Genomic barcoding for clonal diversity monitoring and control in cell-based complex antibody production.

Engineered mammalian cells are key for biotechnology by enabling broad applications ranging from in vitro model systems to therapeutic biofactories. Engineered cell lines exist as a population containing sub-lineages of cell clones that exhibit substantial genetic and phenotypic heterogeneity. There is still a limited understanding of the source of this inter-clonal heterogeneity as well as its implications for biotechnological applications. Here, we developed a genomic barcoding strategy for a targeted integration (TI)-based CHO antibody producer cell line development process. This technology provided novel insights about clone diversity during stable cell line selection on pool level, enabled an imaging-independent monoclonality assessment after single cell cloning, and eventually improved hit-picking of antibody producer clones by monitoring of cellular lineages during the cell line development (CLD) process. Specifically, we observed that CHO producer pools generated by TI of two plasmids at a single genomic site displayed a low diversity (< 0.1% RMCE efficiency), which further depends on the expressed molecules, and underwent rapid population skewing towards dominant clones during routine cultivation. Clonal cell lines from one individual TI event demonstrated a significantly lower variance regarding production-relevant and phenotypic parameters as compared to cell lines from distinct TI events. This implies that the observed cellular diversity lies within pre-existing cell-intrinsic factors and that the majority of clonal variation did not develop during the CLD process, especially during single cell cloning. Using cellular barcodes as a proxy for cellular diversity, we improved our CLD screening workflow and enriched diversity of production-relevant parameters substantially. This work, by enabling clonal diversity monitoring and control, paves the way for an economically valuable and data-driven CLD process.

Burst activity plays no role in the field-to-field variability and rate remapping of grid cells.

Grid cells in rodent medial entorhinal cortex are thought to play a key role for spatial navigation. When the animal is freely moving in an open arena the firing fields of each grid cell tend to form a highly regular, hexagonal lattice spanning the environment. However, firing rates vary from field to field and change under contextual modifications, whereas the field locations shift at most by a small amount under such "rate remapping." The observed differences in firing rate could reflect overall activity changes or changes in the detailed spike-train statistics. As these two alternatives imply distinct neural coding schemes, we investigated whether temporal firing patterns vary from field to field and whether they change under rate remapping. Focusing on short time scales, we found that the proportion of bursts compared to all discharge events is similar in all firing fields of a given grid cell and does not change under rate remapping. For each cell, mean firing rates with bursts are proportional to mean firing rates without bursts. However, this ratio varies across cells. Additionally, we looked at how rate remapping relates to entorhinal theta-frequency oscillations. Theta-phase coding was preserved despite firing-rate changes from rate remapping but we did not observe differences between the first and second half of the theta cycle, as had been reported for CA1. Our results indicate that both, the heterogeneity between firing fields and rate remapping, are not due to altered firing patterns on short time scales but reflect location-specific changes at the firing-rate level.