Help: ECM Extraction from cells grown in Alginate gels - (Dec/25/2006 )
Hi. I was wondering if anyone on this board has successfully extracted ECM proteins from cells grown in alginate beads. I'd also like to hearing pointers any of you may have from any experience in using Alginate with Chondrocytes, or any other cells for that matter.
Anyway....back to my questions. I'm interested in studying matrix secretion in growth plate chondrocytes. My currently evolving model involves growing primary rat costal chondrocytes in 1.2% alginate gels, examining the ECM proteins (collagens) and proteoglycans they produce. Eventually I'd like to get enough to do 2DPAGE and/or immunoblotting, but cant seem to separate the ECM protein from the Alginate.
So far, I've seeded 4x10^4 cells/ml in 100ul beads, solidified in CaCl2 and then allowed them to grow in the gel for 15 days (should I let them go longer?). I then the dissolve 30-50 cell-laden gel beads in a Ca chelating buffer (tried 10 volumes of NaCitrate initially or EDTA more recently, and both work nicely). After disaggregating the gels and centrifuging 10 min at 300xg, I get plenty viable cells, RNA and cellular protein from the pellet to do viability assays/RTPCR/Westerns, respectively, but get no protein when I precipitate the supernatant.
So I think my problem is that I when I try to precipitate the proteins from the pellet supernatant I re-aggregate the alginate. So far I've tried 4 volumes 100% Acetone at -80 and -20`C or 1 volume ice cold 20% TCA and I end up with a huge(~2ml from 35 ml supernatant) gelatinous glob that is diffucult to re-dissolve in SDS PAGE buffer, Urea or Guanidine. The glob is mostly alginate (I think?) but is slightly more dense and only dissolves in the chelation buffers mentioned previously with sonicating. I don't think the glob is genomic DNA, and I have a hard time believing that the glob that size is all protein, considering I get no protein bands on a coomassie stained SDS-PAGE gel.
Now, I'm pretty sure the cells are making ECM: I get good RT-PCR product for Col2, ColX, Aggrecan and other PG's, and can see some bi-refringence under polarized light in sections of FFPE and fresh frozen beads suggesting fibrillar Collagens have been deposited. This is visible both around the cells and their lacuna, as well as in the interterritorial matrix, and no birefringence is evident in the cell-free control beads. Additionally, Alcian blue and SafraninO also both stain strongly in thoughout the beads, but my cell-free control beads also stain similarly. (I don't yet have suitable antibodies yet to do immunohistochemistry 
)
Does anyone have an idea where I'm going wrong, or if maybe I'm fighting an uphill battle? Cheers!-JAH
Hi,
I really want to follow this topic since I'm also working with chondrocytes in alginate beads.
I don't know where you are going wrong, but I can give some suggestions.
I think that 15 days is still a bit early, I would go for 21 days or more, and also consider that most people seed 4x10^6 cells/ml (I know it's hard to isolate that amount of chondrocytes).
I was also thinking on how to isolate proteins from the beads (ecm proteins), because with normal protein precipitation, you also precipitate the alginate. My first thought was to do a first precipitation with 40% acetone and then a second one with 80% acetone (specific for collagen). Only, this does not solve this for the proteoglycans.
I'm still reading articles at this moment, so if I read anything, I will reply it.
And I hope that you will share your experiences with us again!
Not in this field, but I am interested to know-
Is there an good extablished chondrocyte line that maintains most of primary cell properties?
Is there an good extablished chondrocyte line that maintains most of primary cell properties?
Depends on what you call primary cell properties.
There are immortalized chondrocyte cell lines available, only these produce also a lot of collagen type I (far more than a primary chondrocyte), so the question is if you can still call this a chondrocyte cell line. But it is accepted in Literature.
There are also some other cell lines commercially available, but the problem I have with these is that the resource is/stays unclear.
Thanks Aspergillie. I'm currently have some beads set up a week ago that I'm going let go 21 and 28 days. I think I mispoke about my cell number- it's 4x10^4/bead not /ml, but still 10x less than you reccommend. I may try a few beads at that density if my current run does not pan out. I'm also going to try to grow out chondrocytes recovered from the alginate (J Orthop Res. 2003 Jan;21(1):139-48) and see what kind of matrix they produce. As the song goes, 'The wait is the hardest part', but I'll keep you posted.
As for Genehunter1's query... Aspergillie is again right on. We gave up on commercial so-called 'primary' chondrocyte lines early on, because they lost their phenotype quickly. Some people also use various chondrosarcomas for matrix studies too, usually in the context of protein production for structural biology, but you don't want to read too much into normal cartilage biology from these cell types. We've found that isolating our own from weanling rat rib/costal cartilage works best for our studies. This (Bone. 1988;9(3):185-94) is a GREAT paper describing this method in detail especially regarding ECM characteristics. We can maintain growth cells in monloayer for about 15 passages before they get weird. The phenotypic shift is quite dramatic...they become spindly rather than their normal rounded/cuboid morphology and they just suddenly stop proliferating at the usual pace of a 36-48h doubling time to a week or more, so I'm reasonably sure they've changed biologically. This is when I can them and get some more going from the cryo.
I'll keep trying....after all I've got a few years left before my PhD committee kicks me out of here. Thanks again, and of course I'd appreciate any other input. Cheers!