Immobilisation of Delta-like 1 ligand for the scalable and controlled manufacture of hematopoietic progenitor cells in a stirred bioreactor

Abstract

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.

Keywords: Cell culture; Delta-like 1; Ex vivo expansion; Hematopoietic progenitor cell; Immobilised; Manufacturing; Notch; Scalable.

Fig. 1

Delta-Like 1 can be reliably biotinylated and immobilised onto streptavidin coated magnetic micro particles. DL1 can be immobilised using two distinct reaction pathways, a(i) forward, and a(ii) reverse. In the forward pathway DL1 primary amines first react with the NHS functionality of the heterobifunctionalised PEG molecule. Once reacted, streptavidin coated magnetic particles are added allowing the biotin functionality to bind with the streptavidin, resulting in iDL1. In the reverse pathway the heterobifunctionalised PEG molecule first reacts with the streptavidin coated particle, the DL1 is then added and reacts with the NHS functionality to produce iDL1 (n = 3). b DL1 could be immobilised via both pathways, the forward pathway demonstrated no effect of the DL1 solvent. The reverse pathway immobilised most DL1 per particle when DL1 was dissolved in PBS (n = 3). c Optimisation of the reverse pathway for maximum DL1 immobilisation demonstrated a rapid saturation of binding between streptavidin and the heterobifunctionalised PEG molecule during the first stage of the synthesis (n = 3) and d a 2 h optimal incubation time for the second stage (n = 3)

Fig. 2

Surface concentration of immobilised Delta-Like 1 can be controlled during particle synthesis. a Using the forward reaction pathway it is possible to control the presentation of DL1 by regulating the ratio of the heterobifunctionalised PEG molecule to the number of primary amine groups present in DL1 (n = 3). b Using the reverse synthetic pathway, DL1 immobilisation increases with increasing DL1 concentration in the second stage of the process (n = 3). c The use of increased PEG chain lengths within the heterofunctionalised biotin molecule results in a reduction in DL1 immobilisation using the reverse synthetic method (n = 3)

Fig. 3

Investigating manufacturing challenges of the use of immobilised Delta-Like 1. Efficacy of the immobilisation process was evaluated using an ELISA immunoassay. a Efficacy was dependent on the number of particles generated, with up to 96% of the protein immobilised (n = 3). Removal of magnetic particles was evaluated by flow cytometry. b Voltage setting and gating was optimised to assess particle number in culture (i) and after removal from culture (ii), c removal of magnetic particles from cells at the end of the culture period was evaluated, MACS^® columns removed 99.6% of particles with minimal cell loss (<1%) (n = 6)

Fig. 4

Immobilised Delta-Like 1 can induce skewed lineage output of hematopoietic progenitor cells in static culture. CD34⁺ enriched cells (95% CD34⁺) were cultured with iDL1 at three concentrations alongside three controls: untreated, soluble DL1 and blank particles only (n = 5). a FCS files were uploaded into Cytobank and CITRUS cluster trees derived, clustering on CD34, CD33, CD38, CD45ra, CD90, CD133 and CD135 marker intensities. Cluster trees of CD34, CD38, CD90, CD133 and CD135 are shown, identifying the progenitor nodes defined phenotypically as CD34^hiCD38^negCD90^negCD133^hiCD135^hi. b Progenitor nodes (identified in 4A) were evaluated for their percentage abundance at day 6 (i) and day 12 (ii), cells cultured with high levels of iDL1 contained a significantly higher percentage of progenitor cells compared to all controls. All concentrations of iDL1 significantly improved progenitor abundance compared with untreated and soluble DL1 controls at day 12. Further phenotypic analysis was undertaken at day 12 to evaluate the expression of more mature surface markers. FCS files were uploaded into Cytobank and CITRUS cluster trees derived, clustering on CD13, CD14, CD15, CD24, CD33, CD38 and CD123 marker intensities. c Cluster tree of CD14 expression, the parent CD14^hi node was identified. d Evaluation of percentage abundance within the parent CD14^hi node at day 12 identified culture with high and medium levels of iDL1 resulted in significantly less mature CD14^hi cells compared to all controls. e Cluster tree of CD15 expression, the parent CD15^hi node was identified. (F) Evaluation of percentage abundance within the parent CD15^hi node at day 12 identified culture with high levels of iDL1 resulted in significantly less mature CD15^hi cells compared to all controls. All concentrations of iDL1 significantly decreased CD15^hi abundance compared with untreated and soluble DL1 controls. Values were considered (*) statistically significant for P < 0.05, (**) very statistically significant for P < 0.01

Fig. 5

Immobilised Delta-Like 1 induces the same lineage skewing of hematopoietic progenitor cells in stirred bioreactor systems. CD34⁺ enriched cells (95% CD34⁺) were cultured with iDL1, alongside untreated, soluble DL1 and blank particles as controls, in a mechanically agitated suspension bioreactor (n = 3). a As previously shown in the static platform, FCS files were uploaded into Cytobank and CITRUS cluster trees derived, clustering on CD34, CD33, CD38, CD45ra, CD90, CD133 and CD135 marker intensities. Cluster trees of CD34, CD38, CD90, CD133 and CD135 are shown, identifying the progenitor node defined phenotypically as CD34^hiCD38^negCD90^negCD133^hiCD135^hi. b The addition of iDL1 to CD34⁺ enriched cells in a stirred culture system led to the same trend in lineage skewing previously identified in static. Evaluation of the percentage abundance within the progenitor node at day 12 identified high levels of iDL1 resulted in significantly more progenitor cells compared to all controls. c Cluster tree of CD15 expression, the parent CD15^hi node was identified. d Evaluation of percentage abundance within the parent CD15^hi node at day 12 identified, as in the static platform, culture with high levels of iDL1 resulted in significantly less mature CD15^hi cells compared to all controls. Values were considered (*) statistically significant for P < 0.05