Urs Hafeli


Relevant Thesis-Based Degree Programs


Graduate Student Supervision

Doctoral Student Supervision

Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.

Development and evaluation of radiolabeled macromolecular conjugates for targeted delivery of anti-arthritic drugs (2021)

The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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Antibiotic-loaded polymeric microspheres for passive lung targeting after intravenous administration (2020)

Low respiratory tract bacterial infections are currently amongst the leading causes of mortality worldwide. Current treatments consist of oral or intravenous administration of antibiotics. Today´s treatments of pulmonary bacterial infections are often not sufficiently effective due to the difficulty of drugs reaching the infection deep in the lungs, the insufficient drug doses at the site of infection, the development of multi-drug resistance, and the side effects caused by some of the currently used and effective antibiotics. Lung-targeted delivery of antibiotics by using injectable drug-loaded microspheres is a promising alternative to the traditional antibiotic solutions as they achieve local therapeutic concentrations of antibiotics and minimise unwanted off-target effects. This delivery strategy offers potential, especially in the case of patients with compromised lung function or obstruction of the respiratory tract, due to inflammation, infection or significant mucus production, while they still have normal lung perfusion. The aim of this project was to explore the use of polymeric microspheres as carriers for lung delivery of antibiotics to increase the efficacy of these drugs against bacterial respiratory infections, specifically by selectively targeting the lung capillaries after intravenous administration. Biodegradable poly(lactic-co-glycolic) acid (PLGA) microspheres encapsulating levofloxacin were prepared with a flow-focusing microfluidic chip and characterized for their physico-chemical properties, and their in vitro and in vivo performance. The PLGA microspheres were highly homogeneous in size with a mean diameter of ~12 µm and coefficient of variation
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Octadentate chelators for zirconium- and other metal-based radiopharmaceuticals (2019)

Metal-based radiopharmaceuticals are critical in nuclear medicine for non-invasive diagnosis and treatment of serious diseases such as cancer. To be successfully deployed in such radioactive drugs, radiometal ions need to be stably sequestered by a suitable chelating agent. This work investigates a new class of octadentate chelating agents containing four 3-hydroxy-4-pyridinone (3,4-HOPO) entities with a focus on the development of new zirconium(IV) chelates. The diagnostic radionuclide zirconium-89 (89Zr, t1/2 78.41 h, Iβ+ 22.7%, Eβ+ mean 395.5 keV) is of particular interest for antibody-targeted positron emission tomography (immunoPET). The developed tetrakis(3,4-HOPO) chelator is capable of quantitatively sequestering 89Zr within 10 min at ambient temperature. The resultant Zr(IV) complex was found to be of exceptional thermodynamic stability (log β 50.3), showed a favourable pharmacokinetic profile in vivo, and exceeded the current standard Zr-chelator in direct in vitro stability tests. The tetrapodal chelator was derivatized in a multi-step synthesis for covalent attachment to targeting vectors or other carriers. The conjugation and ⁸⁹Zr-radiolabeling of the bifunctional chelator (BFC) was subsequently optimized and investigated in detail with two monoclonal antibodies, a model protein, and with polymeric nanoparticles. The long-term in vivo stability of the ⁸⁹Zr-radiochelate was assessed in a novel way over six days by utilizing long-circulating hyperbranched polyglycerol (HPG) nanoparticles. The radiochelate-nanocarrier conjugates were examined over six days by non-invasive positron emission tomography (PET) and in a biodistribution study. Although in vitro exams demonstrated extended plasma stability, these data revealed a physiologic susceptibility of the tetrakis(HOPO) complex attributed to kinetic lability. In addition to investigations with Zr(IV), the new ligand system was briefly explored with Fe(III), Ga(III), Y(III), Sm(III), Gd(III), Tb(III), Lu(III), and Bi(IIII) and indicated to be a promising chelator for iron(III), gallium(III), yttrium(III), and lanthanide ion coordination.

