Most animal cells are surface-dependent, that is they have to be attached to a surface to be able to survive and/or proliferate. Traditionally this surface has been the interior of glass or plastic flasks. Great difficulties have been involved in culture of cells on a large scale or in implantation of the cells. The size of these cells is 5-20 μm.
Microcarriers are small particles of 0.2 mm diameter, to which the cells can attach and on which they can proliferate (van Wezel, A. L. Nature 216 (1967) 64-65 Growth of cell strains and primary cells on microcarriers in homogeneous culture). These particles have to some extent made it easier to culture surface-dependent cells on a large scale.
The most common type of microcarrier consists of spherical carriers made of dextran and modified by derivatisation with positive groups. This makes the cells adhere to the carriers. Another way of making the cells adhere is either to produce the carriers of gelatin or to link gelatin to the surface of dextran particles. Gelatin is made of collagen which is the substance to which cells normally adhere. The carriers that are currently available for cells are not optimal in every respect. These carriers are often homogeneous, that is the cells can only adhere/grow on their surface. As a result, the surface available for cell adhesion/cell growth will be limited to the surface area of the carriers. Furthermore, the cells can only adhere/grow in two dimensions in comparison to normally three dimensions in vitro. Another limitation of prior-art systems is that when the carriers are used for culture in culture vessels the cells will be damaged by the forces caused by the stirring system.
To some extent this has already been solved by preparing particles having a great number of encased cavities by means of an emulsion method (Kjell Nilsson and Klaus Mosbach, Swedish Patent 8504764-5, Macroporous particles, method for its production and use of the same). This patent specification discloses how particles having a great number of encased cavities can be prepared by adding to an aqueous solution of the matrix material a solid, liquid or gaseous cavity-forming compound. After the particles have formed by dispersion in a water-insoluble dispersing agent, the matrix is made water-insoluble by cooling, covalent crosslinking or polymerisation. The cavity-forming compound is removed to obtain the encased cavities.
The particles can be used as ion exchangers, gel filter media, chromatography media and microcarriers in cell culture. The matrix is made of protein, polysaccharide or polyacrylamide.
The invention according to Swedish Patent 8504764-5 provided particles in which some of the cavities of the particles were available for cell adhesion/cell growth. It has however been found that the thus-obtained particles were not optimal in some respects. Optimally all the cavities are interconnected so that a continuous porous phase and a continuous matrix phase are obtained. In the present invention, this state was unexpectedly obtained in the case of gelatin by a combination of emulsifier and solvent.
In addition, this state has made it possible to pre-pare both particles and other three-dimensional shapes. This phase separation must be stable on a micro-level for the length of time necessary for the preparation of the desirable shapes. Furthermore the phase separation must not result in a separation of the phases on a macro-level since this yields shapes without porosity.
USPTO Backgrounds
USPTO Backgrounds