Cell lines and culture

Human leukemia cell line K562 was cultured in RPMI 1640 (A2494201, Gibco, USA) supplemented with 10% heat-inactivated fetal calf serum (16,000,044, Gibco, USA), 2 mM glutamine, 20 mM Hepes (pH 7.5) and maintained at 37 ℃ under an atmosphere of 95% O2 and 5% CO2. To prepare leukocyte, human whole blood (HUMANWBK2, BIOIVT, USA) was mixed with a separation medium (C-44010, Sigma-Aldrich, USA) and centrifuged at 400 × g for 15 min. Peripheral blood mononuclear cells (PBMCs) were obtained from individuals with chronic myeloid leukemia (PBMNC005C-CML PBMC, BIOIVT, USA) using Institutional Review Board (IBR) approved consent forms and protocols.

Microfluidic device and motion microscopy

Microfluidic devices (Polydimethylsiloxane chip, Microfit, South Korea) were placed on the stage of an inverted microscope and the fluid flow was controlled by individual syringe pumps (BS-9000–12, Braintree scientific, USA). The microfluidic device and syringe pumps were connected by polythene tubing (PE10, Braintree scientific, USA) with an inner diameter of 0.28 mm. Prior to each experiment, isopropanol (W292907, Sigma-Aldrich, USA) was flushed through the whole microfluidic device to remove air bubbles in the channel followed by 1 X PBS (10,010,023, GIbco, USA) wash for 30 min. Leukemia cells or leukocytes were then introduced to the device at a flow rate of 10–30 μm/s and video files were recorded through the inverted microscope at 1200 × 512 pixels and 500 frames per second. The recorded videos were uploaded to lambda vue (https://lambda.qrilab.com/site/) and the magnification type was selected in colour mode, with amplification ratio of 20, and wavelength was selected from 0.1 Hz to 10 Hz in conversion condition.

Quantification of cellular trail intensity

The obtained images were converted to 8-bit format in order to perform uncalibrated optical density. After conversion, the background was subtracted through the rolling ball radius method and cellular trails were individually selected. The area of histograms were obtained and quantified by ImageJ (Java-based image-processing and analysis software). Data were acquired as arbitrary area values.

Optical tomographic microscope

Green light (λ = 520 nm, exposure 0.2 mw/mm2) from a laser diode was splitted into cells and reference beam at Nanolive (3D cell explorer, Switzerland). Cells were illuminated with a laser beam inclined at 45° which rotates around the sample 360°. Holographic images were recorded on a digital camera by combining the beam that had passed through the cells with the reference beam. The 3D cell images were recorded up to 30 μm depth of reconstruction.

Viscosity measurement

Hyaluronic acids (75,043, Sigma-Aldrich, USA) and 1 × PBS (10,010,023, GIbco, USA) were slowly mixed with a blender until completely liquefied. Viscosity for 0.01, 0.02, or 0.05% hyaluronic acids was measured with a cone-and-plate digital viscometer (ASTM D4287, Industrial Physics Inks & Coatings, Netherlands). Shear rates were generated by rotating the brush around 750 rpm and non-newtonian fluid properties were determined.

Cell viability

K562 and leukocytes were treated with 0.01, 0.02, 0.05, or 0.1% of hyaluronic acid for 12 h at 37 ℃ under an atmosphere of 95% O2 and 5% CO2. Using CCK-8kit (ab228554, Abcam, USA), tetrazolium was converted to formazan by dehydrogenase activity from mitochondria of living cells, and cell viability was determined following detection of optical density at 460 nm.

Western blot

Briefly, human leukemic myeloblasts (K562) were homogenized in ice-cold lysis buffer. After centrifugation at 5,000 g for 20 min, protein content of the supernatant was quantified using a Bradford protein assay. Samples were diluted, boiled with sample loading dye, and 100 mg were loaded in SDS-PAGE (4561033EDU, Bio-Rad). After blotting, membranes were blocked in 5% skim milk (70,166, Sigma-Aldrich) in PBS containing 0.1% Tween-20 (P1379, Sigma-Aldrich). Membranes were incubated with antisera directed against CDC42 (1:1000; 2462, Cell Signaling, USA), and then with secondary antibodies (mouse-specific HRP-conjugated antibody or rabbit-specific HRP-conjugated antibody). Bands were visualized using ECL detection kit (32,106, Thermo Scientific) and quantified by densitometry. Blots were stripped and re-exposed to detect TUBB (1:1000; 2125, Cell signaling, USA) as housekeeping protein.

Fluorescence microscope

Briefly, PBMCs were placed in 10% formalin for 3 h and incubated with antisera against FITC-conjugated CD117 (1:400; ab119107, abcam, USA). After washing with PBS, cells were visualized using Zeiss LSM 510 confocal microscope (Carl Zeiss, German).

Statistical analysis

Values are means ± SE. The significance of differences was determined by a two-way analysis of variance (ANOVA), or a one-way ANOVA followed by a Bonferroni post-hoc analysis where appropriate. Differences were considered significant when P < 0.05.