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- Blust, Kelly
(författare)
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Advancing 3D Cell Cultures of Stem-Cell Derived Pancreatic Islets and Breast Cancer Cells Using Recombinant Functionalized Spider Silk : Insights into cellular composition using bioinformatic methods
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The culture of cells in 3D creates a more physiologically relevant cell environment than conventional 2D cultures. Interactions of cells with the extracellular matrix induce important cellular signalling that regulates cell adhesion, migration, proliferation, differentiation, and survival. This is crucial for modelling cell development and disease. This thesis aims to develop and analyse improved 3D cell culture methods for stem cell-derived pancreatic islets (SC-islets) and breast cancer cell lines using a functionalized recombinant spider silk. Spider silk is a natural protein-based material with remarkable mechanical properties of high strength and elasticity. It is also biodegradable and cytocompatible. FN-silk, the recombinant spider silk protein utilized in this thesis, is functionalized with a cell binding motif (RGD) from fibronectin, to improve cell adhesion. Notably, FN-silk self-assembles at the liquid-air interface into a fibrillar structure, making it favourable as support for cell culture. In this thesis, bioinformatic methods were used to discover how the FN-silk supported environment affects the gene expression of cells and the cellular heterogeneity of SC-islets during a differentiation process. Additionally, bioinformatical analysis of the effect of 3D cell culture of breast cancer cell lines in FN-silk networks was performed. The first part of the thesis addresses serval challenges in pancreatic islet transplantation, by presenting an optimized protocol for pancreatic differentiation from human pluripotent stem cells, improving in vitro cultivation, and developing a cryopreservation method for SC-islets. The differentiation protocol presented in Paper 1 resulted in pure endocrine cell populations, avoiding unwanted proliferating and non-endocrine cells. It was also demonstrated that these SC-islets matured in vivo, and could effectively reverse diabetes in a diabetic mouse model. Single-cell RNA sequencing analysis provided new insights into the cellular composition and gene expression of the SC-islets before and after transplantation. In Paper 2, an innovative method for 3D in vitro cultivation of SC-islets using FN-silk networks mimicking the extracellular matrix was established. The FN-silk networks provided structural support for in vitro cultivation and handling during in vivo transplantation. The viability and functionality of free and FN-silk incorporated SC-islets were evaluated and compared. Single-cell RNA sequencing analyses confirmed maintenance of cellular composition, with a slightly improved beta cell maturation for SC-islets supported by FN-silk. In Paper 3, a novel strategy for cryopreservation of SC-islets was explored. The twisted vitrification method, previously employed for 2D cultures, was adapted for 3D cultures by utilizing integration into FN-silk networks to facilitate handling during the vitrification process. The second part of the thesis aimed to develop a method for 3D culture of breast cancer cells to better replicate the complexity of the tumour microenvironment. In Paper 4, FN-silk networks were used to generate a 3D environment for breast cancer cells where crucial cell-ECM interactions can be established. Proliferation rates and key marker expression of the cells cultured in 2D versus in the FN-silk network environment were investigated. Bioinformatic analysis of bulk RNA sequencing data was used to compare breast cancer cells in conventional 2D cell cultures with those cultured in 3D with the support of FNsilk. In conclusion, the work conducted in this thesis presents significant advancements in the development and analyses of 3D cell cultures of both SCislets and breast cancer cell lines, potentially enhancing therapeutic applications, disease modeling, and drug testing.
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| 3. |
- Blust, Kelly, et al.
(författare)
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Cryopreservation of stem cell-derived pancreatic islets using twisted vitrification with a supporting FN-silk network
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Cryopreservation is crucial for making cell therapies more accessible by enabling long-term storage and facilitating transplantation logistics. However, preserving cells in 3D models implies significant challenges, including inhomogeneous penetration of cryoprotectants and non-uniform heat transfer during freezing and thawing. Vitrification is a novel cryopreservation approach that improves the survival of living cells by providing optimal cooling and rewarming rates to eliminate ice crystal-induced mechanical injury. Nevertheless, sterility issues can arise from direct contact with liquid nitrogen (LN2). The twisted vitrification method addresses this by separating the cells and LN2 into two different compartments, ensuring a sterile vitrification process.In this study, we optimized the cryopreservation of stem cell-derived pancreatic islets (SC-islets) by combining twisted vitrification with the supporting biomaterial FN-silk. We compared the viability of free SC-islets to those supported by a FN-silk network during cryopreservation using either conventional slow freezing or twisted vitrification. Our results showed a higher post-recovery for SC-islets incorporated in FN-silk using twisted vitrification, outperforming both conventional slow freezing and twisted vitrification of free SC-islets. Additionally, insulin and glucagon expression were maintained after vitrification within the FN-silk network.
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| 4. |
- Blust, Kelly, et al.
(författare)
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Integration of stem cell-derived pancreatic aggregates into FN-silk network for in vitro cultivation and in vivo transplantation
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Type 1 diabetes is a life-threatening disease characterized by lifelong insulin dependency and a reduced quality of life. Pancreatic islet transplantation is a promising treatment but is limited by donor shortages and significant islet