Abstract
Background
As cell therapies are injected directly into the body, cell authentication is essential. Short tandem repeat (STR) profiling is used for human identification in forensics as well as for cell authentication. The standard methodology (DNA extraction, quantification, polymerase chain reaction, and capillary electrophoresis) takes at least 6 h and requires several instruments to obtain an STR profile. RapidHIT™ ID is a single automated instrument that provides an STR profile in 90 min.
Objective
In this study, we aimed to propose a method to use RapidHIT™ ID for cell authentication.
Methods
Four types of cells which are used for cell therapy or in the production process were used. The sensitivity of STR profiling was compared by the cell type and cell count using RapidHIT™ ID. Moreover, the effect of preservation solutions, pre-treatment with cell lysis solution, proteinase K, Flinders Technology Associates (FTA) cards, and dried or wet cotton swabs (with a single cell type or a mixture of two) were examined. The results were compared to those obtained by the standard methodology using genetic analyzer ThermoFisher SeqStudio.
Results
We accomplished a high sensitivity through our proposed method that can benefit cytology laboratories. Although the pre-treatment process affected the quality of the STR profile, other variables did not significantly affect STR profiling.
Conclusion
As a result of the experiment, RapidHIT™ ID can be used as a faster and simpler instrument for cell authentication.
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Acknowledgements
This research was supported by a grant (20172MFDS194, DY0002258094) from Ministry of Food and Drug Safety in 2020–2021 and the Korea Basic Science Institute (National research Facilities and Equipment Center) and funded by the Ministry of Education (Grant No. 2020R1A6C101A191).
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Supplementary material 1 (DOCX 2078.7 kb)—Fig. S1 Alleles of FLYRD18 at the SE33 marker as per theSeqStudio genetic analyzer. Allele 30.2, which was not called due to the lowpeak height in RapidHIT™ID, was called when analyzed with a genetic analyzer.Allelesof FLYRD18 at the SE33 marker as shown by RapidHIT™ ID.Allele 30.2 was dropped out. The instrument did notcall the allele 30.2 because the peak height of allele 30.2 was less than 21%of the peak height of allele 21.2. Allelesof FLYRD18 at the SE33 marker as per the SeqStudio genetic analyzer. Fig. S2Alleles of FLYRD18 at the SE33 marker as per theSeqStudio genetic analyzer. Allele 30.2, which was not called due to the lowpeak height in RapidHIT™ID, was called when analyzed with a genetic analyzer.Allele30.2, which was not called due to the low peak height in RapidHIT™ ID, was called whenanalyzed with a genetic analyzer. Fig. S3 Celllines 293T and FLYRD18 mixed at a ratio of 1:1.293T allele (minorallele) = *; shared allele = •; non-callingallele = ↓. Fig. S4 Celllines 293T and FLYRD18 mixed at a ratio of 1:4.293T allele (minorallele) = *; shared allele = •; non-callingallele =↓. Fig. S5 Concordance study of STRprofile of cell line 293T. The results of RapidHIT™ ID and SeqStudio geneticanalyzer were consistent. A Results of 293T using RapidHIT™ ID. B Results of293T according to SeqStudio genetic analyzer. C Results of FLYRD18 using RapidHIT™ID. D Results of FLYRD18 according to SeqStudio genetic analyzer. E Results ofNSCs using RapidHIT™ ID. F Results of NSCs according to SeqStudio geneticanalyzer. G Results of iPSCs using RapidHIT™ ID. H Results of iPSCs accordingto SeqStudio genetic analyzer. I Results of blood using RapidHIT™ ID. J Resultsof blood according to SeqStudio genetic analyzer.
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Koh, U.N., Lee, J.H., Kang, H.J. et al. Application of RapidHIT™ ID for cell authentication by fast and convenient STR profiling. Genes Genom 45, 1263–1271 (2023). https://doi.org/10.1007/s13258-023-01388-4
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DOI: https://doi.org/10.1007/s13258-023-01388-4