Isolation, Culture And Identification Of Adult Hepatic Stem Cells In Vitro

Stem cell are cells that have the dividing potential of totipotencyand pluripotentiality. There are many stem cells type because of beingderived from different resource such as embryonic stem cells, adultand so on. Different types of stem cell have a general character.Traditional conception considers the stem cell exist exclusively inthose tissue whose proliferation persists actively, such as bone marrow,epidermis, etc. With the great advance of tissues remodeling, stem cellare found in many other tissues, for example, pancreas, brain and liver.A plenty of evidence have proved that there are facultative stem cellscapable of differentiation into bile ductular cells or hepatocytes inlivers of adult animal and human being. In normal condition, hepaticstem cell lodges to static state, only when the liver has the seriousdamage or the hepatocyte is restrained, like alcoholic cirrhosis,primary biliary cirrhosis, primary sclerosing cholangitis and the tissuenear the hepatic cancer. Untipical proliferation of bile canaliculi canbe seen in the portal area and the nearby connective tissue, named ovalcell. Definition of its position and origin is advantage to the isolatingmore quantities and activated oval cell for people, and provid massivehepatic stem cell origin for the foundation and the clinical practiceresearch. This experiment based on this principle, we gain the hepaticstem cell which can differentiate into bile ductular cells or hepatocytesthrough isolation, culture, and investigation of hepatic stem cell invitro.Method:1. HE staining and immunofluorescin his tochemistry staining ofthe tissue near the hepatic cancer.The tissue comes from operation, after common dehydration,paraffin embedding and HE staining, it is proved that hepatic stem cellexist in the portal area of the atypical tissue near the hepatic cancer.We took this as the origin of hepatic stem cell, then further to culture,isolation and investigation in vitro.2. The primary culture of the hepatic cell First, break the tissue tofragments, then digest in the Ⅳcollagen enzyme. After rinsing andfiltrating, put to the DMEM/F12 culture medium, also add HGF, EGF,FGF, LIA in it. Then culture in 5.0% CO2,37℃ for 3 weeks for latergenerate.3. Immuno-magnetic beads sortingC-kit is considered as the specific surface symbol of Bonemarrou hematopoeitic stem cell. Though C-kit, we gain C-kit+ cell andC-kit-cell. Then vaccinate in the DMEM/F12 culture medium, put inthe 5.0% CO2, 37℃.4. Investigation of C-kit+ cell and C-kit-by immunofluorescinhistochemistry stainingThough the specific anti-AFP and anti-CK19, and continue to addfluorescent antibody, Then set the cell under the laser scanningconfocal microscopy (LSCM), observe.Result:After HE staining of the tissue near the hepatic cancer, atypicalproliferation of bile canaliculi can be seen in the portal area. And alsosmall oval cells can be seen in it. These cell's volume are small, whosediameter approximately are 10-12μm, and both AFP and CK19 arepositive. After the immunofluorescin his tochemistry staining, bothAFP and CK19 are positive in the tissue. We took the tissue as theorigin of the hepatic stem cell, then primary culture in vitro. After 3weeks, the cells grew to cell clones. Mature liver cells can beobviously seen in the early stage, which is easily broken and died.After generation, the cells grew faster than before. Some of the ovalcell clustered together. A plenty of fibroblasts also can be seen, whichare easy to paste the wall .So we need to eliminate them throughgeneration. After 2-3 generation, we gain C-kit+ cell throughimmuno-magnetic beads sorting. Untill now, the specific sign ofhepatic cell hasn't been found, so we choose AFP and ck19 toinvestigate these C-kit+ cell. After immunofluorescin histochemistrystaining, both AFP and CK19 can be seen in the cytoplasm (yellow).Both AFP and CK19 of C-kit-cells culture in vitro are negative.Conclusion:1. Hepatic stem cell exist in the bile canaliculi of the tissue nearthe hepatic cancer.2. After isolation, culture and investigation of the cell in vitro, wegain the hepatic stem cell which have the capable of differentiationinto bile ductular cells or hepatocytes.3. Hepatic stem cell has a close relationship with hepatic cancer.