Cells or tissue coated with non-fibrogenic alginate less than 200 .mu.m thick

• US 5,656,468 A
• Filed: 03/14/1995
• Issued: 08/12/1997
• Est. Priority Date: 05/29/1992
• Status: Expired due to Term

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Cochrum, Kent C., Dorian, Randel E.
• Primary Examiner(s): Naff, David M.
• Application Number:
  US08/403,348
• Time in Patent Office: 882 Days
• Field of Search: 435/175, 435/177, 435/180, 435/182, 435/173.1
• US Class Current: 435/178
• CPC Class Codes: A61K 2035/128 capsules, e.g. microcapsules
• CPC Class Codes: A61K 9/1652 Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin homeopathic globules A61K9/1623
• CPC Class Codes: A61K 9/1694 resulting in granules or microspheres of the matrix type containing more than 5% of excipient
• CPC Class Codes: C12N 11/04 entrapped within the carrier, e.g. gel or hollow fibres
• CPC Class Codes: C12N 2533/74 Alginate
• CPC Class Codes: C12N 5/0012 Cell encapsulation
• CPC Class Codes: C12N 5/0677 Three-dimensional culture, tissue culture or organ culture; Encapsulated cells

Abstract

An electrostatic process is used for coating a biological material with a uniform, continuous polymer layer by discharging a suspension of the biological material in a gelable coating polymer solution in a continuous stream through an orifice into an electrostatic field. The stream is attenuated to form droplets by maintaining an electrostatic voltage between the needle and the gelling solution which is sufficient to maintain an attraction of at least 1×10^-6 newtons on the stream of liquid leaving the needle, and the droplets are collected in a gelling solution. A preferred product is pancreatic islets having a continuous, smooth coating of high polymannuronate non-fibrogenic alginate having a thickness less than 200 μm such as about 20-200 μm. The alginate preferably contains less than 1 wt. % fucose, less than 0.5 wt. % sulfate and less than 0.01 wt. % phloroglucinol, is free of fibrinoogenic concentration of protein, and has a mannuronate to guluronate ratio of from 1.2 to 6. Preferably, the electrostatic field is formed by applying an electrostatic continuous DC voltage to each the needle and gelling solution so that the needle and solution have opposite charges.

Claims

1. Coated cells for transplanting wherein the cells have a smooth, uniform and continuous coating less than 200 μ

m thick, consisting essentially of a non-fibrogenic alginate, said coated cells produced by a process comprising the steps;

(a) dispersing the cells in an aqueous solution of the non-fibrogenic alginate containing less than 1 wt. % fucose, less than 0.5 wt. % sulfate, less than 0.01 wt. % phloroglucinol and being free from fibrinogenic concentration of protein, and having a mannuronate to guluronate ratio of from 1.2 to 6, to form a suspension;

(b) discharging the suspension of step (a) from a reservoir and through an orifice of an electrostatic apparatus in a continuous attenuated stream forming droplets in an electrostatic field created by said electrostatic apparatus, said droplets expelled from the orifice placed above an electroconductive liquid capable of gelling the polymer;

(c) applying a first electrostatic continuous DC voltage on the orifice and a second electrostatic continuous DC voltage on the electroconductive liquid of step (b) generating a continuous constant electrical potential while the suspension is being discharged from the orifice in the continuous stream, the electrical potential being sufficient to continuously attenuate the stream of suspension to form a continuous stream of droplets having the same size; and

(d) collecting the formed droplets in the electroconductive liquid capable of gelling the alginate to form a continuous, smooth and uniform coating on the cells less than 200 μ

m thick resulting in formation of said coated cells;

wherein the alginate solution of step (a) has a viscosity of from about 30 to about 250 centipoises;

wherein the orifice of step (b) has a diameter of from about 0.1 to 2 mm; and

wherein the electroconductive liquid of step (b) comprises an aqueous electrolyte solution containing a concentration of alkaline earth metal ions sufficient to gel the alginate.

