Medium Design for Cell
Culture Processing
Medium, like the food we eat, exerts a fundamental influence on the well-being of cultured cells, profoundly affecting their growth, metabolic activities, and other biological capability. The question of how best to devise culture medium emerges whenever new in vitro cultivation- based science or technology is on the horizon, as occurred three decades ago driven by the revolution in recombinant mammalian cell based biotechnology, and as is occurring now concomitant with the emergence of stem cell science. Cells are the heart of cell technology; however, without proper medium, cell cultivation cannot accomplish process goals. Most cell types share common basic nutritional requirements although their needs for growth factors and cytokines may
Industrial Cell Culture Process 1 . Cell expansion
2 . Production/differentiation
• Cell expansion stages last much longer than production • Medium design for both stages
differ. In the nearly five decades since scientists began to isolate and cultivate cells, the focus of medium design has been to optimize cell growth, maintain growth potential and sustain the differentiated properties in cultures of differentiated cells. With the growing importance of biologics, the focus of medium design has been extended to enhancing production characteristics, such as productivity or product quality. However, even with the focus shifting to production, optimizing medium for cell growth remains important. In fact, the time that cells spend in the production stage in a manufacturing reactor is a comparatively small portion of their life span. A cell spends the majority of its lifespan in growth, yielding progeny to generate a sufficiently large number of cells for producing product. Providing cells with an optimal medium during the expansion stage is critical. Recent advances in stem cell science have spurred renewed interest in elucidating the nutritional needs of cells in culture. Although the fundamental aspects of nutritional requirements of a stem cell are not different from other cell types, their requirements for growth factors, surface matrices, and other microenvironmental factors makes medium design for stem cells far more complex than that for any cell lines used in protein biologics production. Furthermore, stem cell applications require that the stem cell progeny be directed to differentiate to specific lineages, for which the growth factor requirements pose an even greater challenge. Regardless of traditional biologics-based cell technology or stem cell bioprocessing, the culture process will involve both cell expansion and product formation or differentiation. Medium optimization strategy for cell expansion and for production may be rather different. For expansion, the long term healthy state of a cell while proliferating must be safeguarded. In contrast, for production of biologics, the cells are approaching their final stage of utility, and after all products have been released into the medium, cells and product molecules must be separated. Hence, even conditions that might ordinarily hamper
• Classical Medium Design - Optimize Cell Growth • Optimization for production - squeeze cell’s last
productivity out
• Resurgence of media research in stem cell culture • opportunities in growth factors, antogonists, and
growth or harm cells, such as reduced temperature or increased osmolality, are sometimes used. For stem cell processing, or for other cell therapy, the distinction between cell expansion and produc- tion is not as significant. Even though cells are no longer being expanded during the differentiation stage or other final stage of preparation for clini- cal applications, cells must not to be subjected to deleterious conditions. Since the final product is cell mass itself, their survivability and functional capability after the cell culture process is critical. In the following sections we will focus on nutritional needs for cell expansion first, as that is best known. We will then discuss how “optimal” medium for pro- duction compares with that required for growth.
To design an optimal medium for cell growth, it is instructive to examine the chemical environment of their natural niche. The ultimate objectives of medium design are cell expansion, differentiation, and production, not necessarily to reproduce their niche. Understanding their native chemical environment provides us with a starting point from which to devise an environment suited to process goals. The vast majority of cells in the body are not in direct contact with blood, but are surrounded by interstitial fluid. The chemical composition of interstitial fluid, especially the protein and hormone content, varies with tissues. However, the general chemical composition of small molecular weight solutes in interstitial fluid and in intracellular fluid is similar. The low molecular weight solute composition of interstitial fluid bears a few important characteristics. The total osmolarity is around 280- 300 mM (or mOsm). A couple of percentage points of error notwithstanding, the osmolarity can be taken as the sum of molarity of all dissolved species in the fluid. The largest contributor to the final osmolarity is Na+, followed by Cl–. In addition to Na+, other
inorganic species are present; notable are K+, Mg2+,
and Ca2+. However, those positively charged ions are
all present at low concentrations. Since the net charge in a solution must be neutral, the total molarity of positively and negatively charge ions must be equal. In general Cl– concentration is lower than Na+ because
bicarbonate (HCO3–) is also present at ~30 mM
contributing to the negative charge in the solution. For many ion species the interstitial concentration and intracellular concentrations are strikingly different. Both Na+ and Cl– are present outside the cell at a ten-
fold higher concentration than inside the cell, as is Ca2+. In contrast, K+, Mg2+ and PO
43– concentrations
are much higher on the intracellular side. Cells can tolerate deviations from “optimal” conditions for some period of time. The estimated range of non-lethal physiological concentrations of key compounds vary considerably. Keep in mind
Table 1 . Cellular Chemical Environment in vivo
Approximate Concentrations in Cellular Environment Interstitial (mM) Intracellular (mM) Na+ 140 14 K+ 4 .0 140 Ca 2+ 1 .2 0 .01 Mg2+ 0 .7 20 CI-- 108 4 HCO3- 28 .3 10 HPO43-, H 2PO42- 2 11 SO43- 0 .5 1 Amino Acids 2 8 Lactate 1 .2 1 .5 Glucose 56 Protein 02 4
Total Chemical Species
(mmole/L) 301 .8 302 .2
Corrected osmolar activity (mM) 281 .3 281 .3