{"id":702,"date":"2024-09-24T06:53:16","date_gmt":"2024-09-24T06:53:16","guid":{"rendered":"http:\/\/cetaitdemain.org\/?p=702"},"modified":"2024-09-24T06:53:16","modified_gmt":"2024-09-24T06:53:16","slug":"in-contrast-nanoparticles-stabilized-with-peg-sh-with-molecular-weight-of-20000-da-peg20k-gave-negligible-sers-signals-for-non-stem-cells-fig","status":"publish","type":"post","link":"https:\/\/cetaitdemain.org\/?p=702","title":{"rendered":"\ufeffIn contrast, nanoparticles stabilized with PEG-SH with molecular weight of 20,000 Da (PEG20K) gave negligible SERS signals for non-stem cells (Fig"},"content":{"rendered":"

\ufeffIn contrast, nanoparticles stabilized with PEG-SH with molecular weight of 20,000 Da (PEG20K) gave negligible SERS signals for non-stem cells (Fig. safety of hPSC-based therapies Compound 56 [4C10]. For clinical safety, a highly sensitive and specific quality control assay is required to determine the number of undifferentiated cells in hPSC-derived products. In current practice, cell-based assays such as flow cytometry can detect undifferentiated cells when present at ~0.1% or higher in a mixed cell population [11], which is insufficient sensitivity to ascertain that a cell preparation for transplantation contains a number of hPSCs below the threshold for teratoma formation. Compound 56 A study in mice reported that 104 undifferentiated cells were sufficient to produce tumors [2]. Accordingly, if an estimated 109 cells are required for a single transplantation for heart failure [12], the sensitivity of assays used to detect residual undifferentiated cells needs to be 1 stem cell in a background of 105 cells (0.001%), which is unachievable via flow cytometry. A prevailing method to evaluate the risk of teratoma formation is to inject cell products into SCID mice and evaluate tumor formation after at least 3 months [3, 13C15]. While this method may provide a direct assessment of tumorigenicity, it is highly impractical as a quality-control assay due to its non-quantitative, non-scalable, costly, and time-consuming nature. Therefore, an assay that is fast, highly sensitive, and efficient in detecting a trace number of undifferentiated cells is imperative for assessing the safety of hPSC-derived products. Nanoparticle-based surface-enhanced Raman scattering (SERS) technology is gaining momentum in biomedical applications such as molecular multiplex detection, pathogen and cell detection, and imaging [16C21]. When conjugated with biomolecular targeting ligands, Raman reporter-labelled gold (Au) nanoparticles can be used to detect specific molecules with high specificity and sensitivity [19, 21C23]. SERS detection produces a sharp, fingerprint-like spectral pattern that is distinct from other interference patterns in a complex biological environment. This is uniquely advantageous when detecting a low number of cells, since conventional fluorescence signals may be masked by the scattering signals of background cells [20, 21]. In this study, we developed SERS-based assays targeting the hPSC surface markers stage-specific embryonic antigen-5 (SSEA-5) and TRA-1-60 to detect residual undifferentiated hPSCs with high specificity and sensitivity. Using our newly developed assays, we efficiently detected SSEA-5+ and TRA-1-60+ cells at sensitivities several orders of magnitude higher than flow cytometry assays. As such, these assays represent a rapid, efficient, and economic method for assessing the safety of hPSC-based products for pre-clinical and clinical applications. 2. Materials UBCEP80<\/a> and Methods 2.1. Materials Ultrapure water (18 M cm?1) was used to prepare all aqueous solutions. The following chemicals were used without further purification: 60 nm citrate-stabilized gold nanoparticles (2.61010 particles\/mL) (Ted Pella Inc.), black hole quencher (BHQ) (Biosearch Technologies), PEG-SH (MW = 5,000 and 20,000 Da) (Rapp Polymere, Germany), SSEA-5 IgG1 antibody (Stemcell Technologies), and TRA-1-60 IgM antibody (Millipore). All other reagents were obtained from Sigma-Aldrich at the highest purity available. 2.2. BIDI Reporter Molecule The molecular structure of (E)-2-(2-(5′-(dimethylamino)-2, 2-bithiophen-5-yl) vinyl)-1, 1, 3-trimethyl-1H-benzo[e]indol-3-ium iodide (BIDI) is shown here. The synthesis of BIDI will be reported later in another work. BIDI 1HNMR (DMSO, 500MHz): = 8.61C8.64(d, 1H, C10H6), 8.36C8.38 (d, 1H, C10H6), 8.21C8.23(d, 1H, C2H2), 8.15C8.17(d, 1H, C10H6), 8.04C8.05 (d, 1H, C4H2S), 7.97C7.99(1, H, C2H2), 7.74C7.77(m, 1H, C10H6), 7.63C7.66 (m, 1H, C10H6), 7.56C7.57(d, 1H, C4H2S), 7.37C7.38 (d, 1H, C4H2S), 6.84C6.87 (d, 1H, C10H6), 6.16C6.17 (d, 1H, C4H2S), 4.05(s, 3H, Compound 56<\/a> CH3), 3.08(s, 6H, CH3), 1.98(s, 6H, CH3).MALDI-TOF-MS: m\/z433.0 (M-I?). 2.3. Preparation of.<\/p>\n","protected":false},"excerpt":{"rendered":"

\ufeffIn contrast, nanoparticles stabilized with PEG-SH with molecular weight of 20,000 Da (PEG20K) gave negligible SERS signals for non-stem cells (Fig. safety of hPSC-based therapies Compound 56 [4C10]. For clinical safety, a highly sensitive and specific quality control assay is required to determine the number of undifferentiated cells in hPSC-derived products. In current practice, cell-based …<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[48],"tags":[],"class_list":["post-702","post","type-post","status-publish","format-standard","hentry","category-pao","entry entry-center"],"_links":{"self":[{"href":"https:\/\/cetaitdemain.org\/index.php?rest_route=\/wp\/v2\/posts\/702","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/cetaitdemain.org\/index.php?rest_route=\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/cetaitdemain.org\/index.php?rest_route=\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/cetaitdemain.org\/index.php?rest_route=\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/cetaitdemain.org\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=702"}],"version-history":[{"count":1,"href":"https:\/\/cetaitdemain.org\/index.php?rest_route=\/wp\/v2\/posts\/702\/revisions"}],"predecessor-version":[{"id":703,"href":"https:\/\/cetaitdemain.org\/index.php?rest_route=\/wp\/v2\/posts\/702\/revisions\/703"}],"wp:attachment":[{"href":"https:\/\/cetaitdemain.org\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=702"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/cetaitdemain.org\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=702"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/cetaitdemain.org\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=702"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}