Healthcare Engineering Research Group, Centre for Biological Engineering, Wolfson School of Mechanical and Manufacturing Engineering, Loughborough University, Loughborough, Leicestershire, UK.
BMC Biotechnol. 2017 Aug 4;17(1):65. doi: 10.1186/s12896-017-0383-0.
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.
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, CD38, CD90, CD133, CD135 progenitor expansion in both static culture and scalable stirred culture platforms.
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.
脐带血为移植提供了造血干细胞来源，具有免疫优势和可用性优势，优于传统的骨髓来源。脐带血单位的细胞数量有限，植入速度较慢，导致临床开发了固定化 Notch 配体 Delta-Like 1，以促进快速植入细胞群的体外扩增。然而，目前的固定化方法不易以受控的方式进行规模化。
Delta-Like 1 通过异双功能化聚乙二醇连接分子固定在链霉亲和素包被的磁性颗粒上，提供了一种易于操作的表面蛋白呈现形式。用固定化的 Delta-Like 1 颗粒处理 CD34 富集的脐带血细胞，并使用聚类识别、特征描述和回归软件测量免疫表型标记物，以监测群体分布。通过微尺度搅拌槽生物反应器评估了该方法的可扩展性。使用不同的化学计量比试剂可以很好地控制 Delta-Like 1 的表面浓度。蛋白质固定化是一种具有成本效益的过程，并且可以有效地从最终细胞产物中去除颗粒。固定化的 Delta-Like 1 是功能性的，在静态培养和可扩展搅拌培养平台中都能刺激类似的 CD34、CD38、CD90、CD133、CD135 祖细胞扩增。
这种形式的固定化 Delta-Like 1 具有通过与高度控制和表征的悬浮培养系统兼容来提高体外最终扩增细胞产品的制造效率和控制的潜力。
