Bioprocessing Research For Cellular Products

This project aims to develop novel preservation platform technologies required for the successful banking of human cells, an absolute prerequisite for their use as products. Many regenerative medicine products rely on the delivery of live cells to patients. At present this is exemplified by established therapeutic interventions such as bone marrow transplantation, blood transfusion and corneal grafting; future generations of products may include bio-artificial matrices that incorporate donor stem cells, for example bone replacement and repair devices, and artificial 'mini-organs' such as pancreas or liver. Current cryopreservation of stem cell based products results from historic work, using DMSO as a cryopreserving agent which is largely unsubstantiated with respect to final biological activity. DMSO can be toxic to cells, lead to low viabilities post thaw and both genetic and epigenetic instability (i.e loss of pluripotency) over long term culture. Cryopreservation of blood cells has been attempted previously, with limited success due to loss of cell integrity, primarily due to the breakdown of the cell membrane and consequent loss of overall cell structure. A variety of techniques have been investigated for delivering trehalose, a membrane impermeable cryoprotectant, into mammalian cells, including microinjection, ion channel stimulation, pore formation using mutant bacterial toxins, fluid phase endocytosis, and internal trehalose synthesis via genetic engineering but intracellular trehalose concentrations achieved in erythrocytes has not exceeded 50 mM and is therefore below thresholds for cryoprotection. Biopolymer mediated cell loading achieves substantially increased intracellular trehalose concentrations of up to 251 mM and a concomitant improvement of erythrocyte cryosurvival of up to 20.4 % as compared with conventional methods of loading trehalose into cells. The technology utilizes novel amphiphilic biopolymers that interact with the external cell membrane to enable penetration and retention of cryoprotectant agents into the cells. Membrane permeabilisation by these Cell Permeating Polymers (CPPs) is rapid and completely reversible via washing with buffer. Cellular uptake of trehalose is dependent on polymer molecular structure, concentration, pH, external trehalose concentration, incubation temperature and time. Optimization of these parameters imparts cellular osmoprotection. Overall, a total cell recovery through a single freeze-thaw cycle at -80oC of 82.6 % has been achieved, which compares with a recovery of only 0.8 % for cells frozen in PBS. This proposal aims to explore the CPP mediated loading of preservation agents into stem cells, to examine preservation by freezing and dessication and to arrive at integrated processing routes for the preparation of optimally stable stem cells.

The project aims to develop a novel cell preservation platform technology for the successful banking of human cells, an absolute prerequisite for their use as regenerative medicines for improving human health. Current cryopreservation of stem cells results from historic work, using DMSO as a cryopreserving agent which is largely unsubstantiated with respect to final biological activity. DMSO can be toxic to cells, lead to low viabilities post thaw and genetic and epigenetic instability (i.e loss of pluripotency) over long term culture. DMSO has also been linked to stroke and myocardial infarction in stem cell transplant recipients. Trehalose, a hydrophilic disaccharide of glucose accumulated in a wide variety of freezing and desiccation tolerant organisms, is an attractive non-toxic replacement for DMSO. However, trehalose does not normally traffic across the lipid membrane of cells and so to achieve maximum protective effect it must be delivered into cells. Many techniques have been investigated for delivering trehalose into mammalian cells. However, methods yielding sufficient intracellular trehalose for biopreservation (~100-200mM) have utilized hazardous materials, such as mutant bacterial toxins, or have lacked scalability, such as the use of microinjection. Technology developed at Cambridge exploits amphiphilic biopolymers to promote the trafficking of cryoprotectants across erythrocyte cell membranes. By this method it is possible to achieve increased intracellular trehalose concentrations of up to 250 mM and a concomitant improvement of cryosurvival of up to 21%, as compared with conventional methods of loading trehalose into cells. The technology is based on novel, non-toxic cell permeating polymers (CPPs) that insert within the external phospholipid cell membrane to create 'patches' that enable the penetration and retention of bioprotectant within the cells. This project aims to extend the technology to the preservation of human stem cells.

Throughout the project actions will be taken to ensure positive impacts upon biopreservation practices, the competitiveness of BRIC industrial members and UK bioprocessing industries and the training of skilled bioprocessing researchers. These might be: - Facilitating the banking of validated multi/pluripotent cells in sufficient numbers to create reproducible and cost-effective regenerative cell therapy products, so improving quality of life and wellbeing. - Providing BRIC industrial members with a technical competitive edge in supply chain management for regenerative cell therapy products, resulting in financial benefit to the UK bioprocessing sector. - Generating commercially exploitable IP on stem cell preservation technologies, to the potential financial advantage of the academic sector. - Meeting the need of the regenerative cell therapy industries for highly skilled researchers trained in cell culture, biological characterisation and formulation. - Potentially, providing generic technologies that might find beneficial application in the banking of mammalian cells for the manufacture of therapeutic proteins and other biological medicinal products. The team comprises of members representing two leading universities, Cambridge's Bioscience Engineering Laboratory and Loughborough's Centre for Biological Engineering, who will work closely in collaboration. These impacts will be communicated internationally through a wide spectrum of activities; by lectures and displays to specialists (academic and industrial practitioners) and lay audiences, by publications in popular and international scientific journals, by the web sites of the participating institutions, by a blog or Facebook site, by our school outreach programmes and by press releases through the University's Office of External Affairs and Communications.