Novel cell culture supports with particular properties, and production thereof

• US 20030003554A1
• Filed: 04/29/2002
• Published: 01/02/2003
• Est. Priority Date: 10/29/1999
• Status: Abandoned Application

• Current Assignee: 4C BIOTECH
• Original Assignee: Computer Cell Culture Center SA (Thermo Fisher Scientific Incorporated)
• Inventors: Ropson-Kenda, Nathalie Liliane Paule Ghislaine, Miller, Alain Othon Andre
• Application Number:
  US10/133,325
• Publication Number:
  US 20030003554A1
• Time in Patent Office: Days
• US Class Current: 435/174
• CPC Class Codes: C08J 7/18 using wave energy or particle radiation
• CPC Class Codes: C12N 2533/30 Synthetic polymers
• CPC Class Codes: C12N 5/0075 using microcarriers

Abstract

A method for continuously obtaining two-dimensional microcarrier beads (2D-MS) displaying particular functional characteristics for anchorage-dependent cell culture (CAD), includes a succession of steps which consist in: continuously producing polymer film rolls from a specific polymer granules; activating the polymer film by generating reactive groups; covalent grafting between the activated polymer film and a polymer, copolymer or a macromolecule of interest; if required, washing to eliminate the monomers which have not been consumed and fixed on the film, followed by drying the grafted film; and finally cutting the grafted polymer film by continuous punching, the size of the punch being selected according to that of the dimension desired for the two-dimensional microcarrier beads.

Claims

1. A process for continuously producing two-dimensional (2D) supports endowed with particular properties for anchorage-dependent cell culture (ADC), comprising at least the following steps in succession:

continuously producing rolls of polymer film with a thickness of 35 μ

or less and with a density in the range 0.9 to 1.25 g/cm³ from granules of a given polymer;

activating said polymer film using any means for generating reactive groups, in particular radicals and/or peroxide, hydroperoxide or amine functions;

producing covalent grafts between the activated polymer film and a polymer, a copolymer or a macromolecule of interest the property of which is desired, said grafting being achieved by immersing the film in a solution of monomer, copolymerisation being initiated by free radicals created by activation under β

irradiation, the immersion time being directly correlated to the desired thickness of the polymer grafted onto the film;

if necessary, washing to eliminate monomers that are not consumed and not fixed to the film, followed by drying the grafted film;

cutting the grafted polymer film by continuous punching, the size of the punches being selected as a function of the desired size of the 2D supports.

2. A process according to claim 1, in which the contact angle θ

of the polymer film is in the range 30°

to 90°

.

3. A process according to claim 1, in which activation is accomplished by corona discharge, plasma, UV irradiation or electron bombardment.

4. A process according to claim 3, in which the polymer film is electronically activated by bombardment with β

irradiation in an inert atmosphere.

5. A process according to claims 1 to 4, in which the thickness of the polymer layer grafted onto the film surface is determined by a combination of the following parameters:

the total dose of p irradiation received by the film, the concentration of monomer in the grafting tank, the grafting time and the grafting temperature.

6. A process according to any one of the preceding claims, in which the polymer or copolymer of interest is selected from derivatives of poly-N-alkyl(meth)acrylamides, poly-N-isopropylacrylamide (NIPAAm), their respective copolymers, poly-N-acryloyl piperidine and poly-N-acryloyl pyrrolidine the desired property of which is cryosensitivity.

7. A process according to claims 1 to 5, in which the polymer or copolymer of interest is a hydrophilic amine-containing polyethylene oxide PEO type polymer the desired property of which is biocompatibility.

8. A process according to claim 6, in which all traces of stabilising agent are eliminated from the monomer solution.

9. A process according to claims 1 to 5, in which the macromolecule(s) of interest is (are) one or more specific ligands for cell receptors, the desired property of which is the selective adhesion of cells carrying this (these) receptor(s).

10. A process according to claim 6 in which, when the polymer film (substrate) is polystyrene and when the grafted polymer is selected from derivatives of poly-N-alkyl(meth)acrylamides, their respective copolymers, poly-N-acryloyl piperidine or poly-N-acryloyl pyrrolidine, a thickness of 40 to 60 Å

is obtained by a combination of a total dose of irradiation of 50 to 250 Kgrays, and an immersion time and temperature in the monomer solution of 1 to 3 hours and 50 to 70 degrees respectively.

11. A process according to claim 1, in which activation is prior to grafting of the polymer or copolymer of interest with the desired property.

12. Two-dimensional (2D) microsupports endowed with particular properties for mass culture of anchorage-dependent cells (ADC) obtained by a process according to any one of the preceding claims, characterized in that the thickness of the polymer film is in the range 10 to 35 microns, and the thickness of the covalently grafted polymer, copolymer or macromolecule of interest is in the range 1 to 10 nm.

13. Microsupports according to claim 12, in which the grafted polymer is a cryosensitive polymer selected from derivatives of poly-N-alkyl(meth)acrylamides, their respective copolymers, poly-N-acryloyl piperidine and poly-N-acryloyl pyrrolidine.

14. Microsupports according to claim 12, in which the grafted polymer is a hydrophilic amine-containing polyethylene oxide PEO type polymer.

15. Microsupports according to claim 12, in which the grafted macromolecule(s) is (are) one or more specific ligands for cell receptors.

17. A device according to claim 16, in which the film unwinding/winding system can advance said film at a speed in the range 0.05 to 8 m per minute.

18. A device according to claim 16, in which the film unwinding/winding system can advance said film at a speed in the range 5 to 50 m per minute.

16. A device for continuous preparation of a 2D-MS support endowed with particular properties, prepared using the process defined above and employing covalent fixing of a polymer, a copolymer or of biological macromolecules on a substrate constituted by a polymer film, said device comprising:

a system for unwinding/winding a polymer film, the film being entrained at a selected speed for each step;

activation, grafting, punching;

an electron accelerator arranged to continuously activate the rolls of film;

a receptacle for containing the solution of monomers, polymers or macromolecules to be grafted, into which the polymer film continuously passes at a predetermined speed entrained by the winding/unwinding system;

a cutting tool for punching the film as it is unwound.

19. A device according to any one of claims 16 to 18, in which the cutting tool is constituted by a tool block on which are locked, in the longitudinal direction of the film, rows of circular punches of ceramic carbide, and a corresponding recess block.

20. A device according to any one of claims 16 to 18, in which the cutting tool is a laser.

21. A device according to any one of claims 16 to 18, in which the cutting tool is constituted by a rotary cylindrical knife (embossing system).