Human mesenchymal stem cells (hMSCs) are a promising candidate for cell-based therapies given their therapeutic potential and propensity to grow in vitro. However, to generate the cell numbers required for such applications, robust, reproducible and scalable manufacturing methods need to be developed. To address this challenge, the expansion of hMSCs in a microcarrier-based bioreactor system was investigated. Initial studies performed in T-flask monolayer cultures investigated the effect of key bioprocess parameters such as dissolved oxygen concentration (dO2), the level of medium exchange and the use of serum-free media. 20 % dO2 adversely impacted cell proliferation in comparison to 100 % dO2, whilst FBS-supplemented DMEM was found to be the most consistent and cost-effective cell culture medium despite the advances in serum-free cell culture media. Several microcarriers were screened in 100 mL agitated spinner flasks where Plastic P102-L was selected as the optimal microcarrier for hMSC expansion given the high cell yields obtained, its xeno-free composition and effective harvest capacity. The findings from the initial small-scale studies culminated in the successful expansion of hMSCs on Plastic P102-L microcarriers in a fully equipped 5 L stirred-tank bioreactor (2.5 L working volume), the largest reported volume for hMSC microcarrier culture to date. A maximum cell density of 1.68 x 105 cells/mL was obtained after 9 days in culture; further growth was limited by the low glucose concentration and lack of available surface area. A novel, scalable harvesting method was also developed, allowing for the successful recovery of hMSCs. Importantly, harvested hMSCs retained their immunophenotype, multipotency and ability to proliferate on tissue culture plastic.

Regenerative medicine and cell therapies hold great potential to treat a variety of medical conditions. Product characterisation of these therapies is particularly difficult as they pose regulatory challenges due to donor heterogeneity and the lack of standardised lot release tests that can reliably predict in vivo function. Human mesenchymal stem cells (hMSCs), also called multipotent stem cells or mesenchymal stromal cells, are a viable option in cell therapies due to their immunosuppressive and pro-angiogenic functions. Currently there are no standardised methods or potency assays to quantify these properties. To address this, five individual hMSCs lines from different donors were created and characterised based upon growth rate, differentiation capability and extracellular surface protein expression. A novel multiparameter flow cytometry method to characterise the cells based upon extracellular surface markers was developed that supports high-throughput and high-content analyses. Three candidate lines were taken forward and assessed in multiple in vitro bioassays that examined the hMSC immunosuppressive response to a defined inflammatory environment, effect on T-cell proliferation, and effect on a mixed lymphocyte population. Next, the angiogenic properties were assessed using human umbilical vein endothelial cells (HUVECs) tube formation as a model for cardiac regeneration. This involved utilising automated time lapse microscopy techniques coupled with image analysis software to quantify endothelial to tube formation. Further analysis of the hMSC secretome revealed differences in the levels of pro-angiogenic cytokines such as vascular endothelial growth factor, hepatocyte growth factor and IL-8. Significant differences in angiogenic potency were found between the hMSC lines. This thesis highlights the need to develop specific assays that reflect the intended clinical action. Taken together, these quantitative approaches provide valuable tools to measure hMSC quality and potency, and supports continued efforts to improve characterisation strategies for cellular therapies.

