463259 Gentle Microfluidic Cell Capture and Release: PLL Stabilization of the Secondary Anchor Targeted Cell Release System
Microfluidic Cell capture and Release: PLL Stabilization of the Secondary
Anchor Targeted Cell Release System
Ali Ansari, P. Imoukhuede, Ph.D.
University of Illinois at Urbana-Champaign
Introduction: Patients exhibit differential responses to drugs and therapeutics due to their unique genetic, proteomic, and cellular characteristics13. Advancing personalized medicine thereby requires methods of isolating and profiling these differences to individualize therapeutic regimens. However, some current cell isolation techniques are slow, disrupting physiological receptor quantities4 while several of the more rapid lab-on-a-chip methods apply force or chemical digestion to release selected cells59, which causes irreversible changes to biomarkers 6,7,1013. Thus, there is distinct lack of gentle and rapid cell isolation technologies for clinical translation 13. Towards these aims of translatable, fast and gentle cell isolation, we describe adaptation of our novel, Secondary Anchor Targeted Cell Release System14 (SATCR) to microfluidics and optimization of the microfluidic spiral design and buffer solutions to ensure gentle, but effective cell capture.
Materials and Methods: We have previously described the SATCR surface functionalization for selective isolation of cells in suspension14 (Fig. 1A). Briefly, 24-well glass bottom plates are oxygen plasma cleaned, 2% APTES is applied, d-Desthiobiotin (DSB) is solubilized, activated, and dissolved in pH 6.0 MES buffer and mercaptoethanol quenches. Following overnight incubation, excess DSB is washed with PBS, and PBS-reconstituted streptavidin is applied overnight at 4°C. The surface is rinsed with PBS, rewetted, and stored at 4°C until use. Here we integrate the SATCR within a spiral microfluidic device consisting of a polydimethylsiloxane (PDMS) spiral mold (Fig. 1B), cast with a 3D printed master, which is bonded to glass through oxygen plasma treatment (Fig.1A). MCF7GFP cells, a luminal breast cancer cell line, were obtained from Cell Biolabs (San Diego, CA). MCF7GFP cells were grown in high glucose Dulbeccos modified Eagle medium (DMEM) supplemented nonessential amino acids (University of Illinois Cell Media Facility, School of Chemical Sciences, Urbana, IL), 10% fetal bovine serum (Invitrogen, Carlsbad, CA), and 1% PenicillinStreptomycin (Invitrogen). These cells were then fixed with 4% formaldehyde and then labeled with previously biotinylated h-HLA-ABC antibody. FEAP software was used to simulate spiral wall shear stress. Towards optimizing gentleness, buffer osmolarity readings were taken on the Wescor Vapor Pressure Osmometer Vapro 5520 and Human Umbilical Vein Endothelial Cell (HUVEC) sizes and concentrations were measured on the Countess II Automatic Cell Counter. Flow rates for cell capture were 300 µL/min and cells were released via 4 mM biotin solution.
Results: Our computational simulation of the spiral revealed that the experimental flow-rate of 400 µL/min gave a 0.1 mPa wall shear stress (Fig. 1C), a gentle shear stress as it is 4 orders of magnitude below arterial wall shear stress15. The rationale for a spiral design was to increase mixing, and we experimentally observe 12% more cell release for recollection in a SATCR spiral micromixer versus a SATCR straight micro-channel design, indicating the efficacy of the SATCR spiral. We observed a significant, 26% (ANOVA Fisher test, p<0.001), decrease in HUVECs size following capture and release from the SATCR spiral (Fig. 1E). This decrease was not due to the biotin, as increasing biotin concentrations did not affect buffer osmolarity (Fig. 1D). Instead, it was likely due to sequestration of anions, due to an acidic isoelectric point of the SATCR spiral: we observed an entering buffer pH =7.2 and an exit pH =6.4. We determined that buffer supplementation with cationic PLL, stabilized cell diameter: optimal [PLL] = 0.05-0.5 mg/mL (Fig. 1E).
Conclusions: Reducing cell isolation induced stresses will advance personalized therapeutic regimens by retaining physiological biomarkers. Here we have identified optimizations for the adaption of the Secondary Anchor Targeting Cell Release System to capture cells for downstream analysis and recollection by reducing both wall shear and osmolarity induced stresses. These optimizations will allow us to further reduce the stresses experienced by the cells and help maintain physiological receptor and biomarker expression of cells released from the device. Future work will apply these optimizations to spiked blood samples to ensure translatability towards circulating cell capture.
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