Cultivation and Cryopreservation of Induced Human Regulatory T-Cells

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Abstract

This study aimed to evaluate the long-term stability and cryopreservation resilience of human induced regulatory T-Cells (iTregs) in culture. Methods: CD4+ T-Cells were isolated from human peripheral blood mononuclear cells (PBMCs) using magnetic separation. iTreg differentiation was induced by culturing the cells in RPMI‑1640 medium supplemented with TGF-β, IL‑2, and anti-CD3 antibodies. The proportion of cells with a CD4+CD25+CD127low phenotype was assessed by flow cytometry at isolation and on days 7, 14, and 21 of culture, as well as post-cryopreservation. Furthermore, the expression of key Treg markers (CD4, IL2RA, FOXP3, IKZF2) was analyzed at the gene level via total RNA sequencing. Results: A significant increase in the CD4+CD25+CD127low population was observed following induction, reaching a high purity of 96.3±2.5% by day 7, which was maintained throughout the 21-day culture period. This phenotype remained stable after cryopreservation, with no significant loss in cell numbers. RNA sequencing confirmed stable transcriptional upregulation of canonical Treg markers, including FOXP3 and IL2RA. Conclusion: Human iTregs demonstrate remarkable phenotypic and transcriptional stability over 21 days in culture and maintain their defining characteristics following cryopreservation. This confirms their suitability for long-term studies and potential therapeutic applications.

About the authors

L. A Rzhanova

Koltzov Institute of Developmental Biology of the Russian Academy of Sciences

Email: 9303923@gmail.com
ORCID iD: 0000-0003-4082-7662
Moscow, Russian Federation

E. V Kuzmenko

Koltzov Institute of Developmental Biology of the Russian Academy of Sciences; Moscow Institute of Physics and Technology

Email: katya.3734@gmail.com
ORCID iD: 0009-0003-3601-1588
Moscow, Russian Federation; Dolgoprudny, Russian Federation

D. D Zhdanov

Institute of Biomedical Chemistry

Email: zhdanovdd@gmail.com
ORCID iD: 0000-0003-4753-7588
Moscow, Russian Federation

Z. R Starinnov

Koltzov Institute of Developmental Biology of the Russian Academy of Sciences

Email: zakhar3600@gmail.com
ORCID iD: 0009-0001-0233-2231
Moscow, Russian Federation

M. A Machinskaya

Koltzov Institute of Developmental Biology of the Russian Academy of Sciences

Email: maria.machinsky@gmail.com
ORCID iD: 0009-0006-3213-7564
Moscow, Russian Federation

A. S Ryabchenko

Koltzov Institute of Developmental Biology of the Russian Academy of Sciences

Email: anfisafil@gmail.com
ORCID iD: 0000-0003-4998-1367
Moscow, Russian Federation

A. A Ryabinin

Koltzov Institute of Developmental Biology of the Russian Academy of Sciences

Email: andrey951233@mail.ru
ORCID iD: 0000-0002-7337-1166
Moscow, Russian Federation

A. A Permyakova

Koltzov Institute of Developmental Biology of the Russian Academy of Sciences; Lomonosov Moscow State University

Email: dex.winner@gmail.com
ORCID iD: 0009-0009-1821-3454
Moscow, Russian Federation; Moscow, Russian Federation

O. L Cherkashina

Koltzov Institute of Developmental Biology of the Russian Academy of Sciences

Email: olgalcher@gmail.com
ORCID iD: 0000-0002-6798-9365
Moscow, Russian Federation

E. A Vorotelyak

Koltzov Institute of Developmental Biology of the Russian Academy of Sciences; Lomonosov Moscow State University

Email: vorotelyak@yandex.ru
ORCID iD: 0000-0001-5405-0212
Moscow, Russian Federation

E. I Morgun

Koltzov Institute of Developmental Biology of the Russian Academy of Sciences

Email: lady.morgun2016@yandex.ru
ORCID iD: 0000-0002-9082-6206
Moscow, Russian Federation

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