Periodontal disease affects 47% of Americans over 30. Characterized by microbial dysbiosis and unregulated inflammation, severe periodontitis causes degradation of bone and soft tissue around teeth. Current treatments have limited regenerative outcomes and frequent reinfection by harmful bacteria. Human mesenchymal stem cells (hMSCs) have been shown to promote wound healing and tissue regeneration. Many therapeutic benefits of hMSCs are due to their secretome products, like exosomes. Our long-term goal is to develop periodontal therapies with hMSC exosomes. The objectives of this study were to determine the effect of hMSC-derived exosomes on cellular activity of hMSCs and investigate whether hMSC exosome treatment reduces pro-inflammatory cytokine production in LPS-activated RAW264.7 cells. The specific aims of this study were: 1) Determine the characteristics of hMSC-derived exosomes, 2) Determine the biological effect of exosomes on cellular activity of hMSCs, 3) Determine whether exosomes treatment can inhibit cytokine production in activated RAW264.7 cells, and 4) Determine the role of exosomal miRNA in pro-inflammatory cytokine production of RAW264.7 cells. To investigate, exosomes were first harvested from hMSCs culture media through ultracentrifugation. Exosomes were then observed under a transmission electron microscope (TEM) and assessed for surface markers using Western Blot. A transwell migration assay was used to evaluate the chemotactic effect of exosomes. To study the effect of exosomes on stem cell proliferation, exosomes were administered to hMSCs. The immunogenicity of MSC exosome was also evaluated. After 72 hours, cells were lysed and DNA was measured. To study anti-inflammatory effects of exosomes, LPS stimulated RAW264.7 cells were treated with exosomes. Interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFα) levels of supernatant were measured by ELISA. To study exosomal miRNA, exosomal miRNAs were overexpressed in RAW264.7 cells and these cells were stimulated with LPS. IL-6 and TNFα were measured by ELISA. TEM images showed that exosomes are nano-sized vesicles (~100 nm). Western blot images showed that CD63 and CD81 are enriched in exosomes compared to total cell lysates. Exosome treatment increased cell proliferation and migration in hMSCs. At the doses that are chemotactic and mitogenic, MSC exosomes had minimal effect on the inflammatory cytokine IL-6 production. Treatment with exosomes significantly decreased IL-6 and TNFα production in RAW264.7 cells activated by LPS. Transfecting RAW264.7 cells with exosomal miR-760 significantly decreased IL-6 production, but had minimal effect on TNFα. Our results indicate that exosomes have a pleiotropic activity, which includes stimulating stem cell migration and proliferation, and mitigating the inflammatory response. Therefore, hMSC exosome delivery is promising for the treatment of periodontal diseases.

No description available.

L'essor des thérapies régénératives au cours des 10 dernières années a entraîné un effort de recherche important, mais l'obtention des cellules souches humaines en quantité suffisante reste cependant encore problématique, notamment concernant les cellules souches mésenchymateuses (CSM). Ces travaux ont donc mis en œuvre une approche à la croisée de la biologie et du génie des procédés afin d'identifier les verrous limitant la croissance des CSM. L'étude des méthodes d'intensification de culture a été entreprise grâce à l'utilisation de microporteurs et d'une plateforme de minibioréacteurs de 200~mL. Puis le développement d'un milieu de culture sans sérum a été testé dans le but de maximiser la croissance cellulaire dans des conditions biochimiques contrôlées. Les CSM humaines en tant que modèle type en thérapie cellulaire ont été démontrées comme extrêmement sensibles aux phases de congélation/décongélation, aux variations de température, à un vieillissement prématuré et nécessitant un milieu de culture complexe riche en facteurs de croissance et d'adhérence. Suite à cette étude, plusieurs écueils pourront être évités lors de la montée en échelle d'un procédé de culture de CSM afin d'intégrer leurs paramètres biologiques intrinsèques aux paramètres d'ingénierie des bioréacteurs (transfert de chaleur, contraintes hydrodynamiques, surface d'adhérence) / Progress in regenerative medicines over the past ten years have led to an important research mobilisation, but obtaining a sufficient amount of human stem cells remains nonetheless problematic, especially for mesenchymal stem cells (MSC). Hence, this work developed