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

点击翻译按钮获取中文摘要

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