Background: Umbilical cord blood provides a source of hematopoietic stem cells for transplantation with immunological and availability advantages over conventional bone marrow sources. Limited cell numbers and slower engraftment from umbilical cord blood units has led to the clinical development of immobilised Notch ligand Delta-Like 1 to promote ex vivo expansion of a rapidly engrafting cell population. However, current immobilisation methods are not simple to scale in a controlled manner.Results: Delta-Like 1 was immobilised onto streptavidin coated magnetic particles via a heterobifunctionalised polyethylene glycol linker molecule to provide an easily manipulated format of surface protein presentation. CD34(+) enriched cord blood cells were treated with Delta-Like 1 immobilised particles, and immunophenotypic markers measured to monitor population distributions using cluster identification, characterization, and regression software. The amenability of the approach to scalability was evaluated in a micro-scale stirred tank bioreactor. Surface concentration of Delta-Like 1 was well controlled used differing stoichiometric reagent ratios. Protein immobilisation was a cost effective process and particles were efficiently removed from the final cell product. Immobilised Delta-Like 1 is functional and stimulates qualitatively similar CD34(hi), CD38(lo), CD90(lo), CD133(hi), CD135(hi) progenitor expansion in both static culture and scalable stirred culture platforms.Conclusions: Immobilised Delta-Like 1 in this form has the potential to improve the manufacturing efficiency and control of final ex vivo expanded cell product through compatibility with highly controlled and characterised suspension culture systems.

The cell quality following cryopreservation is sensitive to process variation during both cooling and warming. An automated thawer (CellSeal® Automated Thawing System, CookRegentec, Indianapolis, USA) offers the potential to reduce variability in temperature and timing during vial thawing. An experiment was designed to evaluate the capability of this new system to thaw CellSeal vials (CookRegentec) containing cryopreserved human mesenchymal stem cells (RoosterBio Inc. Frederick, Maryland, USA). Post-thaw cell numbers, viability and onward growth capacity from vials of various sizes (2 ml, 5 ml), with different fill volumes (1 ml, 2 ml, 4.7 ml), warmed from different start temperatures (−196°C or −80°C) were determined. The automated thawer demonstrated equivalent performance to a tightly controlled 37°C water bath process in both user-selectable thawing modes (Gentle, Rapid). Furthermore, a simulation of a poorly controlled water bath process with extended incubation in the bath post-thaw demonstrated significant reduction (p = 0.014 and p < 0.001 for 15 mins and 30 mins respectively) in cell recovery and quality, highlighting the risk reduction that could be achieved with automation. Values of post-thaw viability and onward growth of MSCs contained in CellSeal® vials were at the upper end of the range of results typically seen for this cell type.

It is well documented that cryopreservation and resuscitation of human embryonic stem cells (hESCs) is complex and ill-defined, and often suffers poor cell recovery and increased levels of undesirable cell differentiation. In this study we have applied Quality-by-Design (QbD) concepts to the critical processes of slow-freeze cryopreservation and resuscitation of hESC colony cultures. Optimized subprocesses were linked together to deliver a controlled complete process. We have demonstrated a rapid, high-throughput, and stable system for measurement of cell adherence and viability as robust markers of in-process and postrecovery cell state. We observed that measurement of adherence and viability of adhered cells at 1 h postseeding was predictive of cell proliferative ability up to 96 h in this system. Application of factorial design defined the operating spaces for cryopreservation and resuscitation, critically linking the performance of these two processes. Optimization of both processes resulted in enhanced reattachment and post-thaw viability, resulting in substantially greater recovery of cryopreserved, pluripotent cell colonies. This study demonstrates the importance of QbD concepts and tools for rapid, robust, and low-risk process design that can inform manufacturing controls and logistics.