an approach coupling biology and process engineering to identify barriers limiting MSC growth. The study of scaled-up amplification methods was performed using microcarriers and a 200~mL minibioreactors platform. In order to maximise MSC growth in a biochemically controlled environment, a serum free medium development was tested as well. Human MSC as model cell type for cellular therapies have thus been demonstrated as extremely sensitive to freeze/thaw cycles, temperature variations, subject to premature aging and needing a complex medium enriched in multiple growth and adherence factors. Following this study, several pitfalls might be avoided during MSC process scale-up by integrating the cells biology into the bioreactors' process engineering parameters (heat transfer, hydrodamic stress, adhesion surface)

Human mesenchymal stem cells (hMSCs) have been identified as a promising cell-based therapy candidate to treat a number of unmet clinical indications, however, in vitro expansion will be required to increase the available number of cells and meet this demand. Scalable manufacturing processes, amenable to closed, single-use and automated technology, must therefore be developed in order to produce safe, effective and affordable hMSC therapies. To address this challenge, a controlled serum-free end-to-end microcarrier process has been developed for hMSCs, which is amenable to large-scale manufacture and therefore increasing economies of scale. Preliminary studies in monolayer culture assessed the level of variability in growth between five hMSC donors, which was found to have a variance of 25.3 % after 30 days in culture. This variance was subsequently reduced to 4.5% by the development of a serum-free monolayer culture process with the maintenance of critical hMSC characteristics and an increased number of population doublings. In order to transfer this into a scalable system, the serum and serum-free expansion processes were transferred into suspension by the addition of plastic microcarriers in 100 mL spinner flasks without control of pH or dissolved oxygen (DO). This achieved a maximum cell density of 0.08 ± 0.01 · 106 cells.mL-1 in FBS-based medium, 0.12 ± 0.01 · 106 cells.mL-1 in HPL-based medium and 0.27 ± 0.03 · 106 cells.mL-1 in serum free medium after six days. In order to drive consistency and yield into the manufacturing process, a process control system was developed for the FBS-based microcarrier expansion process in a 100 mL DASbox bioreactor platform to control DO, pH, impeller rate and temperature. Reduced impeller rates and DO concentrations were found to be beneficial, with a final cell density of 0.11 ± 0.02 · 106 cells.mL-1 and improved post-harvest outgrowth and colony-forming unit (CFU) potential compared to uncontrolled microcarrier and monolayer culture. This controlled bioreactor expansion process was then applied to the previously developed serum-free microcarrier process, eventually achieving a final cell density of 1.04 ± 0.07 · 106 cells.mL-1, whilst retaining key post-harvest hMSC characteristics. Following the controlled serum-free expansion and harvest of hMSCs, a downstream and cryopreservation process was developed to assess the impact of prolonged holding times and subsequent unit-operations on hMSC quality characteristics. This showed that hMSCs are able to maintain key characteristics throughout the entire end-to-end process, demonstrating their potential for commercial scale manufacture.

Abstract Bone remodelling occurs in humans throughout life, therefore bone is continuously renewed to better respond to changes in weightbearing circumstances. Bone remodelling is extremely vulnerable during fracture healing and integration of prostheses into the surrounding bone. Bone remodelling is a complex system in which many growth factors, cytokines and enzymes, which are essential for the differentiation of osteoblasts and osteoclasts, are involved. Some widely used drugs can affect this sensitive system of remodellation in unexpected manner. Painkillers such as cyclooxygenase (COX) inhibitors have been demonstrated in animal studies to interfere with fracture healing and a few retrospective clinical studies confirm these observations. Bisphosphonates (BP), main target of which is the bone resorbing osteoclast, have been suggested to be the drug of choice to improve periprosthetic bone density and thus prevent aseptic loosening of implants. The exact mechanism of action of clodronate (CLO), a non-amino-BP, which was selected for the study, has not been clarified thus far. In order