Cryopreservation of mammalian cells based on intracellular saccharide delivery using membrane-permeabilising biopolymers
File(s)
Author(s)
Ren, Jie
Type
Thesis
Abstract
Cell cryopreservation is a key process for fields including cell therapy and biobanking. The toxicity concerns around currently standard cryoprotective agents (CPAs) such as dimethylsulfoxide (DMSO) and glycerol are limiting the development of cell cryopreservation. This thesis presents the work on developing and optimising a glycerol-free and DMSO-free cryopreservation method for mammalian cells using saccharides solely as the CPA with saccharides delivered into cells by a novel pH-responsive polymer for the maximal cryoprotection and minimal cell damage.
The polymer PLP-NDA18 displayed negligible cytotoxicity to different mammalian cells after being synthesised and characterised. Different saccharides, including trehalose, sucrose, glucose, and dextran 40 kDa, were delivered into mammalian cells successfully with the assistance of PLP-NDA18. The PLP-NDA18-mediated intracellular saccharide delivery was proven rapid and efficient with negligible cytotoxicity for different mammalian cells.
The cryopreservation process of saccharide-loaded red blood cells (RBCs) was investigated and optimised. Compared with other commonly-used CPAs, saccharides displayed significantly more effective cryoprotection according to the cryosurvival rates and post-thaw cell characterisation.
In addition, the optimal condition for saccharide-loaded RBC cryopreservation was tested on immortalised and primary human T cells. Key parameters of each step were investigated and optimised using immortalised T cells which led to the preliminary success of the primary T-cell cryopreservation using saccharides as the sole CPA without any other excipient.
Overall, intracellular saccharides have been rapidly delivered into RBCs and T cells using the PLP-NDA18 polymer with minimal cell damage. The cryopreservation protocol with saccharides solely as the CPA presenting at both sides of cells has been successfully developed and optimised for RBCs and immortalised T cells, with preliminary success on primary T-cell observed. This DMSO-free and glycerol-free cryopreservation method for mammalian cells can be used as a platform for cellular products to prompt the development of biobanking and wider application of cell therapy.
The polymer PLP-NDA18 displayed negligible cytotoxicity to different mammalian cells after being synthesised and characterised. Different saccharides, including trehalose, sucrose, glucose, and dextran 40 kDa, were delivered into mammalian cells successfully with the assistance of PLP-NDA18. The PLP-NDA18-mediated intracellular saccharide delivery was proven rapid and efficient with negligible cytotoxicity for different mammalian cells.
The cryopreservation process of saccharide-loaded red blood cells (RBCs) was investigated and optimised. Compared with other commonly-used CPAs, saccharides displayed significantly more effective cryoprotection according to the cryosurvival rates and post-thaw cell characterisation.
In addition, the optimal condition for saccharide-loaded RBC cryopreservation was tested on immortalised and primary human T cells. Key parameters of each step were investigated and optimised using immortalised T cells which led to the preliminary success of the primary T-cell cryopreservation using saccharides as the sole CPA without any other excipient.
Overall, intracellular saccharides have been rapidly delivered into RBCs and T cells using the PLP-NDA18 polymer with minimal cell damage. The cryopreservation protocol with saccharides solely as the CPA presenting at both sides of cells has been successfully developed and optimised for RBCs and immortalised T cells, with preliminary success on primary T-cell observed. This DMSO-free and glycerol-free cryopreservation method for mammalian cells can be used as a platform for cellular products to prompt the development of biobanking and wider application of cell therapy.
Version
Open Access
Date Issued
2023-11-23
Date Awarded
01/05/2024
License URL
Advisor
Chen, Rongjun
Publisher Department
Chemical Engineering
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)