Microparticles, preparation thereof and applications thereof in biology, particularly in the culture of human diploid cells

• US 4,373,027 A
• Filed: 12/05/1980
• Issued: 02/08/1983
• Est. Priority Date: 12/05/1979
• Status: Expired due to Term

• Current Assignee: Institut de Technologie Des Surfaces Actives BP 233, Institut Pasteur
• Original Assignee: Institut de Technologie Des Surfaces Actives BP 233, Institut Pasteur
• Inventors: Berneman, Armand, Horaud, Florian, Segard, Emile, David, Alain
• Primary Examiner(s): Warden, Robert J.
• Application Number:
  US06/213,314
• Time in Patent Office: 795 Days
• Field of Search: 435/240, 435/241, 435/284, 435/285, 435/286
• US Class Current: 435/402
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• CPC Class Codes: C12N 5/0075 using microcarriers

Abstract

These microparticles are formed, at least on their surface, from a reticulated protein. (FIG. 1).

Claims

1. Microparticles comprising particles or beads whose surface at least is formed from a reticulated protein, chosen from protein capable of forming a visco-elastic mass which after reticulation has a fibrous-type structure in a mesh network configuration.

2. The microparticles as claimed in claim 1, having at their surface a charge capacity of about 0.5 to 1.8 meq per gram of dry particles.

3. The microparticles as claimed in claim 2, having at their surface a charge capacity of 0.9 to 1.6 meq per gram of dry particles.

4. The mircoparticles as claimed in claim 2 wherein the functional groups of said reticulation agent not engaged in the reticulation reaction are blocked by fibronectine.

5. The microparticles as claimed in claim 1 wherein the protein is gelatin.

6. The microparticles as claimed in claim 1 wherein the protein is fibronectine.

7. The microparticles as claimed in claim 1 wherein the protein is reticulated with a multifunction agent having at least two aldehyde, azo, sulfonic acid, fluoro groups activated by reactive nitro, imine, azide or chloro groups connected with a cyclic structure whereby the chloro groups are sufficiently active to effect a reticulation reaction with protein.

8. The microparticles as claimed in claim 7, wherein the reticulation agent is glutaraldehyde.

9. The microparticles as claimed in claim 7 or 8 wherein the functional groups of the reticulation agent not engaged in the reticulation reaction are blocked by the protein comprising said microparticles.

10. The microparticles as claimed in claim 7, wherein the functional groups of the reticulation agent not engaged in the reticulation reaction are blocked.

11. The microparticles as claimed in claim 1 wherein said microparticles consist substantially of reticulated protein.

12. The microparticles as claimed in claim 1 wherein said particles are comprised of a surface coating of reticulated protein.

13. The microparticles as claimed in claim 12, wherein the reticulated protein coating is fixed on an anchoring nucleus, comprising chemical groups capable of reacting with the functional groups of the protein of the coating to link therewith, and having a charge capacity of about 0.5 to 1.5 meq per gram of dry particles.

14. The microparticles as claimed in claim 13 wherein the anchoring nucleus is formed of reticulated dextran having tertiary or quaternary amino substituent groups in a proportion such that the charge capacity of the anchoring nucleus particles is about 0.5 to 1.8 meq per gram of dry particles.

15. The microparticles as claimed in claim 13, wherein the anchoring nucleus is formed by reticulated dextran.

16. The microparticles as claimed in claim 1 having at their surface a charge capacity of about 1±

0.3 meq per gram of dry particles.

17. The microparticles as claimed in claim 2, 3, 7 or 16 wherein the protein is gelatin.

18. The microparticles as claimed in claim 2, 3, 7 or 16 wherein the protein is gelatin obtained from an alkaline process.

19. The microparticles as claimed in claim 1, 2, 3, or 16, wherein the protein is gelatin and the reticulation agent is glutaraldehyde.

20. The microparticles as claimed in claim 19, wherein the protein is gelatin obtained by an alkaline process.

21. The microparticles as claimed in claim 19, wherein the functional groups of glutaraldehyde not engaged in the reticulation reaction are blocked by gelatin.

22. The microparticles as claimed in claim 2, 3, 7, 8 or 16 in which the protein is fibronectine.

23. The microparticles as claimed in claim 1 wherein the protein is gelatin obtained by an alkaline process.

24. The microparticles as claimed in claim 2, 3, 5, 6, 7, 8, 10, 16 or 23 which consist substantially of reticulated protein.

25. The microparticles as claimed in claim 2, 3, 5, 6, 7, 8, 16 or 23 wherein said microparticles are comprised of a surface coating of the reticulated protein.

26. The microparticles as claimed in claim 1 comprising anchoring nucleus particles comprised of reticulated dextran having tertiary or quaternary amino substituent groups in a proportion such that the charge capacity of the anchoring nucleus particle is about 0.5 to 1.8 meq per gram of dry particles and a surface coating the anchoring nucleus particles comprised of protein reticulated with glutaraldehyde, wherein the protein is selected from gelatin and fibronectine, the functional groups of glutaraldehyde not engaged in the reticulation reaction being blocked by the protein and the microparticles having a charge capacity at their surface of about 0.5 to 1.8 meq per gram of dry particles.

