METHOD 0102

Islet Sheet Medical Useful Methods

Bioinvisible High Mannuronate Alginate

Edited by Randy Dorian, Vice-President

March 31, 1999

Earlier version of method:

"Bioinvisible" high mannuronate alginate 1 U.S. Patent 5,429,821


The alginate described below is the most bioinvisible of those with which we have had experience. The alginate forms a relatively weak gel when contacted with calcium. We recommend it as a coating which will not elicit foreign body response. The calcium gel is stable indefinitely in vivo. The recipe is Islet Sheet's modification of a preparation described in U.S. patent number 5,429,821 by Randel E. Dorian, Kent C. Cochrum and Susan A. Jemtrud, assigned to the Regents of the University of California.

Method

1. Dissolve Keltone HVCR (by Monsanto) 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 0.1 micron.

7. Concentrate 10 fold by ultrafiltration to 10 kD to remove small fragments.

8. Dilute retentate to starting volume with water and reconcentrate 10 fold.

9. Repeat #8.

10. Dilute retentate back to starting volume again with water.

11. Add 1/20 volume 2.5 M NaCl.

12. While vigorously stirring, slowly add an equal volume of ethanol.

13. Spin 10 min at 500 xg. Discard supernatant.

14. Redissolve in 120 mM NaCl, (100 ml per gram starting material).

15. Repeat #12 through #14.

16. While vigorously stirring, slowly add 4 volumes of ethanol.

17. Spin 10 min at 500 xg. Discard supernatant.

18. Thoroughly resuspend pellet in 1 liter ethanol per gram alginate starting material (will not dissolve).

19. Spin 10 min at 500 xg. Discard supernatant.

20. Thoroughly resuspend pellet again in 1 liter ethanol per gram alginate starting material.

21. Collect precipitate on fine mesh stainless steel sieve.

22. Press out excess liquid. Tease with forceps to fluff precipitate. N.B. These final washes extract residual organic impurities, water and salts.

23. 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). You may want to supplement with glucose, amino acids, insulin, transferrin, etc. Sterilize by filtration. Do not autoclave or gamma irradiate, as these sterilization methods will damage the alginate chains.


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Citation

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Sample citation:

Method for purification of high mannuronate alginate:

Dorian, Randy, Ed., Islet Sheet Medical Useful Methods, http://www.isletmedical.com/meth0102 (March 31, 1999).