BUBLES

BUBLES --- Buoyancy enabled Separation. According to the Archimedes law, the flotation of cancer cells in aqueous medium critically depends on the MB size and the number of MBs per cell. For a 20 µm diameter cell, a single 9 µm diameter MB can pull the cell up. Since the median size of our MBs was 5 µm, 5 MBs should be sufficient to lift up a 20 µm cell.

Synthesis & characterization of Ab-coated MB. (A) MBs were conjugated with anti-EpCAM in a two-step process; (B) Images show characteristic rosettes when MBs (left) and magnetic beads (right) were mixed with GFP-4T1 cells after 15-minute incubation. (C) BUBLES demonstration frames from a 1-minute video of a 15-minute procedure (top of page).

Isolation of rare cells with BUBLES. Rare tumor cells were added to plasma-depleted blood, isolated with MBs and counted on a slide. In order to avoid spiking and counting errors, the same number of tumor cells that was added to blood cells prior to isolation (typically in 5–10 µl volume) was placed on a slide and counted in parallel with the isolated sample. (A) Mouse breast cancer GFP-4T1 cells were added to 1 ml blood and isolated with anti-mouse EpCAM MBs (n = 3). (B) Prostate cancer GFP-PC-3 cells were added to 3 ml of plasma-depleted blood and isolated with anti-human EpCAM MBs (n = 3); (C) Pancreatic cancer BxPC3 cells were added to 7 ml plasma-depleted blood and isolated with anti-human EpCAM MBs. Unlike the experiments with GFP-tagged cells, the isolated cells were stained with pan-cytokeratin antibody. A representative microscopic field (20×objective) shows the MB-isolated BxPC3 cells positive for CK (green). Hoechst-positive, CK-negative cells, which are presumably carryover leukocytes, are also visible in the field. Arrow points to a tumor cell cluster; (D) There was a 77% efficiency of isolation of BxPC3 cells from 7 ml (n = 3).

Rare cell isolation from a large volume of blood. Freshly isolated buffy coats (24 ml from 125 ml donor blood) were washed once in PBS and resuspended in 22 ml PBS. The buffy coat (BC) or medium only was spiked with 70 BxPC3 prostate cancer cells and 4x107 MBs were applied. Floating MBs (A) were harvested onto nitrocellulose membrane, stained with pan-cytokeratin antibody and the cells were counted (B).

Circulating tumor cells (CTCs) from two patients with metastatic brain cancers (A and B). In both cases CK+/CD45- cells that are presumably CTCs were identified using 7.5 ml of patient’s blood. Some cells present mitotic figures or multinuclear morphology. CK-/CD45+ cells (presumably leukocytes) are also shown.


C

(A) A patent-pending syringe-like tube is used to isolate microbubbles/cells in a small-volume sample collecting tube.

(B) A benchtop machine, slightly taller than a coffee maker, is used to house the syringe-like tube in the upper compartment. The steps shown in panel A are controlled by a microcontroller to drive a motor in the lower compartment.

(C) A movie is recorded to demonstrate this process. There is a sensor that stops the motor when the liquid reach the top, which allows microbubbles/cells slowly reaching the top surface. This process enables collecting targeted cells in a very small volume.



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