Work package 1: Isolation of Blood Outgrowth Endothelial Cells (BOECs)
Leader: Dr. Joris Braspenning, University Hospital of Würzburg
The main objective of work package 1 is to isolate and start the culture of blood outgrowth endothelial cells (BOECs) from haemophilia A patients’ blood. The cells are expanded and characterized for their cell specific phenotype. If all defined release criteria are fulfilled, the cells are then sent to Partner 3 UPO (WP2) for factor 8 gene correction. The other objectives are to optimize and standardize the process and transfer it to GMP conditions. After establishment of the whole process from the isolation to the cryopreservation of the fully expanded cells (WP1-4) at the respective partners (UKW, UPO and UNILO) all the steps involved in the handling of the cells will be set up and run at UKW to demonstrate transferability and practicability at one site.
Description of the tasks
Based on the results achieved during a previous EU-funded project (Re-Liver) this consortium will establish a GMP compliant isolation and expansion procedure of BOECs from peripheral blood of haemophilia A patients which are then initially expanded, characterized and handed over for correction of the factor 8 gene defect carried out by Partner 3 UPO (WP2).
The procedure will include the following tasks:
Task 1: Isolation and initial expansion of BOECs from haemophilia A patients’ blood.
First, the release criteria for initially expanded BOECs will be defined including cell morphology, growth rate (population doublings / hours), purity (defined by the percentage of CD31 positive and CD133 negative cells), and performance in a standard tube formation assay in matrigel . Peripheral blood from patients with severe haemophilia A will be taken in a vacutainer blood bottle and transported immediately to UKW. UKW will start the isolation procedure of BOECs as soon as samples arrive. In combination with a chemical defined endothelial growth medium developed at UKW these culture conditions lead to a high rate of BOEC colony formation that appear already after around 2 weeks in contrast to protocols reported in the literature that state about 4 weeks for this process (Kolbe et al., 2010; Nuzzolo et al., 2014). After reaching a certain size the colonies are harvested for expansion. These expanded cells will then be analysed for the percentage of CD31 positive and CD133 negative cells and on tube formation in matrigel. If the criteria are met, the cells will be sent to Partner 3 UPO (WP2).
Task 2: Isolation and initial expansion of BOECs under GMP conditions.
GMP standards will be adapted for the process of obtaining patient consent forms, the process of blood collection in the clinic as well as the transport to the facility where the genetic modification and expansion will take place. In addition, regulatory affairs involving German and European officials will be included in WP1 to have as early as possible a clear understanding of their requirements for the product to achieve future human clinical evaluation.
Task 3: Chemical defined medium for the growth of BOECs.
The recently developed chemical defined medium for the growth of BOECs currently works for the initial two-dimensional (2D) expansion in culture dishes. The conditions in the bioreactor from WP3 might require adaptation of the medium composition which will then be tested in close collaboration with Partner 4 UNILO (WP3).
Task 4: Phenotypic characterisation of large scale expanded BOECs.