Stem-like memory T cells are generated during hollow fiber perfusion-based expansion and enriched after cryopreservation in an automated modular cell therapy manufacturing process

Cytotherapy. 2022 Nov;24(11):1148-1157. doi: 10.1016/j.jcyt.2022.07.009. Epub 2022 Aug 26.


Background aims: Modular automation is a flexible and reliable option to build the foundation of a new or evolving process or to introduce automation to a process that is already established. Herein the authors demonstrate that modular automation provides both high-quality and high-yield T-cell products.

Methods: Cells from three individual donors collected on an automated continuous flow centrifugation system were successfully expanded in a functionally closed, automated, perfusion-based hollow fiber bioreactor. These cells were then prepared for cryopreservation in an automated closed-system device that maintains temperature and aliquots a mixed cell product and cryoprotectant into product bags. Cell product bags were thawed and expanded in flasks. Samples taken throughout this manufacturing process were analyzed for cell phenotype, exhaustion markers and functionality. The proportion of CD4+ and CD8+ T cells was maintained through each step, from pre-expansion and post-expansion to immediately after thaw and 24 h after thaw.

Results: Interestingly, phenotypic markers such as CD45RO, CD45RA and CCR7 evolved throughout the process and stem-like memory T cells emerged as the predominant phenotype in the clinically relevant 24-h post-thaw sample.

Conclusions: Modular automation supported the generation of stem-like memory T cells that were not terminally exhausted and were able to produce effector cytokines upon restimulation.

Keywords: Cell expansion; Cell therapy manufacturing; Cryopreservation; Fill and finish; Modular automation; Stem-like memory T cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell- and Tissue-Based Therapy
  • Cryopreservation*
  • Cytokines
  • Leukocyte Common Antigens
  • Memory T Cells*
  • Perfusion
  • Receptors, CCR7


  • Cytokines
  • Receptors, CCR7
  • Leukocyte Common Antigens