Abstract
Cell analysis often requires the isolation of certain cell types. Various isolation methods have been applied to cell sorting, including florescence-activated cell sorting and magnetic-activated cell sorting. However, these conventional approaches involve exerting mechanical forces on the cells, thus risking cell damage. In this study we applied a novel isolation method called buoyancy-activated cell sorting, which involves using biotinylated albumin microbubbles (biotin-MBs) conjugated with antibodies (i.e., targeted biotin-MBs). Albumin MBs are widely used as contrast agents in ultrasound imaging due to their good biocompatibility and stability. For conjugating antibodies, biotin is conjugated onto the albumin MB shell via covalent bonds and the biotinylated antibodies are conjugated using an avidin-biotin system. The albumin microbubbles had a mean diameter of 2μm with a polydispersity index of 0.16. For cell separation, the MDA-MB-231 cells are incubated with the targeted biotin-MBs conjugated with anti-CD44 for 10 min, centrifuged at 10g for 1 min, and then allowed 1 hour at 4°C for separation. The results indicate that targeted biotin-MBs conjugated with anti-CD44 antibodies can be used to separate MDA-MB-231 breast cancer cells; more than 90% of the cells were collected in the MB layer when the ratio of the MBs to cells was higher than 70:1. Furthermore, we found that the separating efficiency was higher for targeted biotin-MBs than for targeted avidin-incorporated albumin MBs (avidin-MBs), which is the most common way to make targeted albumin MBs. We also demonstrated that the recovery rate of targeted biotin-MBs was up to 88% and the sorting purity was higher than 84% for a a heterogenous cell population containing MDA-MB-231 cells (CD44+) and MDA-MB-453 cells (CD44–), which are classified as basal-like breast cancer cells and luminal breast cancer cells, respectively. Knowing that the CD44+ is a commonly used cancer-stem-cell biomarker, our targeted biotin-MBs could be a potent tool to sort cancer stem cells from dissected tumor tissue for use in preclinical experiments and clinical trials.
Highlights
MethodsA solution containing 0.33% biotinylated human serum albumin (HuSA) (w/v), 0.99% unconjugated HuSA (w/v), 5% dextrose (Sigma-Aldrich) (w/v), and perfluorocarbon (C3F8) gas to a final volume of 5 mL was sonicated by a digital sonicator (Model 450, Branson, Danbury, CT) at 200 W for 2 min [27, 28]
Isolating a specific cell type from a mixture of cells is typically the first step in cell analysis and examination, such as isolating circulating tumor cells from blood cells and cancer stem cellsPLOS ONE | DOI:10.1371/journal.pone.0125036 May 20, 2015Buoyancy-Activated Cell Sorting
Since intratumor heterogeneity is a major clinical problem of cancer therapies, the current study focused on buoyancy-activated cell sorting (BACS) based on targeted albumin MBs to isolate different tumor cell subtypes
Summary
A solution containing 0.33% biotinylated HuSA (w/v), 0.99% unconjugated HuSA (w/v), 5% dextrose (Sigma-Aldrich) (w/v), and perfluorocarbon (C3F8) gas to a final volume of 5 mL was sonicated by a digital sonicator (Model 450, Branson, Danbury, CT) at 200 W for 2 min [27, 28]. 5mL biotin-MBs were incubated with 1mL avidin (2 mg/mL) overnight at 4°C and incubated with biotinylated anti-CD44 antibodies (1:10; eBioscience, San Diego, CA) for at least 2 hours at 4°C. Control MBs were produced by sonicating the solution containing 1.32% unconjugated HuSA (w/v), 5% dextrose (w/v), and C3F8 gas in a final volume of 5 mL. Avidin-MBs were produced by adding 2 mg of avidin, 1.2% unconjugated HuSA (w/v), and 5% dextrose (w/v) to the solution prior to the sonication. The avidin-MBs were incubated with biotinylated anti-CD44 antibodies to produce targeted avidin-MBs
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