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Pharmacologic targeting of the cb2 cannabinoid receptor for application in centrally-mediated chronic pain (2019)

Cannabis preparations have been used for millennia for the treatment of pain and various ailments. However, psychotropic effects, mediated by the CB1 cannabinoid receptor in the central nervous system (CNS), limit their therapeutic use. Research to date suggests that selective activation of the CB2 cannabinoid receptor promotes analgesia without the occurrence of psychotropic effects. Cannabinoids, the principal active compounds in Cannabis sativa, lack the ability to selectively activate CB2. Glial cells of the CNS are known to play an important role in mediating certain forms of chronic pain through pro-inflammatory activity. Furthermore, CB2 receptors expressed by glia are now recognized as a potential therapeutic target for such disease states; however, the contribution of glial cell-mediated neuro-inflammation to the pathophysiology of chronic widespread musculoskeletal pain (CWP) disorders, such as fibromyalgia syndrome, remains unclear. An immunohistology investigation within the acidic saline model of CWP revealed significant up-regulation of Iba-1 and GFAP in the lumbar spinal cord, suggesting that gliosis may potentially mediate hyperalgesia in CWP disorders. Although further investigations are required, these data support that targeting of cannabinoid receptors expressed by glia may be a potentially viable approach for addressing CWP. To target cannabinoid receptors expressed by glia while minimizing the potential for CB1-associated CNS effects, compounds with selective CB2 agonist activity were designed, guided by CB2 molecular docking studies in silico, and synthesized for further investigation. β-caryophyllene, a naturally occurring sesquiterpene, along with two novel compounds, DML-3 and DML-4, were found to be full agonists at CB2 with 109, > 40 and >10,000 –fold selectivity over CB1, respectively. Furthermore, all three compounds significantly reduced activation of NF-κB, ERK1/2 and PI3K in U87MG astrocytes. Significant reductions in astrocyte IL-6 and IL-8 secretion occurred following treatment with β-caryophyllene (1 µM) and DML-4 (25 µM), with minor, non-significant reductions observed following treatment with DML-3 (25 µM). Based on the pharmacologic properties determined, each compound may be a potential candidate for therapeutically targeting pro-inflammatory glial cell activity. Further suggested investigations include quantification of β-arrestin recruitment, screening for off-target effects, testing for efficacy in research models of CWP, followed by in vivo pharmacokinetic and toxicological profiling.

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Investigation of the potential application of rhenium in medical imaging (2017)

Many thermodynamically stable coordination compounds have been synthesized using rhenium, a chemically versatile transition metal. Some of these rhenium complexes have been studied for utilization in nuclear medicine. However, many of their applications in medical imaging remain relatively unexplored and require further investigation. A comprehensive study was conducted in preclinical and clinical X-ray equipment to examine the use of rhenium as a contrast agent for X-ray imaging. Usually, there is a trade-off between image quality and radiation dose. This experimental work, along with theoretical Monte Carlo calculations, showed that it is feasible to preserve image quality and minimize radiation dose simultaneously when a rhenium-based formulation is utilized. This research provided thorough evidence of rhenium’s usefulness in X-ray imaging.Another application of rhenium was evaluated by producing a radiopaque, biodegradable electrospun scaffold containing a rhenium complex. Typically, catheterizations are performed under X-ray imaging guidance, but most catheters are radiolucent. After coating catheters with this scaffold, they became strongly radiopaque. Even a thin rhenium-doped coating has the potential of enhancing the contrast during catheterizations, which might be helpful in placing catheters more rapidly and precisely. Not only large medical devices, but also microsized carriers can be made radiopaque. An issue with embolic microspheres is their lack of contrast. To improve their visibility, potentially toxic contrast agents are co-administered in X-ray imaging-guided embolotherapy. Using a microfluidic technology, radiopaque, biodegradable microspheres made of a custom-synthesized polymer containing a rhenium complex were produced. Upon increasing the polymer’s rhenium concentration, these microspheres could be utilized in embolotherapy. Rhenium’s radioisotope Re¹⁸⁸ is a mixed beta and gamma emitter and can thus be exploited in another imaging modality: single photon emission computed tomography (SPECT). The biodistribution of microspheres labeled with Re¹⁸⁸ was evaluated in a hepatocellular carcinoma-bearing rat model. Although challenging in clinical practice, the radiation doses to the tumor and the healthy liver tissue were calculated. The radiation dose from the beta emissions yields these “imageable” microspheres theranostic, with quantifiable cancer radiotherapeutic potential. This work established the foundations to guide further research on the development of biodegradable devices doped with rhenium for medical imaging.

