Centrifugal recovery of embryonic stem cells for regenerative
Doctoral thesis, UCL (University College London).
In order to realise the potential of embryonic stem (ES) cells as a regenerative medicine, it is crucial that economical, robust and scalable bioprocesses be established. Because bioprocesses irrevocably define the safety and efficacy of any biologically derived product, an understanding of the the impact of the engineering environment on ES cells is sought. This thesis uses murine ES cells as a mimic for ES cell types that will be used in cell based regenerative medicine applications to examine the bioprocessing impact of centrifugal recovery cells. A micro scale-down approach was used to examine the e�ffects of centrifugal force, centrifugation time and process temperature on both the yield and biological characteristics of cells subjected to batch centrifugation. When subjected to centrifugation, mES cell loss and cell damage does not appear to occur during the settling or cell pelleting. In general, 5-25% of cells are lost during pellet resuspension to recover the centrifuge cells. The level of cell loss is determined by a combination of centrifugal force, centrifugation time and process temperature. The extent of damage of the remaining cells (i.e. cells not lost during resuspension) is minimised at lower processing temperatures. It is hypothesised that at low processing temperatures, cell loss is minimised due to weak cell-to-cell contact and are thus less susceptible to damage caused by the shear environment generated to disperse the collected cell pellet. The concept of Windows of operations was also applied to evaluate an optimal set of centrifuge operating conditions that results in minimal cell loss and cell damage. The process visualisation tool indicates that operating the centrifuge at 5-9 mins x 300-500 g will result in maximum cell recovery at 4, 21 and 37oC process temperatures. The influence of centrifugation on the biological characteristics of mES cells revealed changes in proliferative capacity, pluripotency and differentiation status when exposed to varying levels of centrifugal force. mES cells exposed to increasing levels of centrifugal force up to 2,000 g progressively lost pluripotency. The pluripotency potential of cells exposed to 3,000 g of centrifugal force was not signi�ficantly di�fferent from un-centrifuged mES cells. Di�fferentiating mES cells exposed to increasing levels of centrifugal force exhibited increased cell proliferation and a possibility of early induction of endoderm and mesoderm di�fferentiation. Although limited in some areas, the results strongly suggest that restricting exposure to no more than low levels of centrifugal force is necessary to safeguard the stability of the desired mES cell characteristics. Overall, the insight gained from the work accomplished serves to create and establish an awareness of the challenges faced within the arena of whole cell bioprocessing for regenerative medicines.
|Title:||Centrifugal recovery of embryonic stem cells for regenerative medicine bioprocessing|
|Open access status:||An open access version is available from UCL Discovery|
|UCL classification:||UCL > School of BEAMS > Faculty of Engineering Science > Biochemical Engineering|
Archive Staff Only