Towards a Chemically Defined Medium for Sf-9 Cell Culture: Micronutrients Reduce Dependence on Yeast Extract

Spodoptera frugiperda clonal isolate 9 (Sf-9) insect cells in conjunction with recombinant baculovirus are an industrially relevant system for producing biologics. Sf-9 cells are capable of robust high-density growth in single cell suspension. However, unlike many other continuous cell lines, Sf-9 cell culture media remains undefined. Typically, the growth medium requires undefined hydrolysate supplementation (most often yeast extract) in order to support cell proliferation. The lack of chemical definition makes medium and process optimization difficult, leads to batch-to-batch variability, and potentially affects downstream processing. This work aims to combine available information on the composition of yeast extract and the composition of media for other cell lines to reduce the concentration of undefined components (yeast extract) in the medium and elucidate the effects of micronutrient compounds. Utilizing an in-house medium based on the classic IPL-41 medium with yeast extract as the only undefined component, several steps were taken towards chemical definition. Through fortifying the trace metal and vitamin content in the medium and the addition of 11 micronutrients, the yeast extract content was successfully reduced 10-fold (from 4 g/L to 0.4 g/L). Without medium fortification and micronutrient addition, the cells were incapable of growth at low yeast extract concentration. Sf-9 cells adapted to this new medium were capable of long-term consistent growth. Micronutrients of key importance in this medium were identified as glycine betaine, ascorbic acid, and the polyamine putrescine. The presence of glycine betaine (1 mM), ascorbic acid (10 uM), and putrescine (10 uM) improved maximum cell density by 32%, 41%, and 28% respectively in the low yeast extract medium. The role of these micronutrients could be properly investigated only after medium enhancement and yeast extract reduction. Further, this medium was found to be cost-effective compared to commercially available alternatives and the potential for added cost-savings related to lipid supplementation was identified. This enhanced low yeast extract medium could allow for micronutrient and other component investigation with less convolution and is particularly applicable to designed compound screening experiments (e.g. Plackett-Burman). Identification and supplementation of additional required components provided solely by the yeast extract could lead to a chemically defined medium

Comprehensive analysis of cancer-associated somatic mutations in class I HLA genes

Detection of somatic mutations in human leukocyte antigen (HLA) genes using whole-exome sequencing (WES) is hampered by the high polymorphism of the HLA loci, which prevents alignment of sequencing reads to the human reference genome. We describe a computational pipeline that enables accurate inference of germline alleles of class I HLA-A, B and C genes and subsequent detection of mutations in these genes using the inferred alleles as a reference. Analysis of WES data from 7,930 pairs of tumor and healthy tissue from the same patient revealed 298 nonsilent HLA mutations in tumors from 266 patients. These 298 mutations are enriched for likely functional mutations, including putative loss-of-function events. Recurrence of mutations suggested that these \u27hotspot\u27 sites were positively selected. Cancers with recurrent somatic HLA mutations were associated with upregulation of signatures of cytolytic activity characteristic of tumor infiltration by effector lymphocytes, supporting immune evasion by altered HLA function as a contributory mechanism in cancer

Antiviral Activity of Contemporary Contact Lens Care Solutions against Two Human Seasonal Coronavirus Strains

Background: Given that reports have suggested SARS-CoV-2 can be transmitted via conjunctiva, the ability of contact lens (CL) care products to reduce the infectiousness of two seasonal human coronavirus (HCoV) (HCoV-229E and HCoV-OC43) surrogates for SARS-CoV-2 was investigated. Methods: Biotrue and Boston Simplus (Bausch&Lomb), OPTI-FREE Puremoist and Clear Care (Alcon), and cleadew and cleadew GP (Ophtecs) were tested. Their ability to inactivate HCoV was evaluated using contact times of 4 and 6 h as well as 1% and 10% of virus inoculum. Results: Non-oxidative systems (Biotrue, Boston Simplus, and OPTI-FREE) did not exhibit a significant log10 reduction compared to controls for the two viral strains for either incubation time (all p > 0.05) when 10% tests were performed. For the 1% test, while Boston Simplus and OPTI-FREE exhibited a significant log10 reduction of both HCoV-229E (after 6 h) and HCoV-OC43 (after either 4 or 6 h incubation), those products showed less than 1 log10 reduction of the two infectious viruses. Oxidative systems based on hydrogen peroxide or povidone-iodine showed a significant log10 reduction compared with the controls for both HCoV-229E and HCoV-OC43 in all tested conditions (all p < 0.01). Clear Care led to virus inactivation to below the limit of quantification for tests performed with 1% of inoculum after 6 h incubation, while cleadew and cleadew GP led to inactivation of the two viruses to below the limit of quantification in all tested conditions. Conclusion: Oxidative CL disinfection systems showed significant virucidal activity against HCoV-229E and HCoV-OC43, while non-oxidative systems showed minimal ability to inactivate the HCoV species examined