Method for cultivating cells in adhesion culture by using a cell culture carrier in capsule form, and cell culture carrier therefor
The invention relates to a method for cultivating cells in adhesion culture, comprising at least the following steps: a) dissolving or suspending a cross-linkable, biocompatible material having adhesion points for cells in a cell culture medium; b) suspending cells in the cell culture medium, which contains the cross-linkable, biocompatible material, or in a medium that contains at least one component that is required for the cross-linking of the cross-linkable, biocompatible material; c) introducing the cell suspension into a medium in drops under conditions that initiate or permit the cross-linking of the biocompatible material, wherein either the cell suspension or the medium into which the cell suspension is introduced in drops contains the cross-linkable biocompatible material; d) forming stable, preferably porous capsules from cross-linked biocompatible material, which capsules contain incorporated adherent cells; e) proliferating the adherent cells in the capsules for a specified time period; f) breaking up the capsule material by means of a physical or chemical stimulus and releasing the cells as a cell suspension. In an especially preferred embodiment of the invention, the method is performed cyclically, in that the cells released in step f) are suspended anew in a cell culture medium that contains a cross-linkable, biocompatible material or in a medium that contains at least one component that is required for the cross-linking of the cross-linkable, biocompatible material and steps c)-f) are repeated at least once.
1. A method for cultivating cells in adhesion culture, comprising at least the following steps:
a) dissolving or suspending a cross-linkable, biocompatible material with adhesion sites for cells in a cell culture medium,
b) providing a cell suspension by suspending cells in the cell culture medium which contains the cross-linkable, biocompatible material, or in a medium which contains at least one component which is required to cross-link the cross-linkable, biocompatible material,
c) introducing the cell suspension in drops into a medium under conditions which initiate or allow the cross-linking of the biocompatible material, wherein either the cell suspension or the medium, into which the cell suspension is introduced in drops, contains the cross-linkable, biocompatible material,
d) forming stable capsules of cross-linked, biocompatible capsule material encapsulating adherent cells, wherein the adherent cells can grow in capsules,
e) proliferating the adherent cells within the capsules for a defined time period,
f) dissolving the capsule material by a physical or chemical stimulus and releasing the cells as a cell suspension,
g) suspending the cells released in step f) in a cell culture medium, which contains a cross-linkable, biocompatible material, or in a medium which contains at least one component which is required to cross-link the cross-linkable, biocompatible material, and
h) repeating steps c)-f) at least once thereby obtaining a suspension of cells.
2. The method according to claim 1 , wherein step c) is carried out under conditions which support pore formation in the capsule material.
3. The method according to claim 2 , wherein a gas is injected into the medium into which the cell suspension is introduced in drops or a gas-forming particulate material, which releases gas after exposure to a physical or chemical stimulus, is incorporated in the capsule material.
4. The method according to claim 1 , wherein the conditions which initiate or allow the cross-linking of the biocompatible material in step c) encompass exposing the biocompatible material to a physical or chemical stimulus.
5. The method according to claim 4 , wherein the physical or chemical stimulus in step c) is a member selected from the group consisting of a temperature change, a pressure change, ultrasound, electromagnetic radiation, a pH change, an action of enzymes, radical starters, addition of water, exclusion of water, and ions.
6. The method according to claim 5 , wherein the physical stimulus in step c) is an ultrasound treatment of the biocompatible material and the chemical stimulus in step c) is a contact of the biocompatible material with a chemical agent selected from the group consisting of an enzyme and a bivalent cation, or comprises said contact.
7. The method according to claim 1 , wherein the stimulus in step f) is a member selected from the group consisting of a temperature change, a pressure change, ultrasound, electromagnetic radiation, a pH change, an action of enzymes, complexing agents, solvents, and combinations thereof.
8. The method according to claim 7 , wherein the stimulus in step f) comprises an action of proteases which are produced by the adherent cells themselves.
9. The method according to claim 1 , wherein the cross-linkable biocompatible material comprises a material which is capable to form a hydrogel.
10. The method according to claim 9 , wherein the hydrogel is a member selected from the group consisting of a protein-based hydrogel, a sugar-based hydrogel, a polyester hydrogel, a polyether hydrogel and a polyalcohol hydrogel.
11. The method according to claim 1 , wherein the cross-linkable, biocompatible material was provided with adhesion sites for cells by a chemical coupling reaction.
12. The method according to claim 11 , wherein the adhesion sites for cells comprise specific or non-specific adhesion or binding motifs for biological cells.
13. The method according to claim 1 , wherein the capsules composed of cross-linked, biocompatible material comprise a material selected from a thermoresponsive material, a polyester, a polyether, a polyalcohol, silk, fibrin and a cross-linked alginate.
14. The method according to claim 11 , wherein gelatin molecules are coupled to an alginate framework structure to provide an alginate-gelatin precursor material, and cross-linking of this alginate-gelatin precursor material is effected by contacting with a bivalent cation.
15. The method according to claim 1 , wherein the capsules composed of cross-linked, biocompatible material further contain immobilized nutrients/growth hormones and/or reporter molecules.
16. The method according to claim 15 , wherein the reporter molecules display a change of at least one parameter in the capsule, by a corresponding verifiable least one member selected from the group consisting of pH, viscosity, CO 2 concentration and O 2 concentration.
17. The method according to claim 15 , wherein the reporter molecules are dyes or fluorescence markers.
18. The method according to claim 15 , wherein the reporter molecules display a change of at least one parameter in the capsule, by a corresponding verifiable change in properties of the reporter molecules.
19. The method according to claim 18 , wherein verifying the change of the parameter is effected by a spectroscopic or spectrometric method.