(570a) Gentle Microfluidic Cell Capture and Release: PLL Stabilization of the Secondary Anchor Targeted Cell Release System

Gentle
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 characteristics^1–3. 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 cells^5–9, which causes irreversible
changes to biomarkers ^6,7,10–13. 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 Dulbecco’s 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% Penicillin–Streptomycin (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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