Hypothermic encapsulation of bone marrow stem cells in alginate hydrogel beads

Robinson, Conor James (2026) Hypothermic encapsulation of bone marrow stem cells in alginate hydrogel beads. PhD thesis, University of Glasgow.

Abstract

The bone marrow niche microenvironment (BM) is highly complex. It is home to haematopoietic stem cells (HSCs) and, most importantly, long-term HSCs (LT-HSCs) reside here in close proximity to osteoblasts and are responsible for producing all blood and immune cells (Nombela-Arrieta et al., 2013). Mesenchymal stem/stromal cells (MSCs) that form all bone, cartilage, muscle and fat cells, also reside in BM (Fink & Zachar, 2011; Gang et al., 2004; B. Johnstone et al., 1998; Nishimura et al., 2015; Mathew et al., 2011). Cytokines expressed by MSCs are known to regulate HSC quiescence – a low metabolic state adopted for long-term survival –, cell cycle entry and differentiation (Fereydani et al., 2024). HSCs are at the forefront of medical science. They have been used for decades in stem cell transplants and remain the most effective method of treating damaged bone marrow (Khaddour et al., 2023). Due to the increasing average lifespan of the population, the natural occurrence of malignant mutations in HSCs and the resultant blood disorders are ever more present in our global society (Jaiswal & Ebert, 2019). HSC genetic damage also occurs as a side effect of many widely used drugs, such as chemotherapeutic agents, and while we know this damage leads to leukaemia, we currently have no curative treatments, and prognosis is poor; further emphasising the need for research into the area (Ezoe, 2012). Unfortunately, HSCs are notoriously sensitive to cryopreservation and culture under traditional 2D culture methods (Branco et al., 2024; Rimac et al., 2023). Combined with being a very rare cell type – 0.01% of the total blood population – this makes them incredibly expensive and therefore relatively inaccessible to many institutions for research purposes (Lee-Six et al., 2018). Further, much of the research into culturing HSCs aims to expand the population and encourages an activated cell-state (Cheng & Scadden, 2009). This is not comparable to how the most clinically relevant LT-HSC population reside in vivo, and therefore, a way to maintain quiescent HSCs and present them for drug testing should be of utmost importance to give insight into this critical yet under-researched cell type and the diseases that impact them. There is also now a societal push towards improved ethical standards and reduced environmental impact of research, which threatens to move beyond a point where current and developing techniques can keep up. For example, while not legally required, the medicines and healthcare products regulatory agency requires animal testing for medicines before human clinical trials (The Use of Animals in Pharmaceutical Research, ABPI, 2025). This is largely due to a lack of adequate and well-tested alternatives, and therefore, foundational research on how human models could be created, such as this project, is necessary to meet phase-out targets by the current government (Labour-Party-Manifesto-2024, 2024.). Work produced by the LifeTime centre for doctoral training intends to discover and develop non-animal technologies in collaboration with industry, charities and the NHS with real-world medical applications (About Us – LifETIME CDT, 2025). The goal of this thesis was to develop a methodology which could improve access and reduce the cost of rare and expensive bone marrow stem cells to enable their use more widely in clinical and research applications and in turn, aid advances in this area of work. More specifically, hypothermic temperatures and alginate hydrogels were used to encapsulate human primary stem cells and induce the low-metabolic state of quiescence. The experimental methodology and optimum temperature for single MSCs were first confirmed to be 15 °C using viability staining as proposed by Atelerix, which is currently used as an environmentally friendly and animal-free preservation alternative (Swioklo et al., 2016). Following this, the surface marker expression of monolayer and encapsulated MSCs was compared. CD34+ haematopoietic cells were then encapsulated, stored, and their surface marker expression analysed to determine the optimum temperature, media and media supplementation for encapsulation. Donor variability prevented meaningful conclusions; however, haematopoietic stem and progenitor cells (HSPCs) were maintained more effectively than others. Raising questions about the applicability of using CD34+ cells as ‘HSCs’, for only a very small number of these cells have functional properties of stem cells. Next, MSC spheroids were encapsulated and their viability and surface marker expression compared to non-encapsulated and monolayer controls. The optimum media and temperature were analysed, with similar trends observed between conditions. Surface marker expression indicated that aggregation may induce phenotypical changes, but this was not enhanced by encapsulation. Further, the surface stiffness of gels without cells, with cells, with spheroids, and with cells and spheroids was analysed, and a stiffness in line with that used to induce osteogenic differentiation in literature was observed (Chaudhuri et al., 2016). Finally, a more extensive panel was used to analyse CD34s to determine which subsection of HSPCs was maintained under encapsulation, and colony-forming assays were performed to assess functional capacity. The core findings of the study showed that MSC spheroids are viable for longer than single MSCs when encapsulated in alginate at hypothermic temperatures, and that aggregation induces surface marker changes that indicate low motility and immunosuppressive abilities. Further, this process does induce quiescence within the quiescence-capable subpopulations of HSPCs and does so less favourably for more mature subpopulations. Therefore, this method could potentially be used for the storage and transport of this cell type, in addition to alternate uses such as a scaffold with which to present HSPCs or MSC spheroids in a simple model for research, drug testing or as a ready-to-use cell therapy. The project exclusively uses directly applicable human primary cells, uses no animal products and is fully biocompatible. Cell-laden alginate has wide-reaching applications; however, this work could be used specifically to improve access to stem cells for future research and, therefore, assist in the development and reduce costs of non-animal testing methods and models, whilst itself being more environmentally friendly than current alternative transport and storage methods.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Supported in part by the EPSRC.
• Subjects: Q Science > QR Microbiology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Funder's Name: EPSRC
• Supervisor's Name: Dalby, Professor Matthew, Salmeron-Sanchez, Professor Manuel and Tsimbouri, Dr. Monica
• Date of Award: 2026
• Depositing User: Theses Team
• Unique ID: glathesis:2026-86010
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 16 Jun 2026 12:39
• Last Modified: 16 Jun 2026 12:40
• Thesis DOI: 10.5525/gla.thesis.86010
• URI: https://theses.gla.ac.uk/id/eprint/86010