to gain a deeper understanding of the role of the COX enzyme in the differentiation of osteoblasts we studied human mesenchymal stem cell (hMSC) cultures in the presence of different COX-inhibitors; indomethacine, parecoxib and NS398, a specific COX-2 inhibitor. We used the liposome encapsulated CLO metabolite (AppCCl2p) to study in detail the mechanism of BP induced apoptosis in osteoclast. The effects of different BPs CLO, pamidronate (PAM) and zoledronic acid (ZOL), on the differentiation of osteoblasts and osteoclasts were tested in vitro. The optimal concentration for in situ CLO rinsing in clinical study was found. Finally, the effects of in situ and per oral CLO on the periimplant bone density and integration of prostheses were studied in vivo. All tested COX-inhibitors significantly inhibited osteoblast differentiation from hMSCs and stimulated the differentiation of adipocytes. It was also demonstrated that AppCCl2p inhibits mitochondrial function by a mechanism that involves competitive inhibition of ADP/ATP translocase. In the comparison of BPs, ZOL seemed to posses the properties of both non-amino- and amino-BPs and it thus belongs to a new class of BPs. Peroral and in situ CLO seemed to have different mechanisms of action. Peroral CLO delayed the integration of prosthesis to the bone and increased peri-implant osteolysis while is situ CLO accelerated integration. In conclusion, we can alter normal bone remodellation during fracture healing and prosthesis integration. On the other hand, we can also improve the circumstances for the integration of implant to the surrounding bone by in situ BP rinsing, thus creating a better environment for bone ingrowth. / Tiivistelmä Läpi elämän luustossa tapahtuu uudelleenmuotoutumista, remodelaatiota, jonka seurauksena luu pystyy paremmin vastaamaan muuttuneisiin kuormitusolosuhteisiin. Remodelaatioprosessi on hyvin haavoittuvainen murtuman luutumisen aikana sekä proteesin kiinnittyessä ympäröivään luuhun. Luun remodelaatioon osallistuvat kasvutekijät, sytokiinit ja entsyymit, jotka puolestaan ovat välttämättömiä osteoblastien ja osteoklastien erilaistumiselle. Monet lääkeaineet voivat yllättävällä tavalla vahingoittaa tätä herkkää remodelaatiosysteemiä. Kipulääkkeet, kuten syklo-oksygenaasi (COX) estäjät, voivat häiritä murtuman luutumista aikaisempien eläintöiden ja muutamien retrospektiivisten potilastutkimusten mukaan. Lisäksi bisfosfonaatit, joiden päävaikutuskohde on luuta hajoittava osteoklasti, voisivat olla lupaavia lääkkeitä myös parantamaan proteesia ympäröivän luun laatua ja siten estämään aseptista implantin irtoamista. Tutkimuksen yhtenä tarkoituksena oli selvittää klodronaatin, ensimmäisen polven typpi-ryhmää sisältämättömän bisfosfonaatin tarkka vaikutusmekanismi. Viljelemällä ihmisen luuytimen kantasoluja indometasiinia, parekoksibia tai spesifistä COX-2 estäjää NS 398:a, sisältävässä kasvatusliuoksessa selvitettiin COX-entsyymin merkitys osteoblastien erilaistumiselle. Liposomien sisälle pakattua klodronaatin metaboliittia (AppCCl2p) käytettiin tutkittaessa millä vaikutusmekanismilla klodronaatti aiheuttaa osteoklastien apoptoosin. Bisfosfonaattien; klodronaatin, pamidronaatin ja tsoledronaatin vaikutusta osteoklastien ja osteoblastien erilaistumiseen tutkittiin soluviljelmämallissa ja määritettiin kliinisessä potilastyössä paikallisesti käytettävän klodronaattiliuoksen pitoisuus. Lopuksi potilastyössä selvitettiin paikallisen klodronaattihuuhtelun ja suun kautta annostellun klodronaatin vaikutus proteesia ympäröivän luun tiheyteen ja proteesin kiinnittymiseen ympäristöönsä. Tutkimukseen valitut COX-estäjät vähensivät ihmisen kantasolujen erilaistumista osteoblasteiksi ja lisäsivät erilaistumista rasvasoluiksi. Lisäksi todettiin, että AppCCl2p estää mitokondrioissa tapahtuvaa hengitystä estämällä ADP/ATP-vaihtajan toiminnan, saaden aikaan solukuoleman. Vertailtaessa bisfosfonaatteja, tsoledronaatilla vaikutti olevan sekä ensimmäisen, että kolmannen polven (sisältää typpi-ryhmän) bispfosfonaattien vaikutuksia, joten tsoledronaatti kuuluu aivan uuteen bisfosfonaattiryhmään. Potilastutkimuksessa suun kautta ja paikallisesti reisiluun ytimeen annostellulla klodronaatilla oli täysin erilainen vaikutus. Suun kautta syötynä klodronaatti hidasti proteesin kiinnittymistä ja aiheutti osteolyysiä. Sen sijaan paikallinen klodronaatti nopeutti merkittävästi proteesin kiinnittymistä ympäröivään luuhun. Näiden tutkimustulosten perusteella voidaan olettaa, että COX-estäjät, samoin kuin peroraalinen bisfosfonaatti, voivat tahattomasti häiritä luun remodelaatiota.

