Chemistry: molecular biology and microbiology – Carrier-bound or immobilized enzyme or microbial cell;... – Enzyme or microbial cell is immobilized on or in an organic...
Patent
1994-06-22
1996-06-25
Naff, David M.
Chemistry: molecular biology and microbiology
Carrier-bound or immobilized enzyme or microbial cell;...
Enzyme or microbial cell is immobilized on or in an organic...
424 937, 435182, 43524022, 5361231, 536127, C12N 1110, C12N 1104, C07H 100, C07H 106
Patent
active
055299130
DESCRIPTION:
BRIEF SUMMARY
This invention concerns a method of making biocompatible capsules containing cells for implantation into a body which lacks the normal functioning of those cells.
A common problem caused by the implantation of foreign cells and tissue is that of immunological rejection by the recipient. Attempts to overcome this problem commonly involve treating a patient with immunosuppressive drugs. However, this is known to be dangerous as these types of drugs have many side effects. Alternatively, the tissue may be treated in some way so as not to provoke an immune response.
With regard to the latter, means of encapsulating cells, particularly islet cells, have been developed as a method of immunoprotection, using relatively nonantigenic material (Lim and Sum, 1980, Science, 210, 908-910; Lim, U.S. Pat. No. 4,409,331). In their method, a suspension of islet cells in sodium alginate solution was formed into droplets and coated with poly-1-lysine to form a membrane permeable to small molecules such as glucose and insulin, but impermeable to large molecules such as immunoglobulins and the cells of the immune system.
The method was modified by O'Shea et al (1984, Biochimica et Biophysica Acta, 804, 133-136) by coating the capsules with an additional outer layer of sodium alginate in order to further minimise the inflammatory response.
Although some transplantation of encapsulated cells has been moderately successful showing normoglycaemia in diabetic mice two weeks after transplantation of encapsulated islet cells (Calafiore et al, Diabetes Research and Clinical Practice, 1988, 5 suppl 1, S334; Tze and Tai, 1982, Transplantation, 33, 563-564; Ricker and Stockberger, 1986, Diabetes, 35, Suppl 1, 61A), the capsules, on removal, showed infiltration of monocytes and macrophages, ie. severe inflammatory reaction.
A recent study (Clayton et al, Diabetes Research 1990 14 127-132) testing the immune response of rats to implanted empty capsules showed that the composition of the outer layer of the capsule affects the severity of the response, with M-alginate (high mannuronic acid sodium alginate), provoking the least immune response. However, even this reduced response resulted in macrophage and fibroblast infiltration after only 3 weeks.
The present invention overcomes, or at least relieves, the problems aforesaid.
According to the present invention there is provided a method of making biocompatible capsules containing cells comprising the steps: water soluble gum which has plural anionic moieties but is substantially protein free; compatible cations to gel the droplets, encapsulating said cells; which cross link with said anionic groups to form a semi-permeable membrane; water soluble gum.
The water soluble gum may be sodium or other water soluble alginate, such as potassium alginate.
The alginate may be predialysed with phosphate buffered saline (PBS) containing dithiothreitol (DTT), which helps break down any associated protein by breaking the disulphide bonds present therein. The alginate may be predialysed with PBS containing any chemical capable of reducing disulphide bonds in associated protein, for example, dithioerythritol, 2-mercaptoethanol, and the like.
"Substantially protein free" in this context does not necessarily mean that protein associated with the alginate or other gum is totally removed. Unexpectedly, it is found that it is sufficient if disulphide bonds in the associated protein are broken.
The dithiothreitol concentration may be greater than 0.1 mg/ml, preferably 0.6 mg/ml, other chemicals in corresponding amounts.
The alginate may be dialysed for more than 1 hour, preferably twice for 2 hours against PBS containing DTT, or any chemical capable of reducing disulphide bonds, and against PBS alone for more than 65 hours, to ensure efficient removal of contaminating antigenic protein.
The cells may be suspended in normal saline containing the purified alginate.
The cell suspension may contain more than 1% w/v of the pre-dialysed, purified alginate, preferably 1.5% w/v.
The cells to be encapsulated may be mammal
REFERENCES:
Stryer, Lubert, Biochemistry, 2nd ed. Walt Freeman & Co., N.Y., 1981, pp. 18-38.
Pedersen, et al: "Purification, Characterization, and Immunological Cross-Reactivity of Alginates Produced by Mucoid Pseudomonas Aeruginosa from Patients with Cystic Fibrosis", J. Clin. Microbiol. vol. 27, No. 4, 1989, pp. 691-699.
Goosen et al: "Optimization of Microencapsulation Parameters:Semipermeable Microcapsules as a Bioartificial Pancreas", Biotechnology and Bioengineering, vol. 27, 1985, pp. 146-150.
Clayton Heather A.
James Roger F. L.
London Nicholas J. M.
Naff David M.
University of Leicester
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