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Skin mechanics, intradermal delivery and biosensing with hollow metallic microneedles (2017)

Microneedles (MNs) have gained significant attention over the past decade in drug delivery and biosensing due to their minimally-invasive and less painful nature of use compared to intramuscular/subcutaneous injections, and significant biological benefits. Several fundamental processes enabling MN functionality have not been completely understood, including mechanical interaction between MNs and skin for targeted depth penetration; and precise quantification of fluid delivery in the skin. This thesis presents novel materials, and methodologies for evaluating MN interactions with skin, and investigates the performance of hollow MNs in both intradermal fluid drug delivery and biosensing.A micromechanical comparison between human skin and porcine skin was performed using to determine their mechanical behavior affecting MN insertions. Stratum corneum (SC) of human skin was significantly stiffer (117 ± 42 MPa) than porcine skin (81 ± 32 MPa), requiring higher force of MN insertion to rupture the SC in human skin (107 ± 17 mN) than porcine skin (96 ± 23 mN). An artificial mechanical skin model was developed layer-by-layer to simulate tough human skin (MN insertion force 162 ± 11 mN) and to study the dynamics of MN insertion. Key factors that affected MN insertions into skin, including velocity of impact and total energy delivered to the skin, were identified. ID fluid delivery by hollow MNs was assessed using a novel method involving the low-activity radiotracer technetium-99m pertechnetate (⁹⁹mTcO₄₋). Its delivery allowed accurate quantification of fluid delivered into the skin, back-flowed to the skin surface, and total fluid ejected from the syringes via ID devices with sub-nanoliter resolution. Hollow MNs performed more accurate ID injections than conventional needles (93% vs. 69-87% of fluid per 0.1 mL injection volume).A MN-optofluidic biosensing platform capable of eliminating blood sampling was developed with MNs that can access dermal interstitial fluid that contains numerous drugs at concentrations comparable to blood. The MN lumen was functionalized to collect, trap and detect drugs in 0.6 nL of sample. The optofluidic components provided specific high-sensitivity absorbance measurements for drug binding using enzyme-linked assays. Streptavidin-horseradish peroxidase (LoD = 60.2 nM) and vancomycin (LoD = 84 nM) binding validated this point of care system.

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Design and Optimization of a Microfluidic System for the Production of Protein Drug Loadable and Magnetically Targetable Biodegradable Microspheres (2016)

The overall goal of this dissertation was to develop a microfluidic system for the production of magnetic and non-magnetic drug-loaded polymer microspheres with narrow size distribution. The size of microspheres is a crucial parameter for their application, as the rate of drug release from the microspheres, the rate of microsphere degradation, and the microsphere biodistribution all correlate with particle size and size distribution. Conventional microsphere production methods generally lead to the formation of particles with broad size distribution. In this work, the conventional emulsification-evaporation method for the production of microspheres was miniaturized into microfluidic flow focusing system. Using a self-designed flow focusing chip, quasi-monodisperse poly (lactic acid) microspheres were prepared. The spherical shape and narrow size distribution of the initial polymer-chloroform droplets generated at the orifice were retained throughout their movement in the channels. After complete solvent removal, the droplets turned into quasi-monosized microspheres with a coefficient of variation between 2% and 16%. To better understand the influence of different parameters on droplet generation, the behavior of the disperse phase and the continuous phase were simulated in a 3D computational multiphase droplet generation model. The experimentally determined droplet sizes correlated well with the computational model and never digressed more than 17% from the simulations. The flow focusing system was then also used directly, for the first time, to produce superparamagnetic microspheres containing 15% magnetic nanoparticles. The microspheres’ thermal properties showed their suitability for magnetic hyperthermia of large tumors in cancer therapy. The microfluidic chip was then further altered into a novel device that is able to encapsulate proteins into the polymer microspheres by integrating flow focusing and passive droplet break up systems into one-chip which contained sections of differing surface properties. With this design, bovine serum albumin-loaded microspheres could be prepared with a protein encapsulation efficiency of up to 96%. The work presented in this dissertation is the first to show that a microfluidic system can be used for the continuous production of quasi-monodisperse magnetic microspheres and protein-drug-loaded polymer microspheres. These microspheres will be useful in hyperthermia treatment, diagnostic imaging, and the targeted and controlled delivery of protein based drugs.