Les phosphates de calcium tels que le β-TCP sont utilisés depuis des décennies comme substitut osseux synthétique. Leurs bonnes propriétés chimiques et leur comportement analogue au tissu osseux in vivo et in vitro peuvent être améliorés par la technique de mise en forme employée. Il est aujourd'hui largement admis qu'une architecture poreuse optimisée aura un impact positif sur la bioactivité du matériau. Cette étude vise à étudier les liens existant entre une structure macroporeuse en β-TCP et la prolifération et différenciation cellulaire. Le β-TCP est fabriqué par précipitation aqueuse. Les paramètres de synthèse sont optimisés afin d'avoir un produit répondant aux normes ISO 13175 et 13779. Trois méthodes de mise en forme ont été choisies pour leur aptitude à générer une macroporosité originale. L'imprégnation d'une structure polymérique par une suspension génère un réseau de pores sphériques (PS), la stéréolithographie génère des pores cubiques interconnectés (3D) et la congélation orientée produit un réseau de pores tubulaires ellipsoïdaux parallèles au sens de la congélation (CO). Deux tendances émergent des cultures de cellules souches mésenchymateuses humaines: PS et 3D favorisent la prolifération alors que CO favorise la pénétration cellulaire et l'activité de la phosphatase alcaline. Cette dernière est favorisée par le β-TCP et cette aptitude est améliorée par la congélation orientée. Cela pourrait s'expliquer par l'état d'avancement de la différenciation cellulaire: les cellules sur les échantillons CO semblent être à un stade de différenciation plus avancé. Des essais complémentaires sur l'expression de gènes clés sont en cours pour vérifier cette hypothèse. / Calcium phosphates such as β-TCP have been used for decades as synthetic bone substitutes. Its good chemical properties and its similar behavior to that of the bone in vivo and in vitro can be enhanced by the chosen shaping method. It is nowadays largely accepted that an optimized porous architecture will have a positive impact on the material's bioactivity. This study aims at studying the links between a porous architecture and cell proliferation and differentiation. β-TCP was manufactured by aqueous precipitation. Synthesis parameters were optimized in order to get a product complying with ISO 13779 and 13175 requirements. Three shaping methods were chosen for their ability to generate original structures. The impregnation of a polymeric scaffold yields a network of interconnected spherical pores (PS), stereolithography yields a network of interconnected cubical pores (3D) and ice templating yields a network of parallel ellipsoidal channel-like structure (CO). Two different trends emerged from the human mesenchymal stem cell culture: PS and 3D favored cell proliferation whereas CO promoted cell penetration and alkaline phosphatase activity. The latter is stimulated by β-TCP and this ability is enhanced by freeze casting. This could be explained by the state of cell differentiation: cells on CO samples seem to be far more differentiated than the other ones. However the study of key genes expression is needed to confirm this hypothesis.

Directing differential cellular response by manipulating the physical characteristics of the material is regarded as a key challenge in biomaterial implant design and tissue engineering. In developing various biomaterials, the influence of substrate properties, like surface topography, stiffness and wettability on the cell functionality has been investigated widely. However, such study to probe into the influence of substrate conductivity on cell fate processes is rather limited. The need for such an understanding is based on the fact that specific tissues in the body are electrically active in nature, such as in brain, heart and skeletal muscle. These tissues make use of electrical conductivity as an effective cue for tissue homeostasis, development, regeneration and so on. Moreover, understanding the importance of underlying conductivity in basic biological processes is essential in developing electrically conductive biomaterials with the ability to simulate normal electrophysiology of the body by interfacing with bioelectric fields in cells and tissues. Electrical stimulation and charge conduction can regulate numerous intracellular signalling pathways, can interact with cytoskeleton proteins to modulate the morphology, increase protein synthesis and on the more can favor the ECM protein conformational changes. On these grounds, the present dissertation illustrates that persistent electrical activation influences the multipotency of hMSCs and acts like a promoter towards selective differentiation of hMSCs into neural/cardiomyogenic or osteogenic lineage. Besides, continual exposure to electric field stimulated conducting culture environments lead to growth arrest while enhancing differentiation. In total, this dissertation suggests the dominant role of conductivity in inducing my oblast differentiation and hMSc lineage commitment that involves EF stimulated in vitro culture conditions. Also, a knowledge base with qualitative and quantitative understanding of stem cells and their response to substrate physical properties and external field effect was developed through this comprehensive study. Such an improved understanding of the ability of hMSCs in sensing electrical conductivity may lead to the development of culture additives/conditions that better induce directed stem cell differentiation.