Hello Hanie,

I would personally be concerned that your alginate is not cell culture quality or that it has some type of contaminant. When working with cells and agarose, I was not using cell culture quality agarose at first and we would see a similar decrease in viability. I have seen this problem with several other 3 dimensional scaffolds as well.

Chris

- Thank you for your answer. Well, I don't know if SIGMA produces alginate for any other reason than cell culture. I used UV to sterilize the alginate. And what should I do in order to get rid of the contaminants? Is dialyses the answer?

Great question. I would contact Sigma to double check that it is cell culture approved. I would also try dialysis to remove contaminants and possibly working with alginate at lower concentrations.

Chris

- I use 1.2% of alginate in 102mM CaCl2 and that's what everyone used to encapsulate ADMSCs. I'm so fed up with this so called biocompatible proven hydrolgel. At first i thought I am being harsh in the extraction procedure i.e. keeping them for such long time (20 min) in sodium citrate (55 mM), but I found out that I am actually losing my cells in the very first step. (I reduced the 20 min rest in sodium citrate to 10 min, but I didn't get what I was looking for)

Could the problem lay in the fact that alginate is not cell interactive. Since it lacks cell adhesion motives, your cells might experience a non-ideal environment.

Novamatrix supplies ultrapure alginates that are low in endotoxins. Also have 3D kits for cell culture and Novatach peptide coupled alginates to promote cell interaction. I have attached link to the relevant part of the website here:-

http://www.novamatrix.biz/Technology/Alginate/NOVATACHPeptide.aspx

http://novamatrix.biz/Default.aspx?alias=novamatrix.biz/3d

Another way to find out if it's really impurities in the alginate is to add cell-free alginate beads to your cell cultures. If there is a toxin or impurity, you would see cell death or at least a difference in proliferation vs. an untreated control.

Could it be an osmosis or pH issue leading to cell death? Alginate having a negative charge could impart a small but significant effect on pH.

When I used alginate back in the day, I made it in phosphate-buffered saline and then steam autoclaved for sterility. This ensured full dissolution and partially ensured heat-deactivation of any protein-based impurities. But I used primary chondrocytes rather than any MSCs.

The toxicity is probably due to several reasons. First thing is to check the pH of the alginate once you dissolve in buffer or cell culture medium. If the pH is too acidic, it is not good. Even if you dissolve in PBS 7.4, the pH would be around 2-3 which is harmful to cells. Other thing is viscosity. 1.2 % should be good, however just see if you use 1%, will the viability increase? I don't think the cell death is due to sterility issue.

How are you measuring the viability? Could it be a technical issue with the assay, rather than a problem with the material?

In my opinion, a slow decrease in cell viability when changing the 2D culture system with 3D culture system is expected. Although in the next 2-5 days cells would start proliferating! http://www.biomedcentral.com/1472-6750/12/35

MIne wasn't slow, it was fast , very fast!!! in just a few second i lost nearly 50% of my totally viable cells. The problem got solved after i purified my alginate!

If it is acceptable, please, describe me how you cleared the alginate.

Hi Aziz!

Of couse I will. I made the alginate solution; simply the alginate and distilled water. Then I poured the solution in a dialysis membrane and after a 3 days dialyses, I made alginate films with an adequate thickness. Later on, I used an hour of UV sterilization. This worked for me, hope it helps you as well.

Hi Hanie!

Thunk you very much!