Cell banking for regulatory T cell-based therapy: strategies to overcome the impact of cryopreservation on the Treg viability and phenotype

// Karolina Goląb 1 , Randall Grose 2 , Veronica Placencia 3 , Amittha Wickrema 3 , Julia Solomina 1 , Martin Tibudan 1 , Evelyn Konsur 1 , Kamil Cieply 1 , Natalia Marek-Trzonkowska 4 , Piotr Trzonkowski 5 , J. Michael Millis 1 , John Fung 1 and Piotr Witkowski 1 1 Department of Surgery, University of Chicago, Chicago, IL, USA 2 South Australian Health and Medical Research Institute, University of Adelaide, SA, Australia 3 Department of Medicine, Hematology-Oncology, Cancer Research Center, University of Chicago, Chicago, IL, USA 4 Department of Family Medicine, Medical University of Gdansk, Gdansk, Poland 5 Department of Clinical Immunology and Transplantology, Medical University of Gdansk, Gdansk, Poland Correspondence to: Piotr Witkowski, email: pwitkowski@surgery.bsd.uchicago.edu Keywords: regulatory T cells (Tregs); cell banking strategies; effect of cryopreservation; Treg-based therapies Received: February 24, 2017      Accepted: November 10, 2017      Published: January 03, 2018 ABSTRACT The first clinical trials with adoptive Treg therapy have shown safety and potential efficacy. Feasibility of such therapy could be improved if cells are cryopreserved and stored until optimal timing for infusion. Herein, we report the evaluation of two cell-banking strategies for Treg therapy: 1) cryopreservation of CD4 + cells for subsequent Treg isolation/expansion and 2) cryopreservation of ex-vivo expanded Tregs (CD4 + CD25 hi CD127 lo/- cells). First, we checked how cryopreservation affects cell viability and Treg markers expression. Then, we performed Treg isolation/expansion with the final products release testing. We observed substantial decrease in cell number recovery after thawing and overnight culture. This observation might be explained by the high percentage of necrotic and apoptotic cells found just after thawing. Furthermore, we noticed fluctuations in percentage of CD4 + CD25 hi CD127 - and CD4 + FoxP3 + cells obtained from cryopreserved CD4 + as well as Treg cells. However, after re-stimulation Tregs expanded well, presented a stable phenotype and fulfilled the release criteria at the end of expansions. Cryopreservation of CD4 + cells for subsequent Treg isolation/expansion and cryopreservation of expanded Tregs with re-stimulation and expansion after thawing, are promising solutions to overcome detrimental effects of cryopreservation. Both of these cell-banking strategies for Treg therapy can be applied when designing new clinical trials.