The alternative approach is to engineer the fermentation process so that cells can attach themselves to the surface of the
culture vessel or some other support.
Anchorage-dependent cell culture
, as it is called, takes a number of forms. In the simplest forms, cells are attached to beads or enclosed in microcarriers
that are allowed to float free in the medium. In other cases, cells might be attached to the wall of the vessel, to a matrix,
gel, ceramic cartridge, or to some other structure (such as the tubes of a hollow-fiber system).
Confining cells to a support protects them from mechanical stresses. Sometimes other sorts of cells can be added to the support
to provide catalytic activity. The chemical composition of solid supports can create favorable microenvironments. Animal cells
can achieve higher densities in attachment culture than in suspension.
Cells can be immobilized in several ways. Collagen-based beads or polymer agents protect against shearing. Gels or solid matrices
can simplify downstream processing, and in stirred or aerated systems, they can offer some protection from air sparging.
At the laboratory scale, major concerns are cell viability, mass transport, and the size of membrane pores. At the industrial
scale, issues such as price, complexity, and reliability come up. Problems can include leakage and polymer toxicity.
Perfusion culture is made simpler when cells are retained in place using a mesh screen. Some perfusion cell culture systems
are based on ceramic matrices that immobilize cells. The technique can immobilize nonadherent cells, protecting them from
. Cell-containing beads are a useful immobilization method for both anchorage-dependent and independent cells because they
provide sparge protection. Encapsulated cells can hold and concentrate product, but beads may not retain their integrity for
long-term culture. Although capsule membranes allow small molecules such as nutrients and oxygen to diffuse through, limitations
in mass transfer can lower cell viability and contaminate or degrade products. High-molecular-weight products are kept within
the capsules, and low-molecular-weight products diffuse out. The cost of encapsulation can be a disadvantage, as can oxygen
transfer limitations at large scales. And encapsulated cells may not receive optimal nutrients.
are commonly used in attached cell systems. Compared to microcapsules, microporous beads (to which cells attach themselves)
are easier to use and scale up. Adsorption is the simplest system for attaching cells to a support. Cells are mixed with beads
(for example) and attach themselves to their surfaces. But adsorbed cells are not protected from shear forces.
There are several ways to attach cells to a support. Covalent attachment involves a chemical bond between the cells and their
support. Leakage is minimized, but chemicals can affect cell viability. Again, no shear protection is provided. With ionic
to covalent crosslinking, a cell suspension is treated with polymers that form bridges between the cells, making them aggregate
loosely. The resulting cloudy flocs are not particularly stable, and cell leakage is still a problem, but additives can improve
the situation. Entrapment offers a gentle solution to many attachment problems. Cells are mixed with polymers or monomers
to form a gel that encases them. Leakage is reduced, and many cells can be loaded.
A Medium for Growth
Cells deteriorate, die, and disintegrate (lyse) when they get too few nutrients. Nutrients are provided to cultivated cells
in the form of a medium.
Different kinds of cells require different media, and vendors offer preformulated media designed for all of the cells widely
used in bioprocessing. In addition to the nutritive elements, media sometimes contain additives designed to improve the fermentation
process. Pluronic F68, for instance, is used to make cell membranes more resistant to shear forces. Polyethylene glycol, polypropylene
glycol, or silicon-based surfactants may be used to reduce foaming.