Page 87 - IJB-9-4

International Journal of Bioprinting Laser transfer for CTC isolation

0.05% trypsin, and used only when its viability exceeded stained with 0.05 µg (2 µL) of α-CD45/PE for 30 min at
90% as assessed by Trypan Blue exclusion. 4°C in the dark. After that, cells were washed two times
with 200 µL 1× PBS, resuspended in a suitable volume of
3.2. Analytical blood samples 1× PBS, and stored shortly at 4°C before use.
Analytical samples were prepared to simulate cancer
samples in experiments that rely on knowing the starting 3.5. Donor substrate preparation
concentration of target cells. Peripheral blood was Donor substrate was made from a soda lime glass
obtained from healthy donors. To collect their samples as microscope slide (Thermo Scientific AAAA000001##02E)
part of the C.0005266 collection (“Research in Cancer”) and laser-cut to a final dimension of 12.5 × 12.5 mm each.
for biomedical research purposes, the informed consent Two layers of commercial polyimide tape were adhered to
was previously approved by the corresponding Ethics and the slide covering all of its surfaces: the first one (Caplinq
Scientific Committees, obtained from the donor prior to PIT0.5S-UT/19) with a thickness of 30 µm and the second
specimen collection. one (Caplinq PIT1S/19) with a thickness of 70 µm on
Blood was drawn into EDTA tubes (Vacutainer BD, the top. Once both tapes were added to the square slide,
Franklin Lkes, NJ) and subsequently spiked with on cancer a 5-mm diameter hole was laser-cut at the center of the
cell lines. MDA-MB-231 was used both for analytical topmost tape and carefully removed to leave a hole with a
instrument performance validation experiments and for depth equal to the thickness of the outermost tape (70 µm).
TP53 and BRAF assay validations because this line harbors This hole worked as a microwell to contain the liquid with
a known homozygous TP53 c.839G>A p.R280K mutation the cells. All slides were cleaned thoroughly with 70% v/v
and heterozygous BRAF c.1391G>T p.G464V mutation. ethanol and left under UV light before used. Slides were
Cells suspended in a stock solution were first counted on stored inside a class II biological safety cabinet kept in a
Neubahuer chamber and then spiked into 7.5 mL whole sterile environment.
blood. To assure a homogenous and thin layer of liquid in
the circular well, and due to the wettability properties of
3.3. Sample preparation polyimide, a surface treatment to improve hydrophilicity
Whole blood samples were added with different was needed before the liquid deposition that happened
concentrations of MDA-MB-231 cell line (1000, 10,000, prior to the laser transfer process. Therefore, the sample
100,000, and 1,000,000 cells). was placed inside a standard vacuum desiccator (Duran
These blood tubes were processed to recover the 247824604) with both the lid and stopcock sealed with high
fraction of peripheral blood mononuclear cells from vacuum grease (Dow Corning). Immediately after that, air
where the MDA-MB-231 cell line was also identified was evacuated using a rotary vane pump (Busch 016-112)
by density. LeucoSep tubes (Greiner Bio-One, Monroe, in 12 s to a pressure of approximately 1000 mTorr. The
NC) were prepared by adding 15 mL of Ficoll-Paque Plus desiccator was placed inside a microwave source of 2450
(d = 1.077gr / mol; GE Healthcare, Pittsburgh, PA). The MHz and a power of 700 W (LG MS-1924W/02), letting
peripheral blood mononuclear cell fraction, together the air plasma formed act for 10 s on the sample. Plasma-
with the MDA-MB-231, was recovered and resuspended activated donor substrate was left to cool down for 30 s,
in 500 µL of MACS buffer. Cell count was performed and 1.5 µL of spiked sample (MDA-MB-231+PBMCs) was
and the cell pellet was resuspended in 80 µL of buffer placed inside the circular well and spread evenly along the
MACs and 20 µL of CD45 MicroBeads per 10 total cells. whole area. Donor substrate was then turned upside down
7
The mixture was incubated for 15 min at 4°C. Then, and placed on top of a 0.2-mL PCR tube (Fisherbrand™).
the sample was added to MS column (MACS, Miltenyi The tube was fixed on a 96-well PCR plate and the sample
Biotec). In this study, the relationship between MDA- was kept on its place using a 3D-printed fixture that adapt
MB-231 and PBMC does not reflect the concentration to the 96-well plate. Finally, the sample was taken to the
of CTC in the blood (1 CTC / 10 –10 PBMCs), because laser system for processing.
9
6
as proof of concept, we only wanted to confirm that the
technology is able to discriminate between different 3.6. Laser system
subpopulations. Laser system comprises a UV laser (Crylas FTSS355-Q4)
emitting 1.3 ns single pulses at demand with a wavelength
3.4. Staining of 355 nm. Laser pulse energy was controlled in the range
Cells were stained with a phycoerythrin-conjugated of 1 to 23 ± 2 µJ by means of a half-wave plate and a beam
monoclonal antibody against CD45 (α-CD45/PE, polarizer. Laser beam was focused onto the sample down
Biolegend, 368510). Cells were washed two times using to a beam waist at focus of 10 µm using a 10× microscope
200 µL 1× phosphate-buffered saline (PBS), and then objective.

Volume 9 Issue 4 (2023) 79 https://doi.org/10.18063/ijb.720