Patric Wallin

Continuous Lipid Bilayers Derived from Cell Membranes for Spatial Molecular Manipulation

Progress with respect to enrichment and separation of native membrane components in complex lipid environments, such as native cell membranes, has so far been very limited. The reason for the slow progress can be related to the lack of efficient means to generate continuous and laterally fluid supported lipid bilayers (SLBs) made from real cell membranes. We show in this work how the edge of a hydrodynamically driven SLB can be used to induce rupture of adsorbed lipid vesicles of compositions that typically prevent spontaneous SLB formation, such as vesicles made of complex lipid compositions, containing high cholesterol content or being derived from real cell membranes. In particular, upon fusion between the moving edge of a preformed SLB and adsorbed vesicles made directly from 3T3 fibroblast cell membranes, the membrane content of the vesicles was shown to be efficiently transferred to the SLB. The molecular transfer was verified using cholera toxin B subunit (CTB) binding to monosialoganglioside receptors (G(M1) and G(M3)), and the preserved lateral mobility was confirmed by spatial manipulation of the G(M1/M3)-CTB complex using a hydrodynamic flow. Two populations of CTB with markedly different drift velocity could be identified, which from dissociation kinetics data were attributed to CTB bound with different numbers of ganglioside anchors.

A method to integrate patterned electrospun fibers with microfluidic systems to generate complex microenvironments for cell culture applications

The properties of a cells microenvironment are one of the main driving forces in cellular fate processes and phenotype expression invivo. The ability to create controlled cell microenvironments invitro becomes increasingly important for studying or controlling phenotype expression in tissue engineering and drug discovery applications. This includes the capability to modify material surface properties within well-defined liquid environments in cell culture systems. One successful approach to mimic extra cellular matrix is with porous electrospun polymer fiber scaffolds, while microfluidic networks have been shown to efficiently generate spatially and temporally defined liquid microenvironments. Here, a method to integrate electrospun fibers with microfluidic networks was developed in order to form complex cell microenvironments with the capability to vary relevant parameters. Spatially defined regions of electrospun fibers of both aligned and random orientation were patterned on glass substrates that were irreversibly bonded to microfluidic networks produced in poly-dimethyl-siloxane. Concentration gradients obtained in the fiber containing channels were characterized experimentally and compared with values obtained by computational fluid dynamic simulations. Velocity and shear stress profiles, as well as vortex formation, were calculated to evaluate the influence of fiber pads on fluidic properties. The suitability of the system to support cell attachment and growth was demonstrated with a fibroblast cell line. The potential of the platform was further verified by a functional investigation of neural stem cell alignment in response to orientation of electrospun fibers versus a microfluidic generated chemoattractant gradient of stromal cell-derived factor 1 alpha. The described method is a competitive strategy to create complex microenvironments invitro that allow detailed studies on the interplay of topography, substrate surface properties, and soluble microenvironment on cellular fate processes.

Kinetics of Ligand Binding to Membrane Receptors from Equilibrium Fluctuation Analysis of Single Binding Events

Equilibrium fluctuation analysis of single binding events has been used to extract binding kinetics of ligand interactions with cell-membrane bound receptors. Time-dependent total internal reflection fluorescence (TIRF) imaging was used to extract residence-time statistics of fluorescently stained liposomes derived directly from cell membranes upon their binding to surface-immobilized antibody fragments. The dissociation rate constants for two pharmaceutical relevant antibodies directed against different B-cell expressed membrane proteins was clearly discriminated, and the affinity of the interaction could be determined by inhibiting the interaction with increasing concentrations of soluble antibodies. The single-molecule sensitivity made the analysis possible without overexpressed membrane proteins, which makes the assay attractive in early drug-screening applications.

