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3D LCMS using C18 functionality in all three dimensions (HFBA-pH10-Formic Acid)

ABSTRACT: 3D LCMS using C18 functionality in all three dimensions (HFBA-pH10-Formic Acid). 1D LC-MS, 90 minutes; 2D LC-MS, 21 fractions x 90 minutes; 3D LC-MS, 126 fractions x 90 minutes. Jurkat cell digest.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Oleg Krokhin

PROVIDER: MSV000079480 | MassIVE | Mon Jan 25 05:24:00 GMT 2016

REPOSITORIES: MassIVE

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Project description:Development of systems allowing the maintenance of native properties of mesenchymal stromal cells (MSC) is a critical challenge for studying physiological functions of skeletal progenitors, as well as towards cellular therapy and regenerative medicine applications. Conventional stem cell culture in monolayer on plastic dishes (2D) is associated with progressive loss of functionality, likely due to the absence of a biomimetic microenvironment and the selection of adherent populations. Here we demonstrate that 2D MSC expansion can be entirely bypassed by culturing freshly isolated bone marrow cells within the pores of 3D scaffolds in a perfusion-based bioreactor system, followed by enzymatic digestion for cell retrieval. The 3D-perfusion system supported MSC growth while maintaining cells of the hematopoietic lineage, and thus generated a cellular environment mimicking some features of the bone marrow stroma. As compared to 2D-expansion, sorted CD45- cells derived from 3D-perfusion culture after the same time (3 weeks) or a similar extent of proliferation (7-8 doublings) maintained a 4.3-fold higher clonogenicity and exhibited a superior differentiation capacity towards all typical mesenchymal lineages, with similar immunomodulatory function in vitro. Transcriptomic analysis performed on MSC from 5 donors validated the robustness of the process and indicated a reduced inter-donor variability as well as a significant upregulation of multipotency-related gene clusters following 3D-perfusion as compared to 2D expansion. The described system offers a model to study how factors of a 3D engineered niche may regulate MSC function and, by streamlining conventional labor-intensive processes, is prone to automation and scalability within closed bioreactor systems. Nucleated cells were isolated from 5 fresh human bone marrow aspirates by means of red blood cells lyses buffer and then were seeded into a 3D perfusion bioreactor system using a pure hydroxyapatite 3D scaffold and in conventional Petri dishes (2D). After culture for 19 days, cells from both systems were enzymatically retrieved and sorted using anti-CD45-coated magnetic beads. Total RNA was extracted from CD45- cells, QCed and hybridized to Affymetrix microarrays.

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3D LCMS using C18 functionality in all three dimensions (HFBA-pH10-Formic Acid)

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