| With the development of the bioartifical liver (BAL) and hepatocytes transplantation recently, cell material, a hard core of BAL, was studied deeply. Porcine hepatocytes are an important cell material in BAL at the present time. The method of effective preserving hepatocytes for long time is desired for the farther clinical research in BAL. Hypothermic preservation, an important means to keep the biological activities of hepatocytes, can be mainly divided into two kinds: one is short storage at about 4℃; another is long cryopreservation below -80℃. Now many researchers over the world adopted the measure of grading freezing and rapidly thawing, but they differed from the concentration of cryoprotective agent (CPA), cryopreservative liquids, concentration of cells and storing time. Aiming at those different opinions, this article is supposed to intensively discuss the multi-factors which affect the hypothermic preservation of porcine hepatocytes, such as preservative temperature, cellular concentration, storage time, the hypothermic preservative medium, the best concentration of dimethyl sulfoxide (DMSO) and the culture of thawed hepatocytes, and it will serve as a help to better keep the activities of liver cells.To explore an optimal method for 4℃ hypothermic preservation of hepatocytes, primary porcine hepatocytes were harvested by modified two-step perfusion collagenase method, then hypothermic preserved for 24,48 or 72 hours before plated in medium. They were stored either in the RPMI-1640 medium with or without polyethylene glycol, or in University of Wisconsin solution, the most effective solution for cold organ preservation. After cold storage, their viability,attachment rate and drug metabolic function were measured, and intracellular ultrastructure changes were observed through phase contract microscope (PCM) and transmission electron microscope (TEM). After being stored at 4℃ in RPMI-1640 medium, cell viability and adherence were significantly reduced. But the hepatocytes maintained in PEG or in University of Wisconsin solution keep their functions well, and their total protein secretion,extracellular LDH and drug metabolic reaction were close to those found in fresh hepatocytes. So primary porcine hepatocytes can be preservedfor 48 hours. Addition of polyethylene glycol to the RPMI-1640 medium resulted in slightly higher viability and function of hepatocytes after cold storage.To determine the best concentration of DMSO for -80℃ cryopreserved suckling porcine hepatocytes, primary porcine hepatocytes were harvested by modified two-step perfusion collagenase method, then stored in the 5%,10%,15% or 20% DMSO cryopreservative liquids. They were cryopreserved for 30 or 60 days before being plated under culture medium conditions. After cold storage, their viability,attachment rate and drug metabolic function were measured, and cellular configuration or intracellular structure changes were observed through PCM and TEM. After being stored in 5%DMSO cryopreservation medium, cell viability and attachment rate were significantly reduced. But the hepatocytes maintained in 15% DMSO cryopreservative solution keep their functions well, and their albumin secretion,extracellular LDH and drug metabolic reaction were close to those found in fresh hepatocytes. The better concentration of DMSO for -80℃ preservation of porcine hepatocytes is 15%, primary pig hepatocytes can be cryopreserved for 60 days in that way. When the suckling primary porcine hepatocytes were cryopreservaed at -196℃, their viabilities, attachment rates, metabolic functions and expression of porcine albumin mRNA were different in the 5%,10%,15% or 20% DMSO cryopreservative medium. After being stored in 5% DMSO preservative medium, cells viabilities were significantly reduced. But the hepatocytes maintained in 15% DMSO cryopreservative solution keep their functions very well, and their porcine albumin mRNA,extracellular LDH and drug metabolic reaction were alike to those found in fresh hepatocytes. So the better concentra...
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