Using QCM-D to study the adhesion of human gingival fibroblasts on implant surfaces

Sealing the soft tissue-implant interface is one of the key issues in preventing transcutaneous implant-associated infections. A promising surface modification for improving osseointegration and possibly soft tissue integration is to coat the implant surface with hydroxyapatite (HA) nanoparticles. When new implant materials are developed, their ability to facilitate cell attachment and spreading are commonly investigated in vitro to establish their potential for good in vivo performance. However, commonly used techniques, such as microscopy methods, are time consuming, invasive, and subjective. This is the first study using quartz crystal microbalance with dissipation monitoring, where the real-time adhesion of biopsy-derived human gingival fibroblasts onto titanium and nanostructured HA was investigated. Experiments were performed for at least 16 h, and we found that cellular attachment and spreading kinetics can be followed in situ by observing the change in dissipation and frequency with time. Interestingly, a correlation between cell coverage and the magnitude of dissipation shift reached at the end of the experiment was found, but no such trend was observed for the frequency. Furthermore, the level of cell coverage was found to influence the cellular attachment and spreading behavior. No difference in cell response to the two surface types, Ti and nanostructured HA, was found.

Linking teaching and research in an undergraduate course and exploring student learning experiences

ABSTRACT In this case study, we first describe how teaching and research are linked in a master’s course on tissue engineering. A central component of the course is an authentic research project that the students carry out in smaller groups and in collaboration with faculty. We then explore how the students experience learning in this kind of discovery-oriented environment. Data were collected through a survey, reflective writing, and interviews. Using a general inductive approach for qualitative analysis, we identified three themes related to the students’ learning experiences: learning to navigate the field, learning to do real research, and learning to work with others. Overall, the students strongly valued learning in a discovery-oriented environment and three aspects of the course contributed to much of its success: taking a holistic approach to linking teaching and research, engaging students in the whole inquiry process, and situating authentic problems in an authentic physical and social context.

Skeletal myoblast differentiation on starch microspheres for the development of cultured meat

