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Tag-free microfluidic separation of cells against multiple markers.

ABSTRACT: Conventional cell separation against multiple markers generally requires the attachment of antibody tags, typically fluorescent or magnetic, to selected cell types in a heterogeneous suspension. This work describes how such separation can be accomplished in a series of microfluidic systems without the need for such tags. Two capture stages containing antibody-functionalized alginate hydrogels are utilized for the isolation of CD34+ and Flk1+ cells from untreated, whole human blood. The capture-release capability of these degradable coatings is harnessed by a mixing chamber and a simple valving system such that the suspension emerging from the first capture stage is prepared for the second capture stage for further enrichment. With this configuration, we demonstrate the isolation of CD34+/Flk1+ endothelial progenitor cells from blood enabled by the depletion of CD34+/Flk1-hematopoietic stem cells population. This ability to achieve isolation of cells against multiple markers in an untagged separation method is of particular significance in applications involving cell implantation-based therapeutics including tissue engineering and molecular analysis.

SUBMITTER: Hatch A

PROVIDER: S-EPMC3352992 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

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Tag-free microfluidic separation of cells against multiple markers.

Hatch Adam A Pesko Danielle M DM Murthy Shashi K SK

Analytical chemistry 20120502 10

Conventional cell separation against multiple markers generally requires the attachment of antibody tags, typically fluorescent or magnetic, to selected cell types in a heterogeneous suspension. This work describes how such separation can be accomplished in a series of microfluidic systems without the need for such tags. Two capture stages containing antibody-functionalized alginate hydrogels are utilized for the isolation of CD34+ and Flk1+ cells from untreated, whole human blood. The capture-r ...[more]

PMID: 22519841

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Project description:BackgroundsDetecting free cancer cells from ascites and peritoneal lavages is crucial for diagnosing gastric cancer (GC). However, traditional methods are limited for early-stage diagnosis due to their low sensitivity.MethodsA label-free, rapid, and high-throughput technique was developed for separating cancer cells from ascites and peritoneal lavages using an integrated microfluidic device, taking advantage of dean flow fractionation and deterministic lateral displacement. Afterward, separated cells were analyzed using a microfluidic single-cell trapping array chip (SCTA-chip). In situ immunofluorescence for EpCAM, YAP-1, HER-2, CD45 molecular expressions, and Wright-Giemsa staining were performed for cells in SCTA-chips. At last, YAP1 and HER-2 expression in tissues was analyzed by immunohistochemistry.FindingsThrough integrated microfluidic device, cancer cells were successfully separated from simulated peritoneal lavages containing 1/10,000 cancer cells with recovery rate of 84.8% and purity of 72.4%. Afterward, cancer cells were isolated from 12 patients' ascites samples. Cytological examinations showed cancer cells were efficiently enriched with background cells excluded. Afterwards, separated cells from ascites were analyzed by SCTA-chips, and recognized as cancer cells through EpCAM+/CD45- expression and Wright-Giemsa staining. Interestingly, 8 out of 12 ascites samples showed HER-2+ cancer cells. At last, the results through a serial expression analysis showed that YAP1 and HER-2 have discordant expression during metastasis.InterpretationMicrofluidic Chips developed in our study could not only rapidly detect label-free free GC cells in ascites and peritoneal lavages with high-throughput, they could also analyze ascites cancer cells at the single-cell level, improving peritoneal metastasis diagnosis and investigation of therapeutic targets.FundingThis research was supported by National Natural Science Foundation of China (22134004, U1908207, 91859111); Natural Science Foundation of Shandong Province of China (ZR2019JQ06); Taishan Scholars Program of Shandong Province tsqn (201909077); Local Science and Technology Development Fund Guided by the Central Government (YDZX20203700002568); Applied Basic Research Program of Liaoning Province (2022020284-JH2/1013).

Project description:Circulating tumor cells (CTCs) have significant implications in both basic cancer research and clinical applications. To address the limited availability of viable CTCs for fundamental and clinical investigations, effective separation of extremely rare CTCs from blood is critical. Ferrohydrodynamic cell separation (FCS), a label-free method that conducted cell sorting based on cell size difference in biocompatible ferrofluids, has thus far not been able to enrich low-concentration CTCs from cancer patients' blood because of technical challenges associated with processing clinical samples. In this study, we demonstrated the development of a laminar-flow microfluidic FCS device that was capable of enriching rare CTCs from patients' blood in a biocompatible manner with a high throughput (6 mL h-1) and a high rate of recovery (92.9%). Systematic optimization of the FCS devices through a validated analytical model was performed to determine optimal magnetic field and its gradient, ferrofluid properties, and cell throughput that could process clinically relevant amount of blood. We first validated the capability of the FCS devices by successfully separating low-concentration (?100 cells per mL) cancer cells using six cultured cell lines from undiluted white blood cells (WBCs), with an average 92.9% cancer cell recovery rate and an average 11.7% purity of separated cancer cells, at a throughput of 6 mL per hour. Specifically, at ?100 cancer cells per mL spike ratio, the recovery rates of cancer cells were 92.3 ± 3.6% (H1299 lung cancer), 88.3 ± 5.5% (A549 lung cancer), 93.7 ± 5.5% (H3122 lung cancer), 95.3 ± 6.0% (PC-3 prostate cancer), 94.7 ± 4.0% (MCF-7 breast cancer), and 93.0 ± 5.3% (HCC1806 breast cancer), and the corresponding purities of separated cancer cells were 11.1 ± 1.2% (H1299 lung cancer), 10.1 ± 1.7% (A549 lung cancer), 12.1 ± 2.1% (H3122 lung cancer), 12.8 ± 1.6% (PC-3 prostate cancer), 11.9 ± 1.8% (MCF-7 breast cancer), and 12.2 ± 1.6% (HCC1806 breast cancer). Biocompatibility study on H1299 cell line and HCC1806 cell line showed that separated cancer cells had excellent short-term viability, normal proliferation and unaffected key biomarker expressions. We then demonstrated the enrichment of CTCs in blood samples obtained from two patients with newly diagnosed advanced non-small cell lung cancer (NSCLC). While still at its early stage of development, FCS could become a complementary tool for CTC separation for its high recovery rate and excellent biocompatibility, as well as its potential for further optimization and integration with other separation methods.

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Tag-free microfluidic separation of cells against multiple markers.

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Tag-free microfluidic separation of cells against multiple markers.

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