27. The microparticles as claimed in claim 13 or 26 wherein the anchoring nucleus is formed of reticulated dextran having diethylaminoethyl or diethyl-2-hydroxypropylaminoethyl substituent groups in a proportion such that the charge capacity of the anchoring nucleus particle is about 0.9 to 1.6 meq per gram of dry particles and wherein the microparticles have a charge capacity at their surface of about 0.9 to 1.6 meq per gram of dry particles.

28. The microparticles as claimed in claim 14 or 26 wherein the charge capacity of the anchoring nucleus particles and the charge capacity at the surface of the microparticles are about 1±

0.3 meq per gram of dry particles.

29. The microparticles as claimed in claim 14 or 26 wherein the reticulated dextran is that commercialized under the trademark Sephadex.

30. In a process for the culture of human diploid cells, comprising forming a suspension of microparticles in a medium suitable for the culture and the growth of the diploid cells, seeding the suspension with normal human diploid cells, subjecting the mixture to incubation under conditions of temperature and duration which allow cellular multiplication and then collecting the cells, the improvement wherein said suspension is comprised of the microparticles as claimed in claim 1, 2, 3, 5, 6, 7, 8, 10, 11, 12, 13, 15, 16, 23, 14 or 26.

31. The process for the culture of human diploid cells as claimed in claim 30, wherein the diploid cells are seeded at the rate of about 10⁵ cells per ml of culture medium containing about 1 to 2 mg, microparticles per ml of culture medium.

32. The process for the culture of human diploid cells as claimed in claim 30 wherein the normal human diploid cells grown on the microparticles are infected with a virus or parasites developing in the human cells for the purpose of preparing antigens of the said virus or said parasites.

33. The process for the culture of human diploid cells in claim 30 wherein the culture medium contains about 1.5 to 1.7 microparticles per ml.

34. The process for the culture of human diploid cells as claimed in claim 30 wherein the culture medium contains about 1.6 mg of microparticles per ml.

35. The process for the culture of human diploid cells as claimed in claim 30 wherein the improvement further comprises prior to forming the suspension,

preincubating the microparticles in a culture medium including the elements required for the culture and growth of human diploid cells.

36. The process for the culture of human diploid cells as claimed in claim 35 wherein the improvement further comprises prior to preincubation,

washing the microparticles with a buffer solution to eliminate impurities without adversely affecting the surface of the microparticles and

sterilizing the microparticles.

37. A process for preparing microparticles or beads useful as microcarriers for the culture of normal human or animal diploid cells or said cells infected by a virus or a cellular parasite comprising contacting particles having a diameter of the order of 40 to 200 μ

m, formed of protein capable of forming a viscoelestic mass which after reticulation has a fibrous-type structure in a mesh network configuration, with a solution of a multifunctional reticulation agent to react with the protein,

separating the thus reticulated protein particles from the solution of reticulation agent and

contacting the reticulated protein particles with said protein to block the functional groups of the reticulation agent not engaged in the reticulation reaction.

39. The process as claimed in claim 37 or 38 wherein the surface charge capacity of the final particles is about 0.5 to 1.8 meq of dry particles.

40. The process as claimed in claim 37 or 38 wherein the protein is gelatin.

41. The process as claimed in claim 37 or 38 wherein the protein is gelatin obtained by an alkaline process.

42. The process as claimed in claim 37 or 38 wherein the protein is fibronectine.

43. The process as claimed in claim 37 or 38 wherein the reticulation agent has a least two aldehyde, azo, sulfonic acid, fluoro groups activated by reactive nitro, azide, imine or chloro groups connected with a cyclic structure whereby the chloro groups are sufficiently active to effect a reticulation reaction with protein.

44. The process as claimed in claim 37 or 38 wherein the reticulation agent is glutaraldehyde.

38. A process for preparing microparticles or beads useful as microcarriers for the culture of normal human or animal diploid cells or said cells infected by a virus or a cellular parasite comprising contacting a suspension of particles having a diameter of the order of 40 to 200 μ

m with a solution of a protein to coat said particles with said protein, wherein said protein is capable of forming a viscoelastic mass which after reticulation has a fibrous-type structure in a mesh network configuration and said particles are formed of material capable of providing an anchoring nucleus for said protein,

contacting the protein coated particles with a solution of a multifunctional reticulation agent to react with the protein coating the surface of said particles,

separating the particles coated with thus reticulated protein from the solution of reticulation agent and

contacting the particles with said protein to block the functional groups of the reticulation agent not engaged in the reticulation reaction.

45. The process as claimed in claim 38 wherein the particles providing an anchoring nucleus are comprised of chemical groups capable of reacting with the functional groups of said coating to link therewith and have a charge capacity of about 0.5 to 1.8 meq per gram of dry particles.

46. The process as claimed in claim 38 wherein the particles providing an anchoring nucleus are formed of reticulated dextran.

47. The process as claimed in claim 38 wherein the particles providing an anchoring nucleus are formed of reticulated dextran having tertiary or quaternary amino substituent groups in a proportion such that the charge capacity of the anchoring nucleus particles is about 0.5 to 1.8 meq per gram of dry particles.

48. The process as claimed in claim 38 wherein the particles providing the anchoring nucleus formed of reticulated dextran having diethylaminoethyl or diethyl-2-hydroxypropylaminoethyl substituent groups.