Scale-up the Culture of Regulatory T Cells (Tregs)

  • Piyanan Boonprasirt

Student thesis: Doctoral ThesisPhD


Regulatory T Cells (Tregs) have been used as a cell therapy to prevent graft rejection in organ transplantation, by expanding populations of these cells in vitro, after selecting them from a patient’s blood sample, and injecting the large population (0.4x106 to 6.4x106 cells kg-1) back to the patient. To date, there is no gold standard method for expansion so the aim of this thesis was to design a fluidized-bed bioreactor (FBB) for ex vivo Treg expansion. The FBB design in this study was divided into three stages. First, the study of cell culture conditions using HL-60 as a cell model. Second, the application of cell culture conditions for 5- and 7-days HL-60 expansion. Third, the application of the FBB for Treg expansion and the development of the Treg expansion protocol. The important parameters during the first stage of the FBB design were the minimum fluidising velocity (umf) and terminal velocity (ut) of the cell culture media. The study of umf and ut found that the values from the experiment were different from the numerical model (Carman-Kozeny equation). The umf from the experiment was 1.44x10-6 m s-1 and ut was 2.88x10-6 m s-1, while the umf from the numerical model was 6.92x10-8 m s-1 and ut was 6.52x10-6 m s-1. Based on the experiment and the correlation, three working velocities (2.41x10-6 m s-1; 3.20x10-6 m s-1 and 4.90x10-6 m s-1) were selected to study the effect of flow velocity on cell proliferation during 48 hours HL-60 cell expansion. The factors that affected HL-60 expansion were the diameter of the FBB, initial cell density, and working velocity. Of the operating conditions tested, a 50 mm cylindrical column at 2.41x10-6 m s-1 with the initial cell number of 5x106 cells provided the highest fold expansion after 48 hours (1.85-fold). Further refinement during the 5- and 7- day HL-60 expansion studies resulted in a revised set-up that used 4.5x104 cell mL-1 at the velocity of 4.9x10-6 m s-1. At these conditions, it was confirmed that glucose was sufficiently supplied (> 700 mg L-1) and lactate and ammonia were lower than the toxic level (< 1800 mg L-1 lactate and < 4 mM ammonia). Based on the data from the HL-60 study, the FBB was utilized for 14 days of ex vivo Treg expansion. The Treg population in this study was CD4+CD25+FoxP3+CD127high which was isolated from the PBMCs. The protocol was developed using the step-wise approach, with the key steps to take forward being an initial cell density of 5x105 cell mL-1 operated at 4.9x10-6 m s-1and 2-days inoculation in well plates. As a limitation of the initial cell number (< 5x105 cell mL-1) during the protocol development, the Tregs were lost from all cell culture devices. This might be due to apoptosis when the cell was cultured at low cell density, lack of cell-to-cell contact and less catalase against oxidative apoptosis and these studies would be part of the future work, alongside repeats and suppression testing to analyse the Treg function. In conclusion, the study found that an FBB has potential for ex vivo Treg cells expansion, however further studies are required before knowing whether it is a better option than current systems.
Date of Award27 Apr 2022
Original languageEnglish
Awarding Institution
  • University of Bath
SupervisorMarianne Ellis (Supervisor) & Stephen Ward (Supervisor)


  • bioreactor
  • regulatory t cell
  • cell expansion
  • fluidized bed
  • organ transplantation

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