Confocal Raman microscopy : applications in tissue engineering

This dissertation describes the use of confocal Raman microscopy and spectroscopy in the field of tissue engineering. Moreover, it describes the combination of two already existing technologies, namely scanning electron microscopy and confocal Raman spectroscopy in one apparatus for the enhancement of the chemical analytical capabilities of the electron microscope

PDB73 – The Expected Value Of Bio-Artificial Pancreas Development In View Of Endocrinologists' And Patients' Preferences

Objectives Islet transplantation is an accepted transplantation method in type I Diabetes Mellitus, yet islet survival is hampered due to an insufficient transplantation site and severe immunological and inflammatory responses. The development of a bio-artificial pancreas (BAP) may contribute to transplanted islet functionality and survival. The objective of this study is to identify the most important transplantation characteristics and to asses patients’ and endocrinologists´ preferences for three potential BAP scenarios in order to guide further development. Methods The current standard of care and characteristics that determine clinical decisions for a particular transplantation method were analysed based on a literature search, semi-structured interviews and focus groups. A decision tree was constructed covering the main attributes effectiveness, patient safety, impact of the treatment for the patient and the required amount of donor material. The analytic hierarchy process was used to obtain the relative weights for each defined attribute in type I DM patients (n=21) and endocrinologists (n=12). Based on these weights, overall preferences for three potential BAP scenarios were calculated and compared to conventional pancreas and islets transplantation. Results The three most important treatment attributes are the effectiveness of the transplant for glucose control, patient safety and the surgical procedure. However, there were considerable differences between patients and endocrinologists in the importance of effectiveness of the transplant (weights were 0.471 and 0.257 respectively) and patient safety (0.331 and 0.423). While considering both endocrinologists’ and patients’ preferences, all three BAP scenarios assessed gained a higher overall preference in comparison to conventional islet transplantation. Conclusions This study indicates the prospects of BAP development. Nevertheless, the study also highlights the discrepancies between endocrinologists’ and type 1 diabetes patients’ preferences. In the future, BAP developers can benefit from this multidisciplinary approach by critically reviewing their BAP design, in view of patient safety and clinical performanc

Fibronectin and Collagen IV Microcontact Printing Improves Insulin Secretion by INS1E Cells

Extracellular matrix (ECM) molecules play significant roles in regulating β-cell function and viability within pancreatic islets by providing mechanical and biological support, stimulating cell survival, proliferation, and their endocrine function. During clinical islet transplantation, the β-cell's ECM environment is degraded by enzymatic digestion. Literature suggests that interactions between islet cells and ECM molecules, such as fibronectin (FN), collagen type IV (Col4), and laminin (LN), are essential for maintaining, or stimulation of islet function and survival, and can effect differentiation and proliferation of the endocrine cells. It is also thought that three-dimensional (3D) culture of β-cells can improve glucose responsiveness by providing a specific niche, in which cells can interact with each other in a more natural manner. Conventional suspension cultures with β-cells results generally in a heterogeneous population with small and large aggregates, in which cells experience different nutrient diffusion limitations, negatively affecting their physiology and function. In this study, we have explored the effect of FN, Col4, and LN111 on INS1E insulinoma cells by using microcontact printing (μCP) to investigate whether a controlled environment and aggregate dimensions would improve their endocrine function. Using this method, we produced a pattern of well-defined circular spots of FN, Col4, and LN111 on polydimethylsiloxane with high spatial resolution. Cell seeding of the INS1E cells on these ECM protein spots resulted in the formation of 3D β-cell aggregates. We show that these INS1E aggregates have very reproducible dimensions, and that the cell culture method can be easily adjusted, leading to a highly accurate way of forming 3D β-cell aggregates on an ECM-functionalized substrate. In addition, we show that ECM molecules can act as anchoring points for β-cells on an otherwise non-cell-adherent material, and this can improve both the endocrine function and viability. We found a significant increase in the secretion of insulin by INS1E cells cultured on μCP FN and Col4 substrates, in comparison to cells that were growing in monolayers on substrates without ECM molecules. Moreover, INS1E cells growing on circular ECM spots in a 3D manner showed improved endocrine function in comparison to their two-dimensional counterparts. This research deals with finding a proper bioengineering strategy for the creation of improved β-cell replacement therapy in type 1 diabetes. It specifically deals with the microenvironment of β-cells and its relationship to their endocrine function

Raman microspectroscopy: A non-invasive analysis tool for monitoring of collagen-containing extracellular matrix formation in a medium-throughput culture system

The three-dimensional environment is known to play an important role in promoting cell–matrix interactions. We have investigated the possibility of using Raman microspectroscopy—which has the great advantage of noninvasive sensing—for in vitro monitoring of extracellular matrix (ECM) formation in a medium-throughput pellet (3D) culture system with soft-litography, agarose-microwell arrays. Chondrocytes were seeded in the agarose microwells in basic or chondrocyte medium. After 3, 7, and 14 days of culture, samples were analyzed for ECM formation by Raman microspectroscopy, histology, and immunofluorescence. ECM formation in the chondrocyte medium-cultured samples was detected by histology and immunofluorescence, and also noninvasively by Raman microspectroscopy. The Raman band of collagen found at 937 cm−1 can be used as a Raman marker for collagen-containing ECM formation over time in the chondrocyte pellets. This culture system can be implemented as a medium-throughput platform for Raman applications and screening microtissue formation, since with these agarose-microwell arrays relatively large numbers of cell pellets could be screened in a short time in situ, without having to transfer the pellets onto microscopic slides. Moreover, in this manner the culture system is suitable for long-term, real-time live-cell measurements

