Chondrocyte Isolation - How long do I got? (Feb/14/2008 )
Our group will be isolating chondrocytes from bovine articular cartilage. My question for those out there who are familiar with the technique is: What's the working time frame on isolation from death of the animal to isolation of viable chondrocytes? I've isolated primary lines from pancreas, skin and nucleus pulposus as well as quite a few in botany throughout my many years in the lab, and I find that each one has a different working time from "whole meat" to isolated cells. I just want to make sure I'm within the time frame and don't waste a lot of time and reagents in isolating from something that's "too old". I had one place offer me a knee joint that was 3 days old - don't think that one will work! But, like with pancreatic islets, we had a VERY short working time. I'd assume that chondrocytes are a bit more hardy, but still........just wanted to get some idea of how long I got once they take it out of the animal. Thanks for any info anyone can give!
Dear tphares,
My only experience is with rabbit knee joints, but it still might be useful information:
We only used FRESHLY isolated cartilage from the joints.
Due to the lack of cartilage, pooled isolates were used in order to get enough cells.
The amount of viable chondrocytes within the cartilage is low.
We were doing experiments in 24 well plates, so our experimental protocol to start with required low numbers of cells.
The good thing is that you can obtain pure chondrocyte populations, as the enzymatic digest is LONG and fibroblasts will be killed, and also the tissue is not full of connective tissue and blood vessels.
My advice to you is to go to the abattoir and collect FRESH cartilage.......as much as possible. Remember that any tissue coming from an abattoir is full of possible contaminants.
Rabbit is a cleaner model to start with.....have you considered this?
Kindest regards
Rhombus
Dear tphares,
My only experience is with rabbit knee joints, but it still might be useful information:
We only used FRESHLY isolated cartilage from the joints.
Due to the lack of cartilage, pooled isolates were used in order to get enough cells.
The amount of viable chondrocytes within the cartilage is low.
We were doing experiments in 24 well plates, so our experimental protocol to start with required low numbers of cells.
The good thing is that you can obtain pure chondrocyte populations, as the enzymatic digest is LONG and fibroblasts will be killed, and also the tissue is not full of connective tissue and blood vessels.
My advice to you is to go to the abattoir and collect FRESH cartilage.......as much as possible. Remember that any tissue coming from an abattoir is full of possible contaminants.
Rabbit is a cleaner model to start with.....have you considered this?
Kindest regards
Rhombus
Thanks Rhombus, actually rabbit would be easier to obtain since we have a good relationship here with a medical testing facility in the area. In fact we get quite a lot of rabbit spines for the nucleus pulposus study from them. I was thinking bovine only due to the relatively large amounts of cartilage that you can get from them compared to how many rabbits we'd have to use, but it's still a viable option and I'll put it to the group at the next meeting. Especially since they are reluctant to use any type of antibiotic and the student who will be starting this project is VERY new to even wet lab work, let alone tissue culture or primary isolations - keeping the possibility of contamination to minimum by using a more controlled tissue may be beneficial.
Hope this post is not too late. I constantly work with bovine cartilage tissues. We normally order cow joints that were killed on that day, however, I did notice that some of the the joints were not as fresh as others (from the color of the cartilage, meant and blood). We later figured out that the slaughter house sometimes would save the joints of a killed animal and deliver them when we order them. Despite of all these issues, the cells harvested were still alive, and there were plenty of them for me to work with.