Long-term maintenance of human hepatocytes in oxygen-permeable membrane bioreactor

0. Source and Links

Cohen S, Dvir-Ginzberg M, Gamlieli-Bonshtein I, et al. “Long-term maintenance of human hepatocytes in oxygen-permeable membrane bioreactor” Biomaterials. 2006 September; Volume 27 Issue 27: 4794-4803.

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TWB-4K48M84-1&_user=10&_coverDate=09%2F30%2F2006&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1273475599&_rerunOrigin=google&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=08a6aae39f6c2641d9e139b1c58cca23

1. Introduction

The main goal of studying hepatocytes (liver cells) in bioreactors is to adapt that method into a liver-support device. Previous studies used flat membrane bioreactors to mimic real liver function and structure. This study used flat, O2 permeable polymer membranes to grow their cells. The O2 permeability enables oxygenation of cells attached to membrane and of the medium which stimulates sinusoidal organization (similar to in vivo morphology). The idea is that this will translate into improved function and viability. The end goal of the study is to evaluate in-vitro performance of the new O2 permeable membrane bioreactor in maintaining and differentiating hepatocytes (human).

2. Summary of Methods and Results

The authors began by preparing human hepatocyte culture in hepatocyte culture medium and feeding it growth factors and various nutrients. They established a positive control: a culture on a plastic petri dish. They then seeded the hepatocytes on a gas-permeable membrane under serum-free conditions for the entire culture time.

Figure 1: the experimental bioreactor set up

The oxygen permeable bioreactor was connected to perfusion system and fresh media was sent through the set-up continuously at 0.2 mL/min. According to a rotating schedule, therapeutic molecules were sent through the bioreactor to gather information on their effects. The molecules used were Interleukin 6 (IL-6) which activates certain genes for liver repair and Diclofenac (DIC), an analgesic which can affect metabloic functions (it is digested by proper liver function).

Figure 2: Top: cell morphology throughout the experiment run time Bottom Left: therapeutic molecule schedule and urea synthesis Bottom Right: Albumin synthesis correlated to therapeutic molecules present.

The liver-specific function was measured by gel electrophoresis and quantified – albumin, urea, secretion of total protein. Total protein analysis was carried out by performing SDS-Page on the proteins collected from the outflowing media, which were then stained with Coomassie Blue. The resulting gel was analyzed with Image Quant TL software to estimate protein molecular weights. Albumin content was measured via ELISA method; several biochemical assays were run on samples from inside the bioreactor. Liver function was also related to the degree of diclofenac digestion (measured by HPLC, a chromatography process): functioning liver cells will digest diclofenac into its metabolites 4’ OH diclofenac and 5' OH diclofenac.

Figure 3: total protein analysis using SDS-Page

The results from the study were many-fold: the researchers not only compared their bioreactor performance with a petri dish, but they also compared the effects of the therapeutic molecules they had treated the cells with. With regard to morphology, their cells reached confluency of plates by day 4 out of a total culture time of 32 days. By day 11 the cells were polyhedral with pericellular zones similar to in vivo. With regard to protein production, total protein secretion was compared between collagen-plated and the O2 permeable method and found that liver function and albumin secretion was improved in the O2 plated method. With regard to the effects of the therapeutic molecules, the Urea synthesis decreased with DIC and recovered when it was removed; Albumin expression worsened when exposed to DIC + IL-6 but levels did recover when the drugs were removed.

Figure 4: HPLC results for diclofenac digestion. Samples from inflow and outflow were compared to determine degree of diclofenac breakdown.

Figure 5: Heptocyte functionality with respect to therapeutic molecule influence. These charts compare the protein secretion of the cells under the influence of various combinations of diclofenac and IL-6. The time schedule references the particular therapeutic molecule combinations present that day (see Figure 2 bottom left).

3. Discussion:

In this experimental set up the cells grew on the border between gas and liquid phase (the medium didn’t act as diffusion barrier so the cells were more able to organize themselves in an in vivo fashion). O2 transport is theoretically improved in this set up because O2 is usually poorly solubilized in medium. The fluid dynamics of device was highly characterized so system was controlled. Hepatocytes grown in new device were morphologically similar to those in vivo and functionally similar, as they expressed proteins for an extended period of time It is suspected IL-6 mediates the effects of diclofenac (DIC). DIC is thought to deplete the cell’s supply of ATP and thus interfere with normal protein production levels. This would explain why the damage was not permanent and after the drugs were removed, protein levels recovered. Prolonged exposure to DIC did seem to reduce the metabolic efficiency of the liver cells (better at 14 days than 29 days). The metabolites of DIC are thought to damage cytochrome P450 used in the microsomal hepatic system.

4. Conclusions:

The experimental set up was an improvement over petri dish culture. The bioreactor hepatocytes can be maintained with good functionality preserved over the long-term. The O2 permeable bioreactor has the potential to serve as a good model for drug behavior. The study also found that IL-6 mediates DIC (reduces its harmful effects).

5. Criticisms:

My first criticism is the order in which the researchers ran their therapeutic molecules. The study ran IL-6, IL-6+DIC, DIC in that particular order. They did not consider if there might be any residual effects from running the therapeutic molecules in that order. For example, they don’t know how DIC -> Il-6 would have affected their results.

My second criticism is the study’s control. The study didn’t have a control reactor which ran IL-6 only or DIC only, etc. to compare their results with. They ran all experiments in the same reactor (granted it allowed them to see recovery from previous processes). They shouldn’t have been able to claim that one drug had a particular effect over another since they did not isolate that interaction in its own bioreactor (insufficient controls). Comparing it to the collagen plate wouldn’t be as good as comparing it to an identical reactor under different conditions.

My third criticism is that there was no direct comparison between the O2 permeable method and the rival method mentioned at the beginning of the paper. The motivation for other scientists to care about the author’s work is that the work shows that their novel bioreactor is an appreciable improvement over other methods. Since the authors don’t make this comparison, the paper loses value to its readers.

My fourth criticism relates to the lack of explanation regarding the therapeutic molecules used in the study. The authors mention IL-6 reduces effect of DIC, but IL-6 alone also decreases protein expression of hepatocytes (as seen Figure 2 bottom right): this is not explained. This could mean that IL-6 has some properties that are damaging to protein production and this topic could be further explored.