RESUMEN
Co-transplantation of hepatocytes and hepatic stellate cells has been shown to increase the engraftment of transplanted hepatocytes in the liver. Here, we describe a method for the simultaneous isolation of human primary hepatocytes and hepatic stellate cells from the same donor for co-transplantation or for use in in vitro cell culture models.
Asunto(s)
Separación Celular/métodos , Células Estrelladas Hepáticas/fisiología , Hepatocitos/fisiología , Hígado/cirugía , Perfusión/métodos , Separación Celular/instrumentación , Supervivencia Celular , Trasplante de Células/métodos , Células Cultivadas , Técnicas de Cocultivo/métodos , Criopreservación , Células Estrelladas Hepáticas/trasplante , Hepatocitos/trasplante , Humanos , Hígado/citología , Perfusión/instrumentación , Cultivo Primario de Células/métodos , Donantes de TejidosRESUMEN
The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. As such, hepatocytes are an important model for a large variety of research questions. In particular, the use of human hepatocytes is especially important in the fields of pharmacokinetics, toxicology, liver regeneration and translational research. Thus, this method presents a modified version of a two-step collagenase perfusion procedure to isolate hepatocytes as described by Seglen (1). Previously, hepatocytes have been isolated by mechanical methods. However, enzymatic methods have been shown to be superior as hepatocytes retain their structural integrity and function after isolation. This method presented here adapts the method designed previously for rat livers to human liver pieces and results in a large yield of hepatocytes with a viability of 77±10%. The main difference in this procedure is the process of cannulization of the blood vessels. Further, the method described here can also be applied to livers from other species with comparable liver or blood vessel sizes.