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International Journal of Bioprinting Laser transfer for CTC isolation

Figure 6. Double-immunofluorescence (CK/CD45) in MDA-MB-231 isolated using liquid laser transfer. (A) Cells were double-stained with anti-CK
(clone CK3-6H5) human antibody conjugated with FITC and anti-CD45 (clone 2D1) human antibody conjugated with PE. Original magnification, 100×.
(B) PBMCs were used as CD45 positive control. Abbreviation: CK, cytokeratin.

(WGA) from single cells and very small samples, using 4.2. Proliferation assays
multiple displacement amplification technology, which Finally, in order to assess that after the transfer process,
carries out isothermal genome amplification utilizing a the cells were viable for in vitro experiments, transferred
uniquely processive DNA polymerase capable of replicating pellets of single cell and also transferred groups of 25 cells
up to 100 kb without dissociating from the genomic DNA were stained with Trypan Blue, a dye to selectively stain
template. In the first step of the procedure, the cell sample dead tissues or cells blue. In all cases, the observed cells
was lysed, and the DNA was denatured. Once denaturation were alive. Also, to ensure CTCs viability preservation after
was stopped by the addition of neutralization buffer, a the process, a proliferation assay was performed. CTCs
master mix containing buffer and DNA polymerase was were isolated in 96-well plates (see Methods section), and
added. The isothermal amplification reaction proceeded images were taken daily. After 9 days, the proliferation
for 8 h at 30°C. rate was lower than that of the control cells. After 14 days,
however, confluence was reached (Figure 8).
To confirm the origin of the isolated DNA, exon 9
of TP53 gene and exon 11 of BRAF gene (mutations 5. Conclusion
described in this cell line) were amplified by PCR.
Then, we sequenced the amplicons through Sanger This work demonstrates that it is possible to preserve
sequencing [76] . Since DNA sequencing is considered maximum CTCs integrity with negative selection. We
the gold standard for detecting mutations, in this case, believe that this approach opens up a new path not
we did not use next-generation sequencing, because only for single-cell isolation but also for CTCs sorting,
we intended to look for specific point mutation if the appropriate staining and imaging strategies are
characteristic of the cell line used in the assay. As shown adapted and as this has been discussed for other cell lines
[73]
in Figure 7, the expected bands were confirmed for in a previous work . Although relevant aspects like
TP53 exon 9 and BRAF exon 11. Single PCR bands for throughput, process automation, etc. are not investigated
each exon verified the specificity of the amplification in this paper, we must highlight the fact that the transfer
before sequencing. A known homozygous TP53 exon process is based on a particular adaptation of a laser direct
9 mutation, R280K, and a heterozygous BRAF exon 11 write technique, in many aspects which is a standard
mutation, G464V, were identified in replicate samples laser material processing approach. This undoubtedly
of MDA-MB-231 cells (positive control) [77] but were not offers the possibility of future use of the technique with
detected in PBMCs (wild type/negative control). all the potential of laser technology, in terms of precision,
repeatability, throughput, and ease of integration in
We also transferred a sample of previously fixated MDA- already existing platforms, as has been proven in medical
MB-231 cells. After the transfer process, cells were stained and industrial fields. Regarding clinical applications, this
with CK (α-CK/FITC, clone CK3-6H5, Miltenyi Biotec), new system would bring us closer to “real-time biopsy”
CD45 (α-CD45/PE, clone 2D1, Biolegend) and DAPI based on CTC molecular characterization. Our technique
for visualization and confirming that the subpopulations is promising and this approach remains a key area of study
isolated correspond to MDA-MB-231. for further intensive research. Since this novel technology

Volume 9 Issue 4 (2023) 83 https://doi.org/10.18063/ijb.720