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Hydroxyethyl piperazineethanesulfonic acid(cas 7365-45-9)

Establishment of a new method of four-step perfusion, in order to obtain high-quality liver cells to provide a new way. Materials and methods: Neutral protease (Dispase), DNase and bovine serum albumin (BSA) were purchased from Roche; type IVcollagenase, Glutamine was purchased from Sigma, USA; Walliams (E) was purchased from Gibco, Glucose, HEPES(cas 7365-45-9), NaHCO3, NaCl, KCl, Potassium Dihydrogen Phosphate, (MgCl 2 · 6H 2 O), magnesium sulfate (MgSO4 · 7H2O), trypsin and B 2 (NH 2+) (FBS) was purchased from Hyclone Inc. of the United States; heparin was purchased from Shanghai First Biochemical Pharmaceutical Co., Ltd.; culture flask and culture plate were made by Danish Nunc Co., Ltd. (PTA) from Shanghai Bioengineering Co., Ltd. Products; 0.22μm filter for the German company Millipore products. Chinese miniature pig is purchased from Beijing Agricultural University Experimental Pig Farm. 

Experimental equipment including Olympus inverted phase contrast microscope, Japan HITACHI H-600A transmission electron microscope, the United States Forma carbon dioxide incubator, the United States Thermo liquid nitrogen storage tank, Germany Sartorius acidity and electronic balance, Germany Millipore ultra water meter, Germany Scotsman ice machine, Japan HITACHI vertical large-capacity low-temperature centrifuge, Japan Tomy steam disinfection pot, Germany Eppendorf peristaltic pump, Shanghai Jinghong Experimental Equipment Co., Ltd. Electric thermostat water bath, Hangzhou Instrument Motor Factory magnetic heating mixer, 80 mesh and 150 mesh stainless steel mesh Screening from the East China Pharmaceutical Co., Ltd., in vitro perfusion device designed. The liver cells of Chinese miniature pig were treated with sterile saline containing EDTA and collagenase. The same method to take the liver, the liver on the stainless steel basin, gently squeeze the liver, with 4 ℃ pre-cooling perfusion buffer (PB) 500ml as much as possible to the liver outside the blood and blood vessels left the blood. The first step of the peristaltic pump to the liver continuous infusion of normal temperature of EDTA with perfusion buffer (PBE) 300ml, preheated to 39 ℃ PBE solution 700ml; the second step preheated to 39 ℃ containing HEPES(cas 7365-45-9) PB solution 500ml; Discard the above perfusate, the liver into the new sterile stainless steel basin, the third step perfusion preheated 39 ℃ Dispase-containing perfusion buffer (PBD) 500ml, the fourth step to abandon about 200ml of perfusate, 500 ml of perfusion of 500 ml of PBC fluid and about 300 ml of PBD solution was continued with the preheated to 39  ℃ of collagenase-containing perfusion buffer (PBC) until the liver capsule was removed; the gallbladder, the acute tearing of the liver capsule, the fibrous connective tissue was removed and the hepatocytes were filtered under 80  ℃ and 150 mesh stainless steel screens under ice bath and the hepatocytes were collected. The hepatocytes collected by two perfusion methods were washed and trypan blue staining to calculate the viability and yield of hepatocytes. The liver cells were cultured with William's Medium E to calculate the adherence rate of hepatocytes for 24 h.



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