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Sampling and Pharmacokinetics of Skin Interstitial Fluid for Therapeutically Monitored Drugs (2016)

To guide therapeutic decision making, the pharmacokinetics (PK) of certain toxic drugs are typically studied in blood. A drug’s blood concentration is thus acting as a surrogate for its target site concentration. However, a drug’s target is often extravascular and measuring tissue concentrations would be more meaningful. Furthermore, blood sampling is painful and can be challenging for some patients such as children, seriously ill, and old patients; it is, however, currently the standard method for drug testing.Current research suggests that a tissue fluid called interstitial fluid (ISF) can be sampled in minimal amounts without pain and can be used to quantify certain drugs. However, to successfully use this fluid for therapeutic drug monitoring, researchers still face three main challenges: sampling ISF, determining the concentrations and PK of drugs in ISF and their relation to blood concentrations, and quantifying drugs in very small volumes.To improve these challenges, we studied all three areas. First, we reviewed and evaluated methods of extracting ISF. Second, we studied ISF and blood concentrations of 13 drugs in a rabbit model to evaluate their PK. And third, we developed a method for quantifying a drug in just 2 µL of serum from rabbits. Currently available methods need larger sample volumes, whereas ISF is only available in small amounts. We found that many of the drugs we tested in a single-dose study were readily detectable in ISF (vancomycin, gentamicin, methotrexate, cisplatin, carboplatin, valproic acid, phenobarbital, mycophenolic acid and theophylline) and their PK parameters were determined using non-compartmental analysis. Furthermore, steady-state concentrations were predicted from the single-dose study for blood and ISF. At equilibrium, ISF drug concentrations were higher (vancomycin and gentamicin) and more stable compared to blood concentrations. For vancomycin these predictions were confirmed in an additional in vivo study. We further found that the concentration vs. time course of some drugs (vancomycin, gentamicin, methotrexate, valproic acid, phenobarbital, mycophenolic acid, digoxin and theophylline) could be well described by compartmental models. This study shows that ISF can be a valuable matrix for therapeutic drug monitoring and merits further studies to ascertain its clinical utility.

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Molecular Imaging of Mesothelin Expressing Cancers (2011)

Mesothelin is a cell surface glycoprotein highly expressed in mesothelioma, ovarian cancer, pancreatic cancer and some other malignancies. It is a promising candidate for tumour specific therapy and diagnosis, given its limited expression in normal tissues. The purpose of the work presented in this dissertation is to develop and characterize a molecular imaging bioprobe that targets mesothelin. We radiolabelled fab and f(ab´)₂ fragments of the anti-mesothelin antibody mAbK1 with ⁹⁹mTc- tricarbonyl core using a histidine-modified tridentate ligand, while whole mAbK1 was radiolabelled with ⁹⁹mTc using a direct labelling approach. In vivo evaluation of these ⁹⁹mTc labelled radioimmunoconjugates in mesothelin expressing NCI-H226 tumour model, revealed low mesothelin specific tumour uptake. These findings were attributed to low expression of mesothelin on NCI-H226 cells as well as to the low affinity of mAbK1. An anti-mesothelin antibody mAbMB, with higher mesothelin affinity than mAbK1 was labelled with ¹¹¹In and evaluated in A431K5 tumour model which expresses clinically relevant levels of mesothelin. Biodistribution studies and SPECT imaging revealed specific localization of ¹¹¹In-mAbMB in mesothelin expressing A431K5 tumours. An interesting finding with ¹¹¹In-mAbMB was its preferential localization in spleen, which suggests a role of circulating mesothelin antigen in forming immune complexes with ¹¹¹In-mAbMB. In comparison, control studies with ¹¹¹In-mAbK1 revealed low specific uptake into A431K5 tumours. These studies provided evidence that ¹¹¹In-mAbMB is a better choice than ¹¹¹In-mAbK1 for imaging mesothelin expression in tumours. A dual-modality SPECT/MR imaging bioprobe was further developed by conjugating ¹¹¹In-mAbMB with SPIONs (superparamagnetic iron oxide nanoparticles) which demonstrated specific targeting and MR imaging capability in A431K5 tumour bearing mice. The work in this dissertation for the first time demonstrates successful SPECT imaging of mesothelin expressing cancers using radiolabelled antibodies. The radiopharmaceutical ¹¹¹In-mAbMB developed in this work holds promise for clinical use as a radioactive imaging bioprobe. Additionally, bioconjugates of ¹¹¹In-mAbMB and SPIONs are promising as dual-modality SPECT/MRI imaging bioprobes, which may be beneficial in improving the imaging outcomes of these difficult to treat tumours. In conclusion, our studies demonstrate that molecular imaging agents targeting mesothelin have a role to play for the detection and monitoring of mesothelin expressing cancers.

