive capacity of mitochondria of isolated hepatocytes using in situ and in vitro liver perfusion. Hepatocytes were isolated by the two-staged Seglen method by in situ and in vitro liver perfusion. Isolated hepatocytes, after 15-minute incubation in the medium without addition or with respective oxidative substrate – glutamine, pyruvate, succinate, monomethyl succinate, α-ketoglutarate, dimethyl-α-ketoglutarate (at a concentration of 2 mM) or glucose (10 mM) – were added into the respiratory chamber and FCCP was added in increasing concentrations. It was established that at in situ liver perfusion maximal rate of uncoupled respiration and the optimal concentration of FCCP was higher than at in vitro liver perfusion. Addition of exogenous substrates to a medium increased the respiration rate of hepatocytes. Upon in situ liver perfusion maximal uncoupled respiration rate increased at all causes except glucose, and at in vitro liver perfusion – only when dimethyl-α-ketoglutarate, succinate and monomethyl succinate were used. The optimal concentration of FCCP at in vitro liver perfusion increased due to the addition of glutamine, pyruvate and monomethyl succinate to the medium, and at in situ liver perfusion – only upon glucose oxidation. In both perfusion methods, the highest maximal rate of uncoupled respiration is with the use of monomethyl succinate and the optimal FCCP concentration – upon pyruvate oxidation. Therefore, in situ liver perfusion is better method to obtain stable and metabolically active hepatocytes in support respiratory processes at a high level then in vitro perfusion.

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Hepatocyte Isolation, Culture, and Its Clinical Applications

Cited by 9 publications

References 48 publications

Kliniczne metody wspomagania niewydolnej wątroby

Kliniczne metody wspomagania niewydolnej wątroby

An Efficient Protocol for Deriving Liver Stem Cells from Neonatal Mice: Validating Its Differentiation Potential

Dependence of the adaptive capacity of liver mitochondria on preparation method

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