Project description:Biotechnology-derived therapeutics manufacturing is highly regulated to assure product quality, safety and efficacy. Process conditions are closely monitored as they can influence product characteristics. Culture of mammalian cells at different scales is a vital part of biomanufacturing. Currently, scale up of bioreactors is largely done based on engineering parameters such as oxygen transfer and mixing characteristics. There is a lack of genomic translational studies in mammalian cell culture scale-up that can help delineate measurable cellular attributes for improved process understanding. These could be used for quantifying physiological response of cells due to changes in bioreactor environment. In this study, we identified 18 process-sensitive sentinel genes using microarray statistical analysis, which we further verified by qRT-PCR. These were differentially expressed transcripts between a typical 5 liter bench-scale bubble-aerated and impeller-agitated bioreactor and novel 35 mL high-throughput minibioreactors. Using expression changes and biochemical-pathway guidance of these sentinel genes, we were able to tune engineering parameters such that the improved scale-down resulted in both process parameter and sentinel gene profile convergence between the two systems, further solidifying the functionality evidence of these sentinel genes. This study serves as a starting point for developing qRT-PCR assays as comparability metrics that could be performed near-at-line during the cell culture process itself leading to enhanced process control. Additional broad applications of sentinel genes could be to validate different manufacturing facilities for the same product, and allow better process definition by fingerprinting the manufacturing process for more rapid biogenerics and vaccine approvals. We ran consecutive bioreactor (5L and HTCB) runs, each with an independent vial thaw, to achieve multiple biological replicates per time-point. Bioreactors were sampled approximately every 12 hours for RNA extraction. For the 5L bioreactors, microarray samples were run for day 1 (n=2), day 2 (n=2), day 3 (n=3), and day 3.5 (n=3). Here 2 or 3 of the three biological replicates run for each time-point were included in the analysis, based on >70% genes found. For the High-Throughput Controlled Minibioreactors (HTCB), microarray samples were run for day 1 (n=3), day 2 (n=3), day 3 (n=3), day 4 (n=2), and day 4.5 (n=2). Here 2 to 4 of the 4 biological replicate runs were included in the analysis, based on >70% genes found. We define early exponential as day 1, peak exponential as day 2 and day 3 and late stationary as day 3.5.

Project description:Cutaneous melanoma first metastasizes into sentinel lymph nodes that control the lymphatic drain from the area of the primary tumor. This observation is used clinically for melanoma patients with primary melanomas thicker than 1mm (tumor stage ≥T2a), for these patients sentinel lymph node biopsy has become an important and routinely performed diagnostic procedure. The importance of sentinel node analysis is reflected by a significant better prognosis of melanoma patients with tumor free sentinel nodes compared to patients with metastatic sentinel nodes. Although intensively studied, not much is known about mechanisms responsible for the development of melanoma metastasis.To analyze gene expression in mouse SLNs of M24met tumor bearing animals as compared to tumor free control animals, SLNs were taken at different time points and analyzed for the presence of human M24met to classify SLNs into control, negative, macro metastatic SLN. After categorized SLNs were subjected to microarray analysis. To analyze gene expression in mouse SLNs of human M24met tumor bearing animals as compared to tumor free control animals, SLNs were taken at different time points and analyzed for the presence of human M24met to classify SLNs into control, negative, macro metastatic SLN. Briefly, explanted SLNs were stored in RNA later (Ambion, Austin, TX) and DNA as well as RNA was extracted using AllPrep DNA/RNA Mini Kit and RNeasy Micro Kit (Qiagen, Valencia, CA) according to manufacturer’s instructions. To detect human cells in mouse SLNs we used a polymerase chain reaction method for the detection of a human-specific 850-bp fragment of the alpha-satellite DNA on human chromosome 17. For analysis of gene expression RNA from control SLN from tumor free animals (control), RNA from tumor negative SLN (negative), and RNA from macro metastatic SLN (positive) from tumor bearing animals was used to analyze gene expression on Mouse Genome 430A 2.0 Arrays (Affymetrix, Santa Clara, CA)

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Sentinel REACT

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