Adequate cellular in-growth into biomaterials is one of the fundamental requirements in regenerative medicine. Type-I-collagen is the most commonly used material for soft tissue engineering, because it is nonimmunogenic and a highly porous network for cellular support. However, adequate cell in-growth and cell seeding has been suboptimal. Different densities of collagen scaffolds (0.3% to 0.8% (w/v)) with/without polymer knitting (poly-caprolactone (PCL)) were prepared. The structure of collagen scaffolds was characterized using scanning electronic microscopy (SEM) and HE staining. The mechanical strength of hybrid scaffolds was determined using tensile strength analysis. Cellular penetration and interconnectivity were evaluated using fluorescent bead distribution and human bladder smooth muscle cells and urothelium seeding. SEM and HE analysis showed the honeycomb structure and the hybrid scaffolds were adequately connected. The hybrid scaffolds were much stronger than collagen alone. The distribution of the beads and cells were highly dependent on the collagen density: at lower densities the beads and cells were more evenly distributed and penetrated deeper into the scaffold. The lower density collagen scaffolds showed remarkably deeper cellular penetration and by combining it with PCL knitting the tensile strength was enhanced. This study indicated that a 0.4% hybrid scaffold strengthened with knitting achieved the best cellular distribution.Human adult heart harbors a population of resident progenitor cells that can be isolated by Sca-1 antibody and expanded in culture. These cells can differentiate into cardiomyocytes and vascular cells in vitro and contribute to cardiac regeneration in vivo. However, when directly injected as single cell suspension, the survival rate and retention is really poor, less than 1% of injected cells being detectable in the hosttissue within few weeks. The present study aimed at investigating the possibility to produce scaffoldless, thick cardiac progenitor cell-derived cardiac patches by thermo-responsive technology. Human cardiac progenitors obtained from the auricles of patients were cultured as scaffoldless engineered tissues fabricated using temperature-responsive surfaces obtained by poly-N-isopropylacrylamide (PNIPAAm) surface immobilization. In the engineered tissue, progenitor cells established proper three-dimensional intercellular relationships and produced abundant extracellular matrix, while preserving their phenotype and plasticity. Cell phenotype and viability within the 3D construct were followed for 1 week, showing that no significant differentiation or apoptotic events occurred within the construct. After engineered tissues were leant on visceral pericardium, a number of cells migrated into the myocardium and in the vascular walls, where they integrated in the respective textures. The study demonstrates the suitability of such approach to deliver stem cells.Spinal cord injury and repair is one of the important focus areas in tissue regeneration. Mechanical trauma caused due to factors such as contusion, compression or involuntary stretching induce post-traumatic secondary tissue damage in many Spinal Cord Injury (SCI) patients. Therefore, there is a need for scaffolds that provide a conducive threedimensionsal (3D) environment for injured cells to attach and grow. In this study we propose to synthesize 3D polymeric scaffolds in order to study the mechanical and adhesive properties & the nature of the interactions between hyaluronan-based (HY) biomaterials and cells and tissues both in vitroandin vivo. Here we have synthesized 3D HY-based hydrogels with robust mechanical and adhesive properties and demonstrate the use of this material for neuronal-related applications such as the treatment of SCI. Cell culture and survivability studies were done with NSC-34 cells. Live/Dead assay performed on the cells revealed significant differences in the staining of live cells and showed increased viability and proliferation. The number of live cells in the HY-based hydrogels with 0.1% collagen showed higher cell numbers compared with the other hydrogels. In this study we show that Injectable HYbased hydrogels with high elasticity, comparable to the mechanical properties of nervous tissue have been used in this study to study their biocompatibility and neuroprotective properties and they show better affinity for neuronal cells.Calcium phosphates (CaP) obtained by biomineralisation in Simulated Boby Fluid have been used for decades to assess the mineralisation capability of biomaterials. Recently, they have been envisioned as potential agents to promote bone formation. In this study, we have fabricated and coated with calcium phosphate melt electrospun scaffolds whereby macropores permit adequate cell migration and nutrient transfer. We have systematically investigated the effect of coating and osteoinduction onto the response of ovine osteoblasts and we observed that the coating up-regulated alkaline phosphatase activity regardless of the in vitro culture conditions. Micro Computed Tomography revealed that only scaffolds cultured in an osteoinductive cocktail were capable of depositing mineralised matrix, and that CaP coated scaffolds were more efficient at promoting mineralisation. Theses scaffolds were subcutaneously implanted in athymic rats and this demonstrated that the osteoinduction was a pre-requisite for bone formation in this ectopic model. It showed that although the bone formation was not significantly different after 8 weeks, the CaP coated scaffolds were superior at inducing bone formation as evidenced by higher levels of mineralisation at earlier time points. This work demonstrated that CaP coating is not sufficient to induce bone formation; however the combination of osteoinduction and CaP coating resulted in earlier bone formation in an ectopic model.Introduction: Bladder regeneration using minced bladder mucosa is an alternative to costly and time-consuming conventional in vitro culturing of urothelial cells. In this method, the uroepithelium ...

Entry points when undergraduate research mentors reflect on their role: a qualitative case study

Abstract Graduate students and postdoctoral researchers are increasingly taking on mentoring roles in undergraduate research (UR). There is, however, a paucity of research focusing on how they conceptualize their mentoring role. In this qualitative interview study, we identified three entry points that mentors reflect on to define their role: (1) What are the goals of UR? (2) What do the students expect from me? and (3) How should I use my expert knowledge? We discuss how academic developers can use these entry points together with a set of reflective lenses to stimulate critical reflection on the mentoring role and help the mentors to define their role and help the mentors to define their role.

The reflective diary as a method for the formative assessment of self-regulated learning

ABSTRACT An increasingly desired outcome of engineering education is the ability to engage in self-regulated learning (SRL). One promising method for the formative assessment of SRL is the reflective diary. There is, however, a paucity of research on the use of reflective diaries in engineering education. To mitigate this gap, we report on a case study where reflective diaries were implemented in a master’s course on tissue engineering. The objective of this paper is to explore the potential of reflective diaries for the formative assessment of three central aspects of SRL: conceptions of knowledge, conceptions of learning, and strategies for monitoring and regulating learning. Based on a theoretical thematic analysis of the diary entries, we show that reflective diaries can be used to assess these three aspects of SRL. We discuss ways of providing feedback to students, with a focus on dialogic feedback.