Hydrogels have been widely explored for cell therapy applications due to their favourable biochemical and mechanical properties. However, the dimensions of bulk hydrogels limit the diffusion of nutrients to cells and cell products to the surrounding environment, negatively affecting cell viability and the therapeutic potential of the encapsulated cells. In addition, invasive procedures are often required for the administration of bulk hydrogels into patients that pose a practical barrier to cell therapy. To address these issues, micrometer sized hydrogels (microgels) have been designed with controlled shapes, sizes, and structures to enable sufficient biomolecule diffusion and injectable administration. In this thesis, in situ gelling poly(oligoethylene glycol methacrylate) (POEGMA) and zwitterionic microgels are fabricated based on delayed dynamic hydrazone crosslinking between cell friendly functionalized polymers without the need for any additional crosslinking agents. Two microgel fabrication strategies were explored: (1) droplet-based microfluidics and (2) droplet extrusion printing. In the first case, microgels with controlled degrees of porosity were fabricated via the incorporation of a non-toxic evaporable porogen into a microfluidic device. Porous microgels had significantly improved diffusion of small molecules compared to nonporous microgels, and cells encapsulated in the porous microgels showed significantly increased viability over 10 days. In the second case, droplet extrusion printing was employed to print a bioink on a hydrophobic surface, resulting in the fabrication of disk-shaped microgels with a height below the maximum pathlength of oxygen and nutrient diffusion. Cells encapsulated in the microgels maintained high viability, with the microgels also supporting effective cell proliferation over 10 days. Overall, the work presented in this thesis poses solutions to challenges around nutrient/cell product diffusion and the invasive procedures typically associated with hydrogel-based cell therapy, providing potentially new translatable therapeutic options for disease treatment. / Thesis / Master of Applied Science (MASc) / Cell therapy is used to improve or replace the function of damaged cells or tissues that currently exist in the body by delivering healthy cells and the therapeutic products they naturally produce to the site of interest. Delivering these cells to the body has many challenges, including attacks from the immune system and substantial cell death caused by mechanical forces applied upon injection. To overcome these problems, the cells can be loaded into hydrogel-based microparticles (microgels), highly hydrated polymer networks that can protect the encapsulated cells from the immune system and mechanical forces while providing an environment that can support cell viability and growth. This thesis is focused on designing microgels with suitable dimensions and structures that allow for nutrients to flow from the environment to the cells and wastes/cell products from the cells to the environment while also supporting long-term cell viability, allowing the therapeutic molecules the cells produce to potentially treat diseases.