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Master's Student Supervision

Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.

Development of immunocompetent and immunosuppressed orthotopic rat model of hepatocellular carcinoma (2021)

Hepatocellular carcinoma (HCC) accounts for over 500,000 deaths per year and is the most common form of primary liver cancer. It has a very dismal prognosis with incidence rates almost equal to prevalence rates. In North America, incidence is on the increase due to increasing hepatitis C virus infections. More than 75% of patients diagnosed with HCC do not qualify for potentially curative therapies such as surgical resection, radiofrequency ablation and liver transplantation, and thus require management by trans-catheter procedures like radioembolization. Radioembolization is performed in more than 75% of patients and involves injecting radioactive microspheres through a catheter directly into the vessel that supplies blood to the tumor. Since the tumor and healthy liver tissue get their blood supply from two different sources, this procedure preferentially irradiates the tumor while sparing the healthy liver tissue.Therefore, we hypothesize that an orthotopic rat model of hepatocellular carcinoma can be used for non-invasive monitoring of tumor growth and development. Rat hepatoma cells (N1S1) were transfected by a lentiviral method and injected into the liver of Sprague Dawley and Rowett Nude (RNU) rats using ultrasound guidance and an alternative method respectively. Longitudinal tumor growth was followed by bioluminescence imaging (BLI). In both models, tumors were visible within 3 to 4 days post cell inoculation by BLI. Tumor take rates were 52% and 73% for male and female Sprague Dawley rats, respectively, and 100% for RNU rats. Day 12 and 15 post inoculation, we recorded complete tumor regression in all Sprague Dawley rats but continuous tumor growth for up to 21 days post induction in the RNU rats.The immunocompetent Sprague Dawley rat model is not ideal for investigation of catheterization techniques like radioembolization. The RNU rat model, however, is characterized ivby consistent and reliable tumor growth and thus a desirable method for future therapeutic investigations.

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Production, characterization, and biodistribution of lignin-capped silver nanoparticles to combat multidrug resistant bacteria In vitro and In vivo (2018)

One of the most important issues in healthcare today is the development of bacterial resistance to antibiotics which has created a generation of bacteria known as multidrug resistant (MDR) bacteria. Due to antibiotics’ inability to treat these MDR bacteria metal and metal oxide nanoparticles have been gaining interest as antimicrobial agents. Among those, silver nanoparticles have been used extensively as broad spectrum antimicrobial agents. Here we describe the production and characterization of silver nanoparticles made from the wood biopolymer lignin as a reducing and capping agent with excellent antimicrobial activity against MDR bacteria both in vitro and in vivo. We describe and compare the productions of these lignin-capped silver nanoparticles (L-AgNPs) both through a standard heating procedure and through a microwave-assisted synthesis. The L-AgNPs have been radioactively labeled using both iodine-123 and the novel radioisotope silver-111 to determine their biodistribution by SPECT/CT imaging after subcutaneous injection or intratracheal instillation. The particles were then tested for in vitro antimicrobial activity by broth dilution against a variety of Gram-positive and Gram-negative MDR clinical isolate bacterial strains and ATCC strains. They were also tested for efficacy in an in vivo cutaneous infection (abscess) model caused by biofilm-forming MDR bacterial strains. The particles were produced using a simple, one-pot synthesis method and characterized by ultraviolet-visual spectroscopy, dynamic light scattering, x-ray diffraction, and scanning transmission electron microscopy. Characterization of the lignin-capped silver nanoparticles shows uniform spherical nanoparticles with a silver core and a lignin coating with a diameter of about 50 nm for both synthesis methods, but the microwave method was significantly faster (10 min vs. 3 days). The particles radioactively labeled with silver-111 were visible on SPECT/CT and were labeled with high efficiency, but produced a poor size distribution. The L-AgNPs radioactively labeled with iodine-123 and injected subcutaneously remained at the injection site up to 48 hours post-injection. The in vitro minimum inhibitory concentration (MIC) of L-AgNPs was ≤5 µg/mL for all tested bacterial strains, and a significant decrease in both abscess size and bacterial load was observed against in vivo infections caused by MDR strains of S. aureus and P. aeruginosa.

