Cryopreservation timing is a critical process parameter in a thymic regulatory T-cell therapy manufacturing protocol

Katherine N. MacDonald, Sabine Ivison, Keli L. Hippen, Romy E. Hoeppli, Michael Hall, Grace Zheng, I. Esme Dijke, Mohammed Al Aklabi, Darren H. Freed, Ivan Rebeyka, Sanjiv Gandhi, Lori J. West, James M. Piret, Bruce R. Blazar, Megan K. Levings

Research output: Contribution to journalArticlepeer-review

Abstract

Regulatory T cells (Tregs) are a promising therapy for several immune-mediated conditions but manufacturing a homogeneous and consistent product, especially one that includes cryopreservation, has been challenging. Discarded pediatric thymuses are an excellent source of therapeutic Tregs with advantages including cell quantity, homogeneity and stability. Here we report systematic testing of activation reagents, cell culture media, restimulation timing and cryopreservation to develop a Good Manufacturing Practice (GMP)–compatible method to expand and cryopreserve Tregs. By comparing activation reagents, including soluble antibody tetramers, antibody-conjugated beads and artificial antigen-presenting cells (aAPCs) and different media, we found that the combination of Dynabeads Treg Xpander and ImmunoCult-XF medium preserved FOXP3 expression and suppressive function and resulted in expansion that was comparable with a single stimulation with aAPCs. Cryopreservation tests revealed a critical timing effect: only cells cryopreserved 1–3 days, but not >3 days, after restimulation maintained high viability and FOXP3 expression upon thawing. Restimulation timing was a less critical process parameter than the time between restimulation and cryopreservation. This systematic testing of key variables provides increased certainty regarding methods for in vitro expansion and cryopreservation of Tregs. The ability to cryopreserve expanded Tregs will have broad-ranging applications including enabling centralized manufacturing and long-term storage of cell products.

Original languageEnglish (US)
Pages (from-to)1216-1233
Number of pages18
JournalCytotherapy
Volume21
Issue number12
DOIs
StatePublished - Dec 2019

Bibliographical note

Funding Information:
ThermoFisher provided in-kind reagents and STEMCELL Technology provided donations to support this project. M.K.L. has received research funding from Bristol Myers Squibb, Takeda, Pfizer, CRISPR Therapeutics and Sangamo for work not related to this project. J.M.P. has received research funding from Amgen, Bayer and Pfizer; he has patents pending with AbCellera and SonoSep Technologies, as well as ownership of SonoSep, all for work not related to this project. B.R.B. receives remuneration as an advisor to Kamon Pharmaceuticals, Five Prime Therapeutics, Regeneron Pharmaceuticals, Magenta Therapeutics and BlueRock Therapeutics, research support from Fate Therapeutics, RXi Pharmaceuticals, Alpine Immune Sciences, Abbvie, Leukemia and Lymphoma Society, Childrens' Cancer Research Fund and KidsFirst Fund and is a co-founder of Tmunity. L.J.W. has received research support from Astellas Canada, Novartis Canada, Roche, BD Biosciences, Canadian Glycomics Network (GlycoNet NCE) and Women and Children's Health Research Institute for work unrelated to this project. This work was supported by grants from the Canadian Institutes of Health Research (CIHR) through the Canadian National Transplant Research Program (TFU 127880 to M.K.L. and L.J.W.), the Stem Cell Network and BioCanRx Network Centres of Excellence (to M.K.L. and L.J.W.), a donation from STEMCELL Technologies (to M.K.L.) and from the National Institutes of Health (R01 HL11879, R01 HL56067, R37 AI 34495 and P01 CA065493 to B.R.B.). M.K.L. K.N.M. and R.E.H. receive salary awards from the BC Children's Hospital Research Institute. K.N.M. is also supported by a CIHR doctoral award. M.G.H. receives a BioCanRx Summer Studentship salary award. KNM: conceived, designed and conducted experiments, analyzed data, and wrote the manuscript. SI: conceived, designed and conducted experiments, analyzed data and critically reviewed the manuscript. KLH: conceived and designed experiments, analyzed data and critically reviewed the manuscript. REH: provided critical feedback throughout the project and critically reviewed the manuscript. MH and GZ conducted experiments and analyzed data. IED: provided critical feedback throughout the project and critically reviewed the manuscript. MAA, DHF, IR and SG: organized tissue collection. LJW: secured funding, provided critical feedback throughout the project and critically reviewed the manuscript. JMP: secured funding, provided critical feedback throughout the project, and critically reviewed the manuscript. BRB: secured funding, conceived and designed experiments and critically reviewed the manuscript. MKL: secured funding, conceived and designed experiments, provided overall direction and interpretation, and wrote the manuscript. The authors thank volunteers, patients and their parents for contribution of samples, as well as the surgical and cardiac clinic staff at the British Columbia Children's Hospital and the University of Alberta Stollery Children's Hospital who made this study possible; special thanks to Melanie Ganshorn, Allison Jamieson, Lyn Nguyen and Colleen Ring. We also thank ThermoFisher Inc for the provision of in-kind reagents.

Funding Information:
ThermoFisher provided in-kind reagents and STEMCELL Technology provided donations to support this project. M.K.L. has received research funding from Bristol Myers Squibb, Takeda, Pfizer, CRISPR Therapeutics and Sangamo for work not related to this project. J.M.P. has received research funding from Amgen, Bayer and Pfizer; he has patents pending with AbCellera and SonoSep Technologies, as well as ownership of SonoSep, all for work not related to this project. B.R.B. receives remuneration as an advisor to Kamon Pharmaceuticals, Five Prime Therapeutics, Regeneron Pharmaceuticals, Magenta Therapeutics and BlueRock Therapeutics, research support from Fate Therapeutics, RXi Pharmaceuticals, Alpine Immune Sciences, Abbvie, Leukemia and Lymphoma Society, Childrens' Cancer Research Fund and KidsFirst Fund and is a co-founder of Tmunity. L.J.W. has received research support from Astellas Canada, Novartis Canada, Roche, BD Biosciences, Canadian Glycomics Network (GlycoNet NCE) and Women and Children's Health Research Institute for work unrelated to this project. This work was supported by grants from the Canadian Institutes of Health Research (CIHR) through the Canadian National Transplant Research Program (TFU 127880 to M.K.L. and L.J.W.), the Stem Cell Network and BioCanRx Network Centres of Excellence (to M.K.L. and L.J.W.), a donation from STEMCELL Technologies (to M.K.L.) and from the National Institutes of Health (R01 HL11879, R01 HL56067, R37 AI 34495 and P01 CA065493 to B.R.B.). M.K.L., K.N.M. and R.E.H. receive salary awards from the BC Children's Hospital Research Institute. K.N.M. is also supported by a CIHR doctoral award. M.G.H. receives a BioCanRx Summer Studentship salary award.

Publisher Copyright:
© 2019 International Society for Cell and Gene Therapy

Keywords

  • Good Manufacturing Practice
  • T-cell manufacturing
  • cell therapy
  • cryopreservation
  • regulatory T cell
  • tolerance

Fingerprint

Dive into the research topics of 'Cryopreservation timing is a critical process parameter in a thymic regulatory T-cell therapy manufacturing protocol'. Together they form a unique fingerprint.

Cite this