Bioprinting, defined as depositing cells, extracellular matrices and other biologically relevant materials in user-defined patterns to build tissue constructs de novo or to build upon pre-fabricated scaffolds, is among one of the most promising techniques in tissue engineering. Among the various technologies used for Bioprinting, pressure driven systems are most conducive to preserving cell viability. Herein, we explore the abilities of a novel bioprinter - Digilab, Inc.'s prototype cell printer. The prototype cell printer (Digilab Inc., Holliston, MA) is an automated liquid handling device capable of delivering cell suspension in user-defined patterns onto standard cell culture substrates or custom-designed scaffolds. In this work, the feasibility of using the cell printer to deliver cell suspensions to biological sutures was explored. Cell therapy using stem cells of various types shows promise to aid healing and regeneration in various ailments, including heart failure. Recent evidence suggests that delivering bone-marrow derived mesenchymal stem cells to the infarcted heart reduces infarct size and improves ventricular performance. Current cell delivery systems, however, have critical limitations such as inefficient cell retention, poor survival, and lack of targeted localization. Our laboratories have developed a method to produce discrete fibrin microthreads that can be bundled to form a suture and attached to a needle. These sutures can then be seeded with bone-marrow derived mesenchymal stem cells to deliver these cells to a precise location within the heart wall, both in terms of depth and surface localization. The efficiency of the process of seeding cells onto fibrin thread bundles (sutures) has previously been shown to be 11.8 ± 3.9 %, suggesting that 88% of the cells in suspension are not used. Considering that the proposed cell-therapy model for treatment of myocardial infarction contemplates use of autologous bone-marrow derived stem cells, an improvement in the efficiency of seeding cells onto the fibrin sutures is highly desirable. The feasibility of using Digilab's prototype cell printer to deliver concentrated cell suspension containing human mesenchymal stem cells (hMSCs) directly onto a fibrin thread bundle was explored in this work, in order to determine if this technology could be adapted to seed cells onto such biological sutures. First the effect of the printing process on the viability of hMSCs was assessed by comparing to cells dispensed manually using a hand-held pipette. The viability of hMSCs 24 hours post-dispensing using the cell printer was found to be 90.9 ± 4.0 % and by manual pipetting was 90.6 ± 8.2 % (p = ns). Thereafter a special bioreactor assembly composed of sterilizable Delrin plastic and stainless steel pins was designed to mount fibrin thread bundles onto the deck of the cell printer, to deliver a suspension containing hMSCs on the bundles. Highly targeted delivery of cell suspension directly onto fibrin thread bundles (average diameter 310 µm) was achieved with the bundle suspended in mid-air horizontally parallel to the printer's deck mounted on the bioreactor assembly. To compare seeding efficiency, fibrin thread bundles were simultaneously seeded with hMSCs using either the cell printer or the current method (tube-rotator method) and incubated for 24 hours. Seeded thread bundles were visualized using confocal microscopy and the number of cells per unit length of the bundle was determined for each group. The average seeding efficiency with the tube rotator method was 7.0 ± 0.03 % while the cell printer was 3.46 ± 2.24% (p = ns). In conclusion, the cell printer was found to handle cells as gently as manual pipetting, preserve their viability, with the added abilities to dispense cells in user-defined patterns in an automated manner. With further development, such as localized temperature, gas and humidity control on the cell printer's deck to aid cell survival, the seeding efficiency is likely to improve. The feasibility of using this automated liquid handling technology to deliver cells to biological scaffolds in specified patterns to develop vehicles for cell therapy was shown in this study. Seeding other cell types on other scaffolds along with selectively loading them with growth factors or multiple cell types can also be considered. In sum, the cell printer shows considerable potential to develop novel vehicles for cell therapy. It empowers researchers with a supervision-free, gentle, patterned cell dispensing technique while preserving cell viability and a sterile environment. Looking forward, de novo biofabrication of tissue replicates on a small scale using the cell printer to dispense cells, extracellular matrices, and growth factors in different combinations is a very realistic possibility.

Endothelial progenitor cells (EPCs) have reduced neovascularization capacity in the context of coronary artery disease (CAD) or cardiac risk factors (RFs). Since, endothelial NO synthase (eNOS) is critical to normal EPC function, we hypothesized that bone marrow cells (BMCs) from rats with RFs and EPCs from humans with CAD and/or RFs show dramatically reduced neovascularization capacity in vitro and in vivo, which can be reversed by eNOS overexpression. BMCs were isolated from rat models of type II diabetes and the metabolic syndrome, and we showed a significant reduction in their ability to stimulate neovascularization in vitro and in vivo. In humans, we isolated circulating ‘early EPCs’ from healthy subjects and patients with CAD and RFs, and transduced them using lentiviral vectors containing either eNOS or GFP (sham). EPCs from patients had reduced in vitro migration in response to SDF-1 or VEGF, which was reversed by eNOS-transduction. In co-culture with human umbilical vein endothelial cells (HUVECs) on Matrigel, eNOS-transduced EPCs contributed to increased and more complex angiogenic tube formation compared to sham-transduced cells. Human EPCs from patients were ineffective in enhancing ischemic hind limb neovascularization and perfusion in a nude mouse, whereas eNOS-transduced EPCs resulted in a significant improvement compared to sham-transduced cells. In a swine model of acute myocardial infarction (MI), eNOS- and non-transfected BMCs both increased left ventricular function compared to sham. However, there was no benefit to eNOS overexpression in this model. Various differences in the models and procedures may explain the incongruous results obtained. Taken together, these results show that eNOS overexpression significantly improves the neovascularization capacity of EPCs of human subjects with CAD and RFs and could represent an effective adjunctive approach for the improvement of autologous cell therapies for cardiovascular disease.

