Investigating bioengineering approaches to produce immuno-modulatory mesenchymal stromal cells and their extracellular vesicle

间充质基质细胞（MSC）是一种罕见的细胞群体，存在于体内的大多数组织中，并且在维持这些结构方面非常宝贵。除了形成骨、肌肉或脂肪组织外，MSC还可以是免疫抑制性的。它们既可以通过调节免疫细胞（ImCs）来解决炎症，也可以通过多种机制（如释放可溶性因子或脂质结合的囊泡-细胞外囊泡（EV））来促进组织修复。目前，MSC正在作为基于细胞的疗法进行试验，然而，需要大量的MSC。EV可以减少这种细胞需求，但患者治疗本身需要大量细胞，因此在输注细胞或EV之前，MSC的扩增是必不可少的。不幸的是，MSC在实验室培养中随着时间的推移自发分化，失去了其关键的幼稚免疫调节（ImmM）能力。此外，对MSC的临床需求仍未得到满足，因为来自老年供体的MSC比来自年轻供体的MSC相对较弱。这限制了目前可以用这种疗法治疗的患者数量，并且能够生长功能性的幼稚MSC，保留其重要的抗炎和组织修复能力仍然是科学研究的关键目标。因此，有必要研究新的方法，以扩大在实验室中的MSC，同时保持他们在一个幼稚的治疗状态。以前的观察已经揭示了改变MSC的生长条件，影响他们的代谢，但保留他们的ImmM和修复性能的潜力。例如，MSC粘附的变化会影响其能量产生;这是MSC如何调节其生理的关键特征。向MSC细胞培养物中补充小分子也可以重现这种效果并维持这些细胞的ImM功能。这些代表了培养个体患者治疗或EV收集所需的大量细胞的新方法。这些培养方法如何影响MSC ImM特性或EV治疗抗炎潜力仍有待确定。因此，使用替代的生长条件可能允许扩增治疗相关的MSC及其EV。将分子从EV递送到细胞可能具有控制免疫系统以获得治疗益处的潜力。虽然活跃生长的细胞不断脱落EV-并且EV含有各种货物，包括可溶性因子，DNA和其他蛋白质- EV治疗炎症条件已被证明与标准药物治疗的毒副作用无关。该项目将建立在初步研究结果的基础上，探索新型培养系统的潜力，以产生用于治疗的功能细胞的大规模培养物。将对基于聚合物的生长表面与生物反应器中的扩增相结合进行评价。MSC将通过基于微载体的生物反应器扩增，最初是小规模的，以优化培养条件。该系统允许MSC的快速生长，比传统的基于实验室的培养更快地增加其数量。扩增的MSC“质量”将通过测量生长、代谢概况、ImC上的ImM功能和MSC EV调节炎症的能力来评估。还将分析在这些培养系统内从MSC释放的可溶性因子。这种方法可能会导致一种新的诊断测试，以筛选大规模的治疗培养物，为患者提供最佳的功能细胞。除此之外，从生物反应器扩增中收集的MSC EV将被检查和表征，并评估其对其他ImCs的影响。总的来说，该项目将为新的生物处理和诊断方法提供信息，以允许MSC培养物的升级，以产生治疗所需的细胞或EV数量。这有可能揭示通过调节其体外生长条件来逆转来自老年供体的MSC功能降低的新策略

Mesenchymal stromal cells (MSCs) are a rare population of cells found in most tissues within the body and are invaluable in the maintenance of these structures. As well as forming bone, muscle or fatty tissues, MSCs can also be immunosuppressive. They can both resolve inflammation by modulating immune cells (ImCs) and promote tissue repair through multiple mechanisms such as the release of soluble factors or lipid-bound vesicles - extracellular vesicles (EVs). Currently, MSCs are being trialed as a cell-based therapy, however, large numbers are required. EVs may reduce this cell requirement, but are themselves needed in high quantities for patient therapy, therefore expansion of MSCs is essential before transfusion of either cells or EVs. Unfortunately, MSCs spontaneously differentiate over time in laboratory culture, losing their critical naïve immunomodulatory (ImM) abilities. Moreover, the clinical demand for MSCs remains unmet as MSCs from older donors are comparatively less potent than those from younger donors. This limits the number of patients that can be currently treated with this therapy and the ability to grow functional, naïve MSCs which retain their important anti-inflammatory and tissue repair abilities remains a key goal for scientific research. Thus, there is a need to investigate new methodologies to expand MSCs in the laboratory whilst maintaining them in a naïve state for therapy.Previous observations have revealed the potential for altering the growth conditions of MSCs which affect their metabolism yet retaining their ImM and repair properties. Changes in MSC adhesion for example, affect their energy production; a key feature of how MSCs modulate their physiology. Supplementation of small molecules to MSC cell culture can also reproduce this effect and maintain the ImM functions of these cells. These represent new approaches to culturing the large number of cells required for individual patient therapy or EV collection. How these culture methods affect MSC ImM properties or EV therapeutic anti-inflammatory potential remains to be defined. Therefore using alternative growth conditions potentially allows expansion of therapeutically relevant MSC and their EVs. The delivery of molecules from EVs to cells may have the potential to control the immune system for therapeutic benefit. While actively growing cells continuously shed EVs - and EVs contain a variety of cargos including soluble factors, DNA and other proteins - EV treatment of inflammatory conditions has been demonstrated not to be associated with toxic side effects of standard drug treatments. This project will build upon initial findings, exploring the potential of novel culture systems to generate large scale cultures of functional cells for therapy. Combining polymer-based growth surfaces with expansion in bioreactors will be evaluated. MSCs will be expanded via a microcarrier-based bioreactor, initially on a small-scale to optimise culture conditions. This system allows the rapid growth of MSCs, increasing their numbers more quickly than traditional laboratory-based culture. Expanded MSC 'quality' will be evaluated by measuring growth, metabolism profiles, ImM function on ImCs, and the MSC EV's ability to modulate inflammation. Released soluble factors from MSCs within these culture systems will also be analysed. This approach may lead to a new diagnostic test to screen large scale cultures for therapy to provide patients with optimal, functional cells. Alongside this, MSC EVs collected from bioreactor expansion will be examined and characterised, with their effects on other ImCs evaluated.Overall, this project will inform on new bioprocessing and diagnostic approaches to allow the upscaling of MSC cultures to generate the required numbers of cells or EVs required for therapy. This has the potential to reveal new strategies for reversing the reduced functionality of MSCs from older donors through modulating their in-vitro growth condition