Page 90 - IJB-9-4

International Journal of Bioprinting Laser transfer for CTC isolation

gene and for the exon 11 of BRAF gene, all of which include Trypan Blue staining were considered viable, whereas
mutational hot spots. those stained with Trypan Blue were considered nonviable.
The primers used were as follows: CTC-stained cells were selected and isolated in 96-well
plates containing RPMI medium (GibcoTM), 10% of FBS
• TP53 forward primer: (Sigma) and 10% of acclimated medium (from established
5ʹ-GGCTTTGGGACCTCTTAACC-3ʹ cultures from which cells were removed), under normal
• TP53 reverse primer: cell incubation environment (37°C, 5% CO ). Proliferation
2
5ʹ-TAACTGCACCCTTGGTCTCC-3ʹ was observed after 9 days and maintained at day 14.
• BRAF forward primer: 4. Results and discussion
5ʹ-CACTTGGTAGACGGGACTCG-3ʹ
4.1. Single CTC isolation using BA-LIFT and SCS
• BRAF reverse primer: Our assessment experiments used a classic approach in
5ʹ-GTTTATTGATGCGAACAGTGA-3ʹ which a CTC was defined as a CK-positive, CD45-negative,
PCR products were analyzed by agarose gel nucleated intact cell. In our case, MDA-MB-231 (cancer
electrophoresis coupled with fluorescence detection. cell line from human breast carcinoma) simulates the
characteristics of a true CTC. As indicated previously, the
3.10. Sanger sequencing first step before CTC isolation comprises the enrichment of
DNA sequencing is considered the gold standard for the sample. This step consists of cell blood subpopulation
detection of mutations. This method allows determination separation by Ficoll density gradient centrifugation; CTCs
of the order of nucleotides in a target DNA sequence. will be present in the peripheral blood mononuclear cell
[74]
The Sanger chain-termination method is commonly used (PBMCs) fraction . Depending on the approach followed
in molecular diagnostic laboratories. In this method, for cell identification, the pellet should be fixated or not and
the incorporation of a chemically modified nucleotide the staining could be used for positive selection (EpCAM,
(dideoxynucleotide) terminates extension of the DNA Vimentin or another specific CTCs markers) or negative
strand at the point of incorporation. This results in a selection (CD45 leukocyte staining) (Figure 6).
mixture of DNA fragments of various lengths. Each In this study, we performed a negative selection using
dideoxynucleotide (A, T, C, or G) was labeled with different a phycoerythrin-conjugated monoclonal antibody against
fluorescent dye, allowing their individual detection. The CD45 (α-CD45/PE, clone 2D1, Biolegend). Nonfixated
newly synthesized and labeled DNA fragments were cells were incubated for 30 min at 4°C. Once the liquid
separated by size through capillary gel electrophoresis. sample has been prepared and deposited in the donor
The fluorescence was detected by an automated sequence substrate, cells to be transferred were identified with the
analyzer (Applied Biosystems 3130xl y 3730xl genetic vision and fluorescence systems previously described.
analyzer) and the order of nucleotides in the target DNA
was depicted as a sequence electropherogram. MDA-MB-231 cells (negative for CD45) were
transferred by shooting a single laser pulse at the
3.11. Immunofluorescence staining cell location with a focused laser beam diameter of
After blocking by 10% FBS for 10 min, cells were approximately 20 µm and an effective laser pulse energy
2
incubated with α-CD45/PE clone (2D1) (Biolegend), then of 5.5 µJ, resulting in a laser fluence of 3.5 J/cm . This laser
permeabilized with 0.2% Triton for 10 min, stained with transfer system allows us to isolate the number of cells
anticytokeratin-FITC human clone (CK3-6H5) (Miltenyi that we determine and collect them in the most suitable
Biotec) for 60 min, and finally mounted with Hoechst platform for the experiments.
33342. For our proof of concept, a known number of MDA-
Trypan Blue (Sigma) staining was used to determine MB-231 cells suspended in PBS were spiked into 7.5 mL
viability of the transferred cells. The cells that are not of whole blood. We performed two types of transfer
stained by Trypan Blue are considered viable, whereas experiments. First, we addressed the problem of isolating
those stained by Trypan Blue were considered nonviable. a single cell in a tube. Second, we isolated 25 cells, which
In this study, we did not perform any other experiments to were transferred one by one into a tube. In both types of
evaluate cell viability. experiments, the collector tubes contain 4 µL of PBS. Once
the cells were transferred, DNA was extracted and amplified
3.12. Cell viability and proliferation assay with REPLI-g Single Cell kit (QIAGEN) , which contains
[75]
Trypan Blue (Sigma) staining was used to determine an optimized Phi29 polymerase formulation as well as
viability right after the transference process. Cells without buffers and reagents for whole genome amplification

Volume 9 Issue 4 (2023) 82 https://doi.org/10.18063/ijb.720