2. The coated cells of claim 1 wherein the voltage potential between said first and second voltages is sufficient to provide a continuous attraction of at least 1×

10^-6 newtons for the liquid leaving the orifice.

3. The coated cells of claim 2 wherein the coated cells are pancreatic islet cells.

4. The coated cells of claim 3 wherein the coating has a thickness of from about 20 to about 200 μ

m and wherein the electrolyte solution is calcium chloride.

5. The coated cells of claim 4 wherein the mannuronate to guluronate molar ratio of the alginate coating is from 1.5 to 2.5.

6. The coated cells of claim 5 wherein the electrostatic continuous DC voltage of step (c) is from 1 to 400 KV.

7. The coated cells of claim 6 wherein the alginate has a molecular weight from about 10 to about 200 kilodaltons.

8. The coated cells of claim 7 wherein a calcium ion concentration is at least 0.5 wt. % of the aqueous solution.

9. A coated pancreatic islet tissue for transplanting coated with a smooth, uniform and continuous coating less than 200 μ

m thick, consisting essentially of a non-fibrogenic alginate, said coating produced by a process comprising the steps;

(a) dispersing the pancreatic islet tissue in an aqueous solution of the non-fibrogenic alginate containing less than 1 wt. % fucose, less than 0.5 wt. % sulfate, less than 0.01 wt. % phloroglucinol and being free from fibrinogenic concentration of protein, and having a mannuronate to guluronate moiety ratio of from 1.2 to 6, to form a suspension;

(b) discharging the suspension of step (b) from a reservoir and through an orifice of an electrostatic apparatus in a continuously attenuated stream forming droplets in an electrostatic field created by the apparatus, said droplets expelled from the orifice placed above an electroconductive liquid capable of gelling the polymer;

(c) applying a first electrostatic continuous DC voltage on the orifice and a second electrostatic continuous DC voltage on the electroconductive liquid of step (b) generating a continuous constant electrical potential while the suspension is being discharged from the orifice in the continuous stream, the electrical potential being sufficient to continuously attenuate the stream of suspension to form a continuous stream of droplets having the same size; and

(d) collecting the droplets in the electroconductive liquid capable of gelling the alginate and forming a continuous, smooth and uniform coating on the pancreatic islet tissue less than 200 μ

m thick resulting in formation of said coated pancreatic islet tissue;

wherein the solution of step (a) has a viscosity of from about 50 to about 150 centipoises;

wherein the orifice of step (b) has a diameter of from about 0.1 to 2 mm; and

wherein the electroconductive liquid of step (b) comprises an aqueous electrolyte solution containing a concentration of alkaline earth metal ions sufficient to gel the alginate.

10. The coated pancreatic islet tissue of claim 9 wherein the electrolyte solution is calcium chloride.

11. The coated pancreatic islet tissue of claim 10 wherein the coating has a thickness of from about 20 to about 200 μ

m.

12. The coated pancreatic islet tissue of claim 11 wherein the coating has a thickness of about 130 μ

m and the electrolyte is calcium chloride.

13. The coated pancreatic islet tissue of claim 12 wherein the voltage potential between said first and second voltages is sufficient to provide a continuous attraction of at least 1×

10^-6 newtons for the liquid leaving the orifice.

14. The coated pancreatic islet tissue of claim 13 wherein the mannuronate to guluronate molar ratio of the alginate coating is from 1.5 to 2.5.

15. The coated pancreatic islet tissue of claim 14 wherein the alginate has a molecular weight from about 10 to about 200 kilodaltons.

16. The coated pancreatic islet tissue is wherein the electrostatic continuous DC voltage of step (c) is from 1 to 400 KV.

17. The coated pancreatic islet tissue of claim 16 wherein a ratio of a current necessary to generate the electrostatic continuous DC voltage of step (c) is from 0.2 to 3.5 μ

A.

18. The coated pancreatic islet tissue of claim 17 wherein a calcium ion concentration is at least 0.5 wt. % of the aqueous solution.