Intracellular degradation of microspheres based on cross-linked dextran hydrogels or amphiphilic block copolymers: A comparative Raman microscopy study

Micro- and nanospheres composed of biodegradable polymers show promise as versatile devices for the controlled delivery of biopharmaceuticals. Whereas important properties such as drug release profiles, biocompatibility, and (bio)degradability have been determined for many types of biodegradable particles, information about particle degradation inside phagocytic cells is usually lacking. Here, we report the use of confocal Raman microscopy to obtain chemical information about cross-linked dextran hydrogel microspheres and amphiphilic poly(ethylene glycol)-terephthalate/poly(butylene terephthalate) (PEGT/PBT) microspheres inside RAW 264.7 macrophage phagosomes. Using quantitative Raman microspectroscopy, we show that the dextran concentration inside phagocytosed dextran microspheres decreases with cell incubation time. In contrast to dextran microspheres, we did not observe PEGT/PBT microsphere degradation after 1 week of internalization by macrophages, confirming previous studies showing that dextran microsphere degradation proceeds faster than PEGT/PBT degradation. Raman microscopy further showed the conversion of macrophages to lipid-laden foam cells upon prolonged incubation with both types of microspheres, suggesting that a cellular inflammatory response is induced by these biomaterials in cell culture. Our results exemplify the power of Raman microscopy to characterize microsphere degradation in cells and offer exciting prospects for this technique as a noninvasive, label-free optical tool in biomaterials histology and tissue engineering

High Throughput Micro-Well Generation of Hepatocyte Micro-Aggregates for Tissue Engineering

The main challenge in hepatic tissue engineering is the fast dedifferentiation of primary hepatocytes in vitro. One successful approach to maintain hepatocyte phenotype on the longer term is the cultivation of cells as aggregates. This paper demonstrates the use of an agarose micro-well chip for the high throughput generation of hepatocyte aggregates, uniform in size. In our study we observed that aggregation of hepatocytes had a beneficial effect on the expression of certain hepatocyte specific markers. Moreover we observed that the beneficial effect was dependent on the aggregate dimensions, indicating that aggregate parameters should be carefully considered. In a second part of the study, the selected aggregates were immobilized by encapsulation in methacrylamide-modified gelatin. Phenotype evaluations revealed that a stable hepatocyte phenotype could be maintained during 21 days when encapsulated in the hydrogel. In conclusion we have demonstrated the beneficial use of micro-well chips for hepatocyte aggregation and the size-dependent effects on hepatocyte phenotype. We also pointed out that methacrylamide-modified gelatin is suitable for the encapsulation of these aggregates

Preferred Islet Delivery Device Characteristics and Implantation Strategies of Patients With Type 1 Diabetes

Islet delivery devices (IDDs) offer potential benefits for islet transplantation and stem cell-based replacement in type 1 diabetes. Little is known about patient preferences regarding islet delivery device characteristics and implantation strategies. Patient preferences for IDDs and implantation strategies remain understudied. We invited patients, parents and caregivers to fill in an online questionnaire regarding IDDs. An online survey gathered responses from 809 type 1 diabetes patients and 47 caregivers. We also assessed diabetes distress in a subgroup of 412 patients. A significant majority (97%) expressed willingness to receive an IDD. Preferred IDD attributes included a 3.5 cm diameter for 37.7% of respondents, while when provided with all options, 30.4% found dimensions unimportant. Respondents were open to approximately 4 implants, each with a 5 cm incision. Many favored a device functioning for 12 months (33.4%) or 24 months (24.8%). Younger participants (16-30) were more inclined to accept a 6 months functional duration ( p < 0.001). Functional duration outweighed implant quantity and size ( p < 0.001) in device importance. This emphasizes patients' willingness to accommodate burdens related to IDD features and implantation methods, crucial for designing future beta cell replacement strategies

A High Cell-Bearing Capacity Multibore Hollow Fiber Device for Macroencapsulation of Islets of Langerhans

Macroencapsulation of islets of Langerhans is a promising strategy for transplantation of insulin-producing cells in the absence of immunosuppression to treat type 1 diabetes. Hollow fiber membranes are of interest there because they offer a large surface-to-volume ratio and can potentially be retrieved or refilled. However, current available fibers have limitations in exchange of nutrients, oxygen, and delivery of insulin potentially impacting graft survival. Here, multibore hollow fibers for islets encapsulation are designed and tested. They consist of seven bores and are prepared using nondegradable polymers with high mechanical stability and low cell adhesion properties. Human islets encapsulated there have a glucose induced insulin response (GIIS) similar to nonencapsulated islets. During 7 d of cell culture in vitro, the GIIS increases with graded doses of islets demonstrating the suitability of the microenvironment for islet survival. Moreover, first implantation studies in mice demonstrate device material biocompatibility with minimal tissue responses. Besides, formation of new blood vessels close to the implanted device is observed, an important requirement for maintaining islet viability and fast exchange of glucose and insulin. The results indicate that the developed fibers have high islet bearing capacity and can potentially be applied for a clinically applicable bioartificial pancreas