Cell line or cell culture expansion involves serial passaging of cell populations from smaller cultures or volumes to larger ones, ultimately arriving at some number of usable cells. The process is similar whether the cells' ultimate destiny is production (e.g. Chinese hamster ovary cells), cell-based assays (which often involved harvested or induced primary cells), or therapy (typically stem cell-derived). Expansion protocols for production- and assay-purposed cells can be well established if these cells are phenotypically and genetically stable, their behavior is predictable, and they are readily expanded into very large volumes and high cell densities. Developers have many additional things to think about with cells ultimately destined for re-infusion into patients.
Establishing, maintaining the phenotype
Cell culture generally has two objectives: Obtaining cells with the desired characteristics and producing enough of them to get the job done. For stock production of cells like CHO, early development work tends to focus on establishing the phenotype through evolutionary processes, cell line engineering, or a combination of the two. Only after the desired characteristics are established and validated do investigators turn to optimizing conditions that promote culture health and expansion.
Establishing and maintaining the phenotype is a greater challenge for cultures derived either from primary cells or from induced pluripotent stem cells. Primary cells are intrinsically limited in terms of expansion, and their donor age or passage number can affect their behavior. Moreover, primary cells are inevitably heterogeneous. The progeny of many colony founding cells compete for dominance. This can be a stocastic process. As a result, reliable manufacturing often requires some form of systematic clone selection. For induced pluripotent stem cell-derived cultures, individual clones are often biologically different, requiring systematic clones selection. Moreover, progenitor cells can take any number of different turns, differentiation becomes the signature event in establishing and maintaining the cell line.
“Control of starting materials through clone selection can sometimes be accomplished using sorting or surface markers. However, often, clone selection for both primary cell cultures or iPS cell sources can only be accomplished by performance-based selection in culture,” says George Muschler, M.D., Chief Science Officer, Cell X Technologies.
Cell X Technologies has developed automated systems that enable non-invasive live cell imaging and automated biopsy, picking or weeding of mixed cell populations during primary culture and downstream expansion, providing controls over source materials and quantitative performance metrics.
"For these types of cells the relevant processes are proliferation to deliver viable quantities, and differentiation or transdifferentiation to establish the cells' distinct therapeutic qualities," says Rodrigo Santos, Director of Cell Technologies at Mogrify. "Each cell line presents its own set of challenges, such as the shortening of telomeres and loss of functionality as a result of proliferation, or low differentiation efficacy and insufficient maturity of product cells."
Mogrify has developed a suite of technologies that identify the transcription factors and culture medium conditions required to directly convert any target cell type from any source cell type, a process known as transdifferentiation. Possibilities include the re-programming of mature cells into other types of mature cell or even back to progenitor status.
Many development-stage cell lines don't get very far along in this process due to fundamental challenges associated with cell culture, for example reproducing the in vivo microenvironment in vitro. "Included in these considerations are the cells' native chemical environment, the effects of neighboring cells, regulatory and self-regulatory signals, and others. Cultures that fail to meet these requirements will fail to thrive," Santos adds. As these products progress toward the clinic, developers will also need to focus on culture conditions that are robust, reproducible, and comply with Good Manufacturing Practices.
It comes down to niche
Cell growth depends on a multitude of variables. "Our bodies, the source of cultured cells, are a symphony of nutrients including salts, minerals, enzymes, hormones, and amino acids, all at their own concentrations and states of composition," says Mary Kay Bates, Senior Global Applications Scientist at Thermo Fisher Scientific. In vivo, cells exist within unique microenvironments as well, with temperature, blood flow, gases, microorganisms, pH, pressure, scaffolding, and chemical signaling all exerting a combined and unique niche. "That is why it took many decades of trial and error before cells were successfully cultured outside the body at all, and why so many factors can negatively affect cell growth."
Examples of common inhibitory factors include fluorescent and UV light, volatile organic compounds, desiccation, vibration, too many cells in a dish, too few cells in a dish, the wrong kind of dish, and, of course, microbiological contamination. "So even when cells are growing nicely and the technician is trained and experienced, factors we may not have identified can cause cell cultures to crash."
Moreover, success in cell culture involves more than just keeping cells alive: "It’s about the goal in growing cells in the first place," Bates adds. "If the goal is to understand biological function under the control of factors that are not easily reproduced in culture, for example blood pressure or signals from distant cells, investigators may not get the answer they are looking for."
For example, if the goal is to produce a protein like insulin or an antibody, expression levels or yields may be affected by these and other elements that are difficult to reproduce in vitro, such as mutations induced by artificial conditions, by the age of the cells, general cell culture conditions. Additional factors include the age or constituents in the growth medium, or stresses cells experience as a consequence of the size and shape of culture vessels or modifications to induce or inhibit cellular attachment.
"There are entire textbooks written on these issues, and often the questions that arise are too broad and involve too much uncertainty to come up with solid answers about what is really happening," Bates says.
Cell therapy today is clearly in its developmental or discovery stage, where the focus is identifying usable therapeutic phenotypes. As more such therapies become commercialized, however, the emphasis will switch to production or manufacturability, the basis of which is expansion.
These expansion protocols will come, but likely at a cost. "Inducing cell proliferation can lead to some loss in functionality and subsequently to a decrease in the cells' quality and viability," Santos says. "Therefore, it is crucial to begin considering both the selection and validation of the optimal cell source (starting materials) and the scalability of the therapeutic product in the earliest development stages."
One strategy Santos suggests is to select a renewable cell source, specifically established iPSC lines for which both differentiation and expansion protocols are now routine. "However, the challenge in bringing stem cell-derived therapies to the clinic is the need to recapitulate the natural biological pathways required to differentiate stem cells into desired cell types."