I am working with 3D cultures in alginate. I encapsulate human neonatal dermal fibroblasts in sodium alginate then crosslink in a 100 mM calcium chloride solution for 10 minutes. Initially, I had no issues with viability. Recently, we started checking the pH of our original solutions and I found that the alginate was originally at a pH of about 8.6. I adjusted this pH to about 7.4, but when I did this I saw a drop in viability. Is there any chance this change in pH could affect the crosslinking step somehow, leading to a decrease in viability?
Dear Margaret,
Calcium chloride solubility varies with the changes in the pH of the solution and so will be the cross-linking ability of alginate with calcium ions. With the variation in pH, the concentration of available calcium ion would be different too. Therefore, a formation of an intermolecular network of alginate of alginate molecules in the presence of calcium ions could be different with different pH. Similarly, –COO– and –COOH groups along the alginate chain can attain different charge densities depending on the pH. Alginate can induce the formation of hydrogels and acid gels in aqueous solution only when a pH value is less than the pKa value of uronic acid residues of alginate. Encapsulation efficiency of alginate has been attributed to the degree of cross-linking. The mechanical properties of formed hydrogels are affected as well and subsequently, there can be distinct differences in cell growth, differentiation, and tissue formation. Thank you.
Dear Margaret,
I would say this could be related to the accuracy of the pH adjustment process. I managed to solve this problem in my work by dissolving the Alginate in Ca free medium. The aliginate source makes a difference as well, mine is from Acros, pH is around 7.2 and it cross links in less than 3 mins in 100mM CaCL. It takes longer with other brands. I followed the cell viability for more than 4 weeks.
Hope you find this helpful
Alginate gelling tends to be favoured at lower pH, while higher pH tends to favour the sol colloidal phase. The (divalent) Ca2+ cations are expected to expand the temperature gap between the gel-sol (higher) and sol-gel (lower) transition temperature, hence stabilizing either the gel or the sol, with a pronounced hysteresis effect, thus inducing a significant thermal history dependency at the sol-gel transition. By increasing the Ca2+ concentration, the sol-gel transition can progressively acquire a thermo-irreversible character. This is a non-linear characteristic that may concomitantly promote a gel syneresis aging effect. Hence, once gel flocs form by cooling the sol, such recently formed gel flocs may loose water by syneresis while they age, rather than to coalesce with adjacent gel flocs to extend the swollen hydrogel network structure. Eventually, the density gradient between the aged gel flocs and the suspending liquid solution may induce flocculation, rather than gelation. This effect would tend to be enhanced by increasing temperature. Agitation tends to oppose gelation, being this effect prolonged for some time after being stopped. Also to be noticed is that is difficult to weight accurately the very hygroscopic and deliquescent CaCl2 salt, even if the hexahydrate is selected. The salt is also prone to react with atmospheric CO2. After agitation, moreover if accompanied by swirling, the salt can become further carbonated, what would also be favoured by prolonged contact with the laboratory atmosphere. The pH of solutions obtained from carbonated salt tends to be lowered, what helps to detect this effect. You may consider to purify this salt by recrystallizing it, after dissolved in absolute ethanol. Also convenient might be to standardize a prior CaCl2 solution, before alginate addition. For comparable results, the ionic strength should be maintained constant. Reproducibility concerns are quite demanding.
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