Drug Discovery
Figure 4 Large-scale stem cell
production requires scalable, optimised solutions that impact every phase of the development pipeline
address each phase of the development pipeline (Figure 4). The physical system in which the cells are grown must also be able to:
l Minimise variability in the cell population. l Enable harvesting and formulation without damaging cells. l Incorporate processes to ensure cell viability during storage, transport and administration to the patient.
An important element in these integrated sys- tems is the substrate upon which the stem cells grow. Anchorage-dependent stem cells must affix to a solid surface when grown in vitro. Currently, microcarriers are the only convenient way to scale up cultures of stem cells in a stirred bioreactor sys- tem. Unfortunately, the use of microcarriers pres- ents a number of challenges. Small particulates or ‘fines’ are often generated during the microcarrier manufacturing process. These fines can end up in the culture system. Fines can also result from beads being crushed during the cell harvest process. Filtration of the stem cell cultures cannot remove these particulates, so they are co-purified with the cells. The presence of foreign particulate matter – such as microcarrier fines – is unacceptable for injectable products.
In addition, the ability of a stem, or derivative, cell, to grow on a microcarrier is influenced by how strongly the cells bind to the surface. If the surface chemistry causes the cells to adhere too tightly to the microcarriers, the cells’ ability to grow and divide may be limited. The strength of adherence can also influence cellular phenotypes, as well as the success of the harvesting process. Cells must be recovered at the end of the scale-up process. The more tightly the cells are bound to the microcarrier surface, the more difficult it can be to remove the cells. Repeated trypsin digests can damage the cells, cause phenotypic changes and reduce viability.
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It is likely that expansion of different stem cell lines will require a slightly different microcarrier surface to ensure optimal growth. A less than opti- mal surface may lead to an undesired cell popula- tion. This is especially true when differentiation is carried out on the microcarrier because the process is quite susceptible to both elasticity and surface chemistry.
A number of technological solutions are being developed including scaffolds made from a variety of biocompatible materials such as polyacrylate, hydroxyapitite and polylactic acid as well as natu- ral materials including silk, chitin, collagen and polyglycolic acids such as hyaluronic acid. Scaffolds made from fibrous mats and hollow fibres, have been shown applicable with stem cells because they afford the ability to culture with per- fusion, better simulating the natural environment. Alternatives to microcarriers are in the early stages of development and are not yet feasible for large-scale cultures. Techniques are being devel- oped to culture these cells in suspension culture without the need for a surface. In these systems, cells clump into small aggregates or are encapsu- lated in gel microdroplets. Cell densities can be increased limited by availability of nutrients. This approach has been demonstrated with murine stem cells, but has yet to be proven with human cells which are less robust.
The final step in the development pipeline – har- vesting and packaging of live cells – is also a focus of intense development efforts.
Existing centrifugation and filtration technologies are not optimised for the harvest and recovery of live cells. Centrifugation is often used to collect cells for research applications. However, centrifugation is not always practical for the collection of large num- bers of stem cells. Centrifugation is typically not a closed system. In addition, shear forces can damage cells. After cells are harvested, they must be rapidly concentrated, the media washed away with buffer solution, and packaged into containers for freezing
Drug Discovery World Spring 2011
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