http://www3.interscience.wiley.com/cgi-bin/fulltext/113391970/HTMLSTART

Growing tissue-like constructs with Hep3B/HepG2 liver cells on PHBV microspheres of different sizes

In this paper, the researchers were interested in the molecular, functional, and proliferative behaviors of liver cells, from the Hep3B and HepG2 hepatoma cell lines, seeded on 8% poly[3-hydroxybutyrate-co-3-hydroxyvalerate] (PHBV) microspheres as compared to the liver cells cultured 2-dimentionally with laminin and poly-L-lysine (positive control). This new cell seeding technique could become a possible cell growing methods in a tissue engineered liver/liver device. They choose PHBV because it is biocompatible, biodegradable, and non-cytotoxic in vivo. The microspheres were made by emulsifying and homogenizing 8% PHBV oil-in-water-in-oil solution, followed by evaporation of the solvent. The researchers considered microsphere size as a parameter and made 3 different sizes of microsphere by changing the homogenizer’s speed. After the microspheres were made, hepatoma cells were seeded onto them and cultured. As positive control, hepatoma cells are cultured two dimensionally with laminin and poly-L-lysine. Polyurethane film with zinc diethydithiocarbamate was used as negative control. At the appropriate time points, the cultured is analyzed. The analysis includes MTT assay for viability, bisbenzimide fluorescent assay for cell proliferation, ELISA assay to quantify albumin (since healthy liver cells make albumin), and EROD assay to test cytochrome P-405 activity (which measure liver cell’s detoxification activity).

The researchers were able to show that cells cultured on the microsphere spread over the microsphere and connect the adjacent microspheres together. There were multiple cells layer formed in the space between the microspheres. More interesting, it was shown that cells seeded on the microsphere had higher proliferation rate and viability, secreted more albumin, and had higher detoxification (cytochrome P-405) activity than the positive control. This means that the cells are able to live longer, divide better, and maintain liver specific activities/functions better. These behaviors of cells cultured on microsphere made this new culturing method a good choice to use in a tissue-engineered liver or liver device.

The reasons I chose the paper are as followed. (1) Liver is a very important organ and many people are waiting for the limited liver transplantation. This paper is attempting to solve this problem by researching tissue engineered liver. (2) Many of the finding in this paper will be useful for a tissue engineered (TE) liver. For example, liver cells grow on microsphere proliferate better and have higher viability. This is extremely important since primary cells used in TE liver are limited, usually not very proliferative, and their viability decrease with time. This new technique could improve, if not solve, these problems. Livers cells seeded on the microsphere secreted higher albumin and have higher P-405 activity, suggesting that they maintain their function and not likely to de-differentiate. This aspect is especially useful in TE liver since we want the cells to maintain their function instead of ceasing to act like liver cells. (3) I think it’s really nice that this paper use many of the methods we learned in class such as trypan blue plus other live/dead assay, and ELISA. I picked this paper because the materials are relevant to the class and tissue engineering.