Islet Sheet Medical Useful Methods
High Guluronate Alginate
Edited by Randy Dorian, Vice-President
March 31, 1999
Earlier version of method:
"Bioinvisible" high guluronate alginate 1 U.S. Patent 5,643,594
This alginate may elicit some foreign body response. The alginate forms a strong gel when contacted with calcium. It is useful for an initial coating, as described in U.S. patent number 5,643,594 by Randel E. Dorian, Kent C. Cochrum and Richard David Antanavich, assigned to the Regents of the University of California. The calcium gel is stable indefinitely in vivo. The recipe is Islet Sheet's modification of methods described in the referenced patent.
1. Dissolve Protanal-HF alginate to 0.1% in 0.5 mM EDTA, 10 mM HEPES, pH 7.0.
2. Filter to 0.45 microns to remove particulates.
3. In a separate flask, bleach 4 gm fine mesh activated charcoal per gram alginate by resuspension to 4% (w/v) in 0.1 M sodium perchlorate.
4. After 30 minutes mixing, wash the bleached charcoal by centrifugation (5 min @ 500 xg) twice with water, 4x with ethanol, 4x with water. Discard the supernatants.
5. Add the bleached activated charcoal slurry to the filtered alginate and stir for 30 min to adsorb organic contaminants.
6. Filter the supernatant to 0.22 micron; then filter at 0.1 micron.
7. Add 10.2 ml 10% MgCl2.5H2O per liter filtrate and mix thoroughly.
8. Gradually add 3.8 ml 34% CaCl2.2H2O while stirring and mix for 30 min to precipitate the higher molecular weight, guluronate-rich chains.
9. Spin 20 min at 2,000 xg. Discard supernatant.
10. Estimate pellet volume and add 2 volumes 0.1 M EDTA, 10 mM HEPES, pH7.0
11. Q.s. H2O to 500 ml per gram alginate starting material. Adjust pH to 7.0 if necessary.
12. Concentrate 10 fold by ultrafiltration to 10 kD to remove small fragments.
13. Dilute retentate to starting volume with water and reconcentrate.
14. Repeat #13.
15. Dilute retentate back to starting volume again with water.
16. Add 1/20 volume 2.5 M NaCl.
17. While vigorously stirring, slowly add an equal volume of ethanol.
18. Spin 10 min at 500 xg. Discard supernatant
19. Redissolve in 120 mM NaCl, 0.5 mM EDTA (200 ml per gram alginate starting material).
20. While vigorously stirring, slowly add 4 volumes of ethanol.
21. Spin 10 min at 500 xg. Discard supernatant.
22. Thoroughly resuspend pellet in 1 liter ethanol per gram alginate starting material (will not dissolve).
23. Spin 10 min at 500 xg. Discard supernatant.
24. Thoroughly resuspend pellet again in 1 liter ethanol per gram alginate starting material.
25. Collect precipitate on fine mesh stainless steel sieve.
26. Press out excess liquid. Tease with forceps to fluff precipitate.
27. Dry at 60 C in vacuo.
Notes: Store the dry purified alginate in a cool, dry place until ready for use. Dissolve to desired concentration in buffer (we use 10 mM HEPES buffered normal saline with 0.5 mM sodium citrate) and sterilize by filtration.
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Method for purification of high guluronate alginate:
Dorian, Randy, Ed., Islet Sheet Medical Useful Methods, http://www.isletmedical.com/meth0202 (March 31, 1999).