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Assessment of the Biodistribution of Aluminum-Based Vaccine Adjuvants using 111/115In-AlO(OH) (2016)

Aluminum-based adjuvants are found in a wide range of non-living vaccines to enhances antibody-mediated immune responses. Alhydrogel is one of the most common aluminum-based adjuvants and consists of 1-12 μm clusters of loosely aggregated aluminum oxyhydroxide (AlO(OH)) nanoparticles. Despite widespread use since in 1930s, the biodistribution of this adjuvant is poorly understood. The current assumption within the field is that AlO(OH) particles remain at the site of injection and slowly dissolve over time. However, rare neurological and muscle adverse effects, such as macrophagic myofasciitis, are thought to be due to the bio-persistence of the adjuvant at the site of injection and the distribution of AlO(OH) nanoparticles throughout the body. The objective of this thesis was to examine if component nanoparticles of Alhydrogel distribute following administration. In order the test this hypothesis, traceable forms of AlO(OH) were synthesized by doping the adjuvant’s crystal structure with different indium isotopes. Various physical, morphological and functional assessments showed that the doped adjuvant closely matched commercial Alhydrogel. Using a radioactive version of the tracer, ¹¹¹In-AlO(OH), the biodistribution of the adjuvant was assessed using serial SPECT/CT imaging. Approximately 20% of the tracer was released from the site of injection over 15 days and could be found in organs like the draining lymph nodes and liver. A novel application of mass cytometry was used to examine the kinetics and cellular distribution of ¹¹⁵In-AlO(OH) particles within the draining lymph nodes; the adjuvant appeared at this site within 1 day and reached peak levels 15 days post-injection. The adjuvant was sequestered into antigen presenting cells and induced the up-regulation of maturation markers in adjuvant-positive dendritic cells within the draining lymph node. Finally, electron microscopy coupled with atomic mapping confirmed the presence of Alhydrogel component nanoparticles in the draining lymph node and liver. This thesis presents the most detailed assessment to date of the biodistribution of aluminum-based adjuvants. Understanding Alhydrogel’s biodistribution can help shed light on the adjuvant’s immunostimulatory mechanisms and adverse events. Lessons learnt from one of the world oldest adjuvants can be applied to the design of next generation of immunopotentiators.

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Droplet Generators for Encapsulation of Neonatal Porcine Islets in Alginate (2015)

For the treatment of type 1 diabetes, islet transplantation has the potential to control blood glucose levels more effectively than daily insulin injections. However, its widespread application is hindered by the limited supply of transplantable human islets and the side effects of life-long immunosuppression. Microencapsulation of islets in alginate can contribute to overcoming these barriers by facilitating the use of alternative sources of islets, such as neonatal porcine islets (NPI), and by reducing the need for immunosuppressive therapy. This work describes a process for immobilization of NPI in 5% alginate capsules by an emulsification and internal gelation approach utilizing droplet generators fabricated via soft lithography or stereolithography. Alginate capsules produced with microfluidic devices fabricated in PDMS had mean diameters between 300 and 600 µm. Capsules generated with 3D-printed droplet generators fabricated via stereolithography were capable of producing more than 30-times the number of capsules per minute than a microfluidic devices, but with approximately 2-fold greater mean bead diameters. These results suggest that 3D-printed droplet generators are better suited for encapsulating NPI for transplantation applications (e.g., future in vivo studies). Furthermore, encapsulation in 3D-printed droplet generators did not alter cell viability after processing unlike the microfluidic devices where the viability decreased 20%. After encapsulation, three different imaging techniques were explored to aid in the quantification of partially encapsulated islets. Overall, the results of this study should be useful for the improvement of immunoisolation approaches employing alginate immobilization.

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