Screening and optimization of chemically defined media and feeds with integrated and statistical approaches.

The majority of therapeutic proteins are expressed in mammalian cells, predominantly in Chinese Hamster Ovary cells. While cell culture media and feed supplements are crucial to protein productivity, medium optimization can be labor intensive and time-consuming. In this chapter, we describe some basic concepts in medium development and introduce a rational and rapid workflow to screen and optimize media and feeds. The major goal of medium screening is to select a base formulation as the foundation for further optimization, but ironically, the most conventional screening method may actually rule out ideal chemically defined medium candidates. Appropriate cell adaptation is the key to identifying an optimal base medium, particularly when cells were originally cultured in serum-free medium containing recombinant proteins and/or undefined hydrolysates. The efficient workflow described herein integrates the optimization of both medium and feed simultaneously using a Design-of-Experiment (DOE) approach. The feasibility of the workflow is then demonstrated with a case study, in which chemically defined medium and feed were optimized in a single fed-batch study using a high-throughput microbioreactor system (SimCell™), which resulted in improving protein titers three- to sixfold.

Paternal and maternal genomes confer opposite effects on proliferation, cell-cycle length, senescence, and tumor formation.

Loss of imprinting is the silencing of active imprinted genes or the activation of silent imprinted genes, and it is one of the most common epigenetic changes associated with the development of a wide variety of tumors. Here, we have analyzed the effects that global imprinted gene expression has on cell proliferation and transformation. Primary mouse embryonic fibroblasts (MEFs), whose entire genome is either exclusively paternal (androgenetic) or maternal (parthenogenetic), exhibit dramatically contrasting patterns of growth. In comparison with biparental MEFs, andro-genetic proliferation is characterized by a shorter cell cycle, increased saturation density, spontaneous transformation, and formation of tumors at low passage number. Parthenogenetic MEFs reach a lower saturation density, senesce, and die. The maternally expressed imprinted genes p57kip2 and M6P/Igf2r retard proliferation and reduce the long-term growth of MEFs. In contrast, the paternally expressed growth factor Igf2 is essential for the long-term proliferation of all genotypes. Increased Igf2 expression in primary MEFs not only stimulates proliferation, but also results in their rapid conversion to malignancy with tumor formation of short latency. Our results reveal that paternally expressed imprinted genes, in the absence of maternal imprinted genes, predispose fibroblasts to rapid transformation. A potent factor in their transformation is IGF2, which on increased expression results in the rapid conversion of primary cells to malignancy. These results reveal a route by which malignant choriocarcinoma may arise from molar pregnancies. They also suggest that the derivation of stem cells from parthenogenetic embryos, for the purposes of therapeutic cloning, may be ineffective.