Endothelial progenitor cells (EPCs) have reduced neovascularization capacity in the context of coronary artery disease (CAD) or cardiac risk factors (RFs). Since, endothelial NO synthase (eNOS) is critical to normal EPC function, we hypothesized that bone marrow cells (BMCs) from rats with RFs and EPCs from humans with CAD and/or RFs show dramatically reduced neovascularization capacity in vitro and in vivo, which can be reversed by eNOS overexpression. BMCs were isolated from rat models of type II diabetes and the metabolic syndrome, and we showed a significant reduction in their ability to stimulate neovascularization in vitro and in vivo. In humans, we isolated circulating ‘early EPCs’ from healthy subjects and patients with CAD and RFs, and transduced them using lentiviral vectors containing either eNOS or GFP (sham). EPCs from patients had reduced in vitro migration in response to SDF-1 or VEGF, which was reversed by eNOS-transduction. In co-culture with human umbilical vein endothelial cells (HUVECs) on Matrigel, eNOS-transduced EPCs contributed to increased and more complex angiogenic tube formation compared to sham-transduced cells. Human EPCs from patients were ineffective in enhancing ischemic hind limb neovascularization and perfusion in a nude mouse, whereas eNOS-transduced EPCs resulted in a significant improvement compared to sham-transduced cells. In a swine model of acute myocardial infarction (MI), eNOS- and non-transfected BMCs both increased left ventricular function compared to sham. However, there was no benefit to eNOS overexpression in this model. Various differences in the models and procedures may explain the incongruous results obtained. Taken together, these results show that eNOS overexpression significantly improves the neovascularization capacity of EPCs of human subjects with CAD and RFs and could represent an effective adjunctive approach for the improvement of autologous cell therapies for cardiovascular disease.

In Form einer Zusammenfassung aus zwei Publikationen und bisher unveröffentlichtem Material stellt die vorliegende Arbeit die Frage, welche der Zellfraktionen (HUCB-MNC, CD45+/CD34+ und CD45+/CD133+) innerhalb des heterogenen HUCB die am effektivsten wirksame in Hinblick auf die Neuroprotektion nach einem experimentellen Schlaganfall ist und welche Mechanismen dabei zum Tragen kommen. Für die Untersuchung der molekularen Mechanismen des Zusammenwirkens zwischen den genannten HUCB Fraktionen und neuronalen Zellen bzw. Gewebe, kamen ein selbst etabliertes Zellkulturmodel neuronaler Hypoxie bzw. OGD geschädigte hippocampale Schnittkulturen zur Anwendung. Die zu untersuchenden HUCB Zellfraktionen wurden direkt oder indirekt appliziert und ihr Effekt wurde über drei aufeinanderfolgende Tage hinweg untersucht. Das molekulare Mikromilieu, welches durch die hypoxisch geschädigten neuronalen Zellen produziert wurde, stimulierte alle untersuchten HUCB Fraktionen zur Sekretion neurotrophischer Faktoren und/oder immunologisch aktiver Mediatoren, die die neuronale Apoptose günstig beeinflussten. HUCB-MNC zeigten, sowohl in der direkten als auch in der indirekten Kokultur mit geschädigten neuronalen Zellen eine sehr überzeugende Fähigkeit zur Neuroproduktion. In den direkten Kokulturen reduzierten sie die Apoptose der neuronalen Zellen sogar bis auf das Niveau der Kontrollen. Dies kann auf den deutlichen Anstieg der von den HUCB-MNC produzierten Chemokine CCL5; CCL3 und CXCL10 zurückgeführt werden. Weiterhin war zu beobachten, dass HUCB-MNC aktiv zu geschädigten neuronalen Zellen migrierten und sich, vorzugsweise unter Ausbildung von direkten Zell-Zellkontakten, an Axonen und Somata anlagerten. Überraschenderweise zeigte die CD45+/CD133- Zellfraktion ein ähnliches Potential wie HUCB-MNC. Für diese nahezu stammzellfreie Fraktion konnten in den indirekten Kokulturen hohe Konzentrationen an CCL3 und neuroprotektiven G-CSF nachgewiesen werden, wobei letzteres für die Aufrechterhaltung des neuronalen Phänotyps verantwortlich gemacht werden kann. CD45+/CD133+ Stammzellen, die aus der HUCB-MNC Fraktion isoliert wurden, konnten die neuronale Apoptose in direkten Kokulturen signifikant reduzieren. Die Konzentration an löslichen Faktoren, die von den Stammzellen produziert wurde, lag dabei unterhalb der Nachweisbarkeitsgrenze. Die Ergebnisse aus den Untersuchungen der hippocampalen Schnittkulturen zeigen, dass HUCB-MNC direkt neuroprotektiv wirken. Dies gilt insbesondere, wenn sie direkt und in ausreichender Konzentration (12.5x104 Zellen pro Schnitt) appliziert werden. In Kokulturen mit der CD45+/CD34+ Stammzellfraktion fand sich eine verringerte Sekretion an Nervenwachstumsfaktor und damit verbunden eine geringere Anzahl degenerierter Pyramidenzellen. In Kokulturen in welchen die stammzellfreie CD45+/CD34- Fraktion verwendet wurde, trat dieser Effekt nicht auf. Die Resultate, die in den beiden hier verwendeten in vitro Modellen gefunden wurden, legen nahe, dass der Einsatz von HUCB-MNC eine stabile Neuroprotektion hervorruft. Im Vergleich der verwendeten Modelle lieferten die Applikationen von verschiedenen Stammzellfraktion keine einheitlichen Ergebnisse. Damit wird eine starke Systemabhängigkeit induziert. Speziell im Hinblick auf den klinischen Einsatz scheint es keinen deutlich überlegenen Vorteil durch die Verwendung reiner, aus der HUCB-MNC Fraktion gewonnener Stammzellfraktionen zu geben, der den Aufwand rechtfertigt eine zahlenmäßig so geringe Zellfraktion aus der HUCB-MNC Fraktion zu separieren.

Malignant melanoma is a common type of solid tumor that causes high cancer-related mortality in young adults of Northern Europe. The incidence of melanoma increases rapidly which renders us a special responsibility to investigate this disease in depth. One recent promising approach to treat malignant melanoma is adoptive cell therapy with tumor-directed autologous T cells. This thesis aims to improve this therapy in four different studies. We first sought to establish a protocol for the assessment of melanoma-specific T-cell cultures in order to screen for optimal specificity and reactivity in a robust, reliable and simple manner. The conclusion was that reactive cells could be found in a majority of patients and could be screened for specificity by stimulation with melanoma cell lines. In the next study, 28 melanoma patients with advanced disease were treated with autologous tumor-infiltrating T cells. Objective responses (18%) including one sustained complete response were observed. This is the first study in cancer patients with autologous T cell transfer combined with low-dose s.c. IL-2 as supportive cytokine. In the following two studies we wanted to improve management and culture conditions of the T cells. When investigating methods for improved handling and preservation of large numbers of T cells, we observed that freeze-thawing of T cells could impair the metabolic activity of the T cells. Another conclusion was that rapid expansion of T cells could lead to loss of antigenic specificity and apoptosis. These adverse effects could be prevented with short time recovery. In order to improve expansion methods, mass expansion of T cells in an automated bioreactor was evaluated. We concluded that the bioreactor is suitable for this task and allows for higher cell densities and absolute cell numbers compared to traditional culturing conditions without influencing cell phenotype or reactivity. Taken together, my current studies present guiding principles and encouragement for the further development of immunotherapies for treatment of patients with malignant melanoma.

The purpose of this study was to compare the effects of two types of stem/progenitor cells on the healing of critical sized bone defects in a rat model. Endothelial progenitor cells (EPCs), a novel cell type with previously demonstrated effects on both osteogenesis and angiogenesis, were compared to both a control group (no cells), and a treatment group of mesenchymal stem cells (MSCs). The hypothesis was that EPCs would demonstrate both superior bone healing and angiogenesis, when compared to MSCs and controls. EPCs, MSCs, or a control carrier were placed in surgically stabilized bone defects in a rat femur and both bone formation and angiogenesis were assessed. EPC treated defects demonstrated significantly more bone formation and angiogenesis at the bone defect site than MSC or control treated defects. These results strongly suggest that EPCs are more effective than MSCs for therapeutic osteogenesis and angiogenesis in a bone defect model.

The purpose of this study was to compare the effects of two types of stem/progenitor cells on the healing of critical sized bone defects in a rat model. Endothelial progenitor cells (EPCs), a novel cell type with previously demonstrated effects on both osteogenesis and angiogenesis, were compared to both a control group (no cells), and a treatment group of mesenchymal stem cells (MSCs). The hypothesis was that EPCs would demonstrate both superior bone healing and angiogenesis, when compared to MSCs and controls. EPCs, MSCs, or a control carrier were placed in surgically stabilized bone defects in a rat femur and both bone formation and angiogenesis were assessed. EPC treated defects demonstrated significantly more bone formation and angiogenesis at the bone defect site than MSC or control treated defects. These results strongly suggest that EPCs are more effective than MSCs for therapeutic osteogenesis and angiogenesis in a bone defect model.

Eine Vielzahl von Studien konnte den nützlichen Effekt einer Knochenmarkzelltherapie nach Schlaganfall an Ratten nachweisen. Diese Ergebnisse ließen sich jedoch in klinischen Studien nicht reproduzieren. Eine mögliche Ursache können die Unterschiede zwischen den eingesetzten Versuchstieren und Zellspendern sowie den Patienten im klinischen Bereich sein. Während die eingesetzten Tiere und Spender meist jung und gesund waren, ist der typische Schlaganfallpatient in der Regel älter und leidet an einer Vielzahl von Begleiterkrankungen. Ziel dieser Studie war es, die Auswirkungen von erhöhtem Alter und Begleiterkrankungen auf die Effektivität einer Knochenmarkzelltherapie des Schlaganfalles zu untersuchen. Zu diesem Zwecke wurde die Arteria cerebri media von 18 Monate alten spontan-hypertensiven Ratten operativ verschlossen. Nach 24 Stunden wurden entweder Zellen eines jungen oder eines alten Spender transplantiert. Tiere, die eine äquivalente Menge an PBS erhielten, dienten als Kontrolle. Im Anschluss wurden das neurofunktionelle Defizit und das Infarktvolumen über einen Zeitraum von 63 Tagen mittels verschiedener Verhaltenstest und magnetresonanztomographischer Bildgebung analysiert. Nach Induktion des Schlaganfalls kam es zu einer Abnahme der funktionellen Leistung in allen Verhaltensversuchen, die sich jedoch bis zu Tag 27 wieder erholte. Im MRT war im gleichen Zeitraum eine Reduktion des Infarktvolumens zu beobachten. In der zweiten Versuchshälfte kam es anschließend zu keinen weiteren Veränderungen, weder funktional noch morphologisch. Ein Einfluss der Therapie auf die Regeneration oder die Größe des Infarkts, war zu keinem Zeitpunkt feststellbar. Anhand dieser Ergebnisse muss in Frage gestellt werden, ob gealterte und komorbide Tiere durch einer Zelltherapie nach Schlaganfall profitieren können und ob sich Zellen älterer Spender als Therapeutikum für einer Knochenmarkzelltherapie des Schlaganfalls eignen.

Regenerative medicine has the potential to establish or restore normal function in defective tissues and organs. The realisation of such therapies is restricted due to costs, lack of scalability and inefficient manufacturing process controls. A major contributor to cost is the use of expensive growth factors supplemented into media at high concentrations. In vivo, growth factors exist in soluble, immobilised and transmembrane forms, expressed in a spatiotemporal fashion within the stem cell niche. In comparison to soluble equivalents, immobilised growth factors exhibit increased potency, distinct functional activities, improved cell phenotypic control and act in synergy with other soluble and immobilised ligands. To date, most research into immobilised growth factors has been restricted to planar cell culture surfaces such as tissue culture plastics which have limited scalability. To address the scalability limitations, a novel growth factor immobilisation technology was developed using magnetic microparticles which can be scaled with respect to surface area to volume ratio in standard stirred tank bioreactors. Three clinically relevant growth factors, SCF, TPO and GM-CSF were immobilised and were shown to remain functionally active where surface concentration could be manipulated in a number of ways. Through a series of experiments, it was demonstrated that immobilised growth factors exhibited ~10-fold increase in potency compared with soluble equivalents and remain stable for up to 192 hours following recycling during multiple media passages. Immobilised growth factors were able to expand more cells over a longer period of time after transient exposure and finally, the immobilisation technique was successfully applied to the expansion of umbilical cord derived haematopoietic stem cells using immobilised SCF. The immobilisation method described here has the potential to significantly reduce media costs in large scale cell manufacturing processes.