Hepatocytes metabolize phenol red to a significant extent. Therefore, there is some concern that phenol red might interfere with certain metabolic processes.

If you search for culture media from commercial sources that are free of phenol red you'll find some that are recommended for hepatocyte cultures.

Hepatocytes metabolize phenol red to a significant extent. Therefore, there is some concern that phenol red might interfere with certain metabolic processes.

If you search for culture media from commercial sources that are free of phenol red you'll find some that are recommended for hepatocyte cultures.

BD Bioscienc and Lifelinecelltechnologies . Some claim phenol red to have estrogenic activty and pro/anti oxidant activity. It may also interfere with colorimetric assays. Good Luck!

Thank you Sieber and Michael sir, I'll go through......

My experience is with human hepatocytes - you don't specify whether you are asking about human or animal. If you are purchasing hepatocytes I would use the media supplied/recommended by the company. For primary hepatocytes that we isolated in our lab we generally used Williams E based media. We use arginine-free media. For long term culture (more than 24-48 hours) I have had good results with the culture media described by Pichard et al in Methods Mol Biol. 2006. For plating we add serum to the media but then change to serum free media after a few hours once cells have attached. In my experience (human) hepatocytes do not do well if left in media containing serum for more than about 4 hours. If you look at my profile you will find 2 publications on hepatocyte isolation which describe/reference these media. Dave

To echo Fritz, phenol red is metabolized to an extent, and can particularly inhibit certain Phase II metabolism in hepatocytes. Depending what assay you may be running, this may or may not have any effect for you. I have performed long term culture with phenol red containing medium with no adverse effects.

I have also used phenol red in media with no adverse effects, at least not in terms of cell viability and their ability to attach in culture. There may well be effects for certain assays as others have said.

Thank you Dr. Bartlett,

I need to establish primary culture of caprine, porcine, bovine and ovine hepatocytes for comparative study of mineral ion uptake and its metabolic pathways. Kindly let me know whether you have any suggestions for hepatocyte from these animals. Also suggest on use of phenol red containing medium for these studies. Please suggest on prospects of use of collagenase and trypsin for perfusion.

I have only had experience of human hepatocyte isolation. I would certainly recommend Liberase (from Roche) rather than crude collagenase. A quick look through their website and a search on google will bring up a few reports with suggestions for specific blends of Liberase.

I kept primary human hepatocytes in culture to collect culture supernatants for urea/albumin synthesis etc, made lysates for Western Blotting, did some flow cytometric analysis and adhesion studies and transplanted them into mice. None of these assays/experiments were obviously affected by the use of phenol red (although without repeating them using hepatocytes cultured in the absence of phenol red I can't be sure). Unfortunately I have no experience of the sort of work you are doing so I would defer to the advice of others.

Dear Dr. Bartlett and friends,

kindly clarify my following doubts

1. Why should perfusion buffer 1 ( Ca and Mg free buffer) should be used in cold condition, what will be if it is in room temperature.

2. Can we use Trypsin-EDTA for passaging the hepatocytes grown on collagen coated plates.

3. How long the primary hepatocytes can cultured and sub cultured ( upto how many passages for sub culture).

Also suggest the references for the same where they are best described.

Again, my experience is only of human hepatocytes.

1. We perfuse with warm fluids. I was isolating hepatocytes from resected liver tissue/explanted livers and unused donor liver unsuitable for transplant. Donor liver was preserved by cold perfusion and placed on ice at the time of retrieval and resected/explanted tissue was kept on ice from the time of resection/explant. However, enzymes will not work effectively at cold temperatures and so all perfusion buffers were warmed to 37 C in a water bath in order to warm the liver prior to and during enzymatic digestion. Following enzymatic digestion, many protocols (including our own) call for suspension of cells in ice cold media and for the centrifugation steps to be carried out in a refrigerated centrifuge. Instinctively I wonder if repeated cooling-warming cycling (the liver is warm at the time of retrieval/resection, then cooled on ice, then warmed for perfusion, then the cells placed in ice cold media, then plated and placed in a warm incubator) may stress the cells. In particular, using ice cold media in a refrigerated centrifuge following enzymatic digestion may not be necessary. Anecdotally we have done this step at room temperature on occasions with good results. The only time we perfuse initially with cold buffers is if there is any delay(more than 1-2 hours) in processing tissue from resected/explanted liver. In this case, we perfuse the tissue with cold fluid to wash out any remaining blood and ensure complete cooling of the liver tissue before placing it back on ice. However, we rarely use such tissue as we have shown that a delay in processing greater than 1-2 hours is associated with much poorer outcomes in terms of cell yield/viability.

2. I have found Trypsin-EDTA to be the best method for detaching hepatocytes from culture on collagen. However, we only did so to remove cells from culture for experimental work. Human hepatocytes do not undergo any meaningful proliferation under standard culture conditions and do not require to be/cannot be passaged. It is important to achieve a confluent monolayer on initial plating, and in our experience a good batch of cells cultured on collagen as a confluent monolayer will look healthy (rather unscientific I know) for up to 7 days. Cells then start to look stressed and some start to detach/undergo apoptosis. The reference I mentioned in my previous post regarding long term culture describes successful culture for longer periods with the maintenance of a differentiated phenotype as indicated by various measures of hepatocyte function, but we have never tried to replicate this.

Hi, Amanda, 

Did you mean it is hard to split the cell for primary murine hepatocytes? I am trying to transfer the cells from 24 well to 96 well plate but the cells look not very happy after the trypsin digestion.