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Sandlin, Rebecca

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Sandlin

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Rebecca

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Sandlin, Rebecca
Author's Publications: search.filters.isAuthorOfPublication.86803a30-5859-4757-9042-3eb6d59c9dda

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    Ultra-fast vitrification of patient-derived circulating tumor cell lines
    (Public Library of Science, 2018) Sandlin, Rebecca; Wong, Keith H. K.; Tessier, Shannon; Swei, Anisa; Bookstaver, Lauren D.; Ahearn, Bennett E.; Maheswaran, Shyamala; Haber, Daniel; Stott, Shannon; Toner, Mehmet
    Emerging technologies have enabled the isolation and characterization of rare circulating tumor cells (CTCs) from the blood of metastatic cancer patients. CTCs represent a non-invasive opportunity to gain information regarding the primary tumor and recent reports suggest CTCs have value as an indicator of disease status. CTCs are fragile and difficult to expand in vitro, so typically molecular characterization must be performed immediately following isolation. To ease experimental timelines and enable biobanking, cryopreservation methods are needed. However, extensive cellular heterogeneity and the rarity of CTCs complicates the optimization of cryopreservation methods based upon cell type, necessitating a standardized protocol. Here, we optimized a previously reported vitrification protocol to preserve patient-derived CTC cell lines using highly conductive silica microcapillaries to achieve ultra-fast cooling rates with low cryoprotectant concentrations. Using this vitrification protocol, five CTC cell lines were cooled to cryogenic temperatures. Thawed CTCs exhibited high cell viability and expanded under in vitro cell culture conditions. EpCAM biomarker expression was maintained for each CTC cell line. One CTC cell line was selected for molecular characterization, revealing that RNA integrity was maintained after storage. A qPCR panel showed no significant difference in thawed CTCs compared to fresh controls. The data presented here suggests vitrification may enable the standardization of cryopreservation methods for CTCs.
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    The Role of Physical Stabilization in Whole Blood Preservation
    (Nature Publishing Group, 2016) Wong, Keith H. K.; Sandlin, Rebecca; Carey, Thomas R.; Miller, Kathleen L.; Shank, Aaron T.; Oklu, Rahmi; Maheswaran, Shyamala; Haber, Daniel; Irimia, Daniel; Stott, Shannon; Toner, Mehmet
    The rapid degradation of blood ex vivo imposes logistical limitations on the utilization of blood-borne cells in medical diagnostics and scientific investigations. A fundamental but overlooked aspect in the storage of this fluid tissue is blood settling, which induces physical stress and compaction, aggregates blood cells, and causes collateral damage due to leukocyte activation. Here we show that the polymer Ficoll 70 kDa stabilized blood samples and prevented blood settling over the course of 72 hours, primarily by inhibiting depletion-mediated red blood cell aggregation. Physical stabilization decreased echinocyte formation, improved leukocyte viability, and inhibited the release of neutrophil elastase—a marker of neutrophil extracellular trap formation. In addition, Ficoll-stabilized blood was compatible with common leukocyte enrichment techniques including red blood cell lysis and immunomagnetic purification. This study showed for the first time that blood settling can be prevented using polymers and has implications in diagnostics.
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    Whole blood stabilization for the microfluidic isolation and molecular characterization of circulating tumor cells
    (Nature Publishing Group UK, 2017) Wong, Keith H. K.; Tessier, Shannon; Miyamoto, David; Miller, Kathleen L.; Bookstaver, Lauren D.; Carey, Thomas R.; Stannard, Cleo J.; Thapar, Vishal; Tai, Eric C.; Vo, Kevin D.; Emmons, Erin S.; Pleskow, Haley M.; Sandlin, Rebecca; Sequist, Lecia; Ting, David; Haber, Daniel; Maheswaran, Shyamala; Stott, Shannon; Toner, Mehmet
    Precise rare-cell technologies require the blood to be processed immediately or be stabilized with fixatives. Such restrictions limit the translation of circulating tumor cell (CTC)-based liquid biopsy assays that provide accurate molecular data in guiding clinical decisions. Here we describe a method to preserve whole blood in its minimally altered state by combining hypothermic preservation with targeted strategies that counter cooling-induced platelet activation. Using this method, whole blood preserved for up to 72 h can be readily processed for microfluidic sorting without compromising CTC yield and viability. The tumor cells retain high-quality intact RNA suitable for single-cell RT-qPCR as well as RNA-Seq, enabling the reliable detection of cancer-specific transcripts including the androgen-receptor splice variant 7 in a cohort of prostate cancer patients with an overall concordance of 92% between fresh and preserved blood. This work will serve as a springboard for the dissemination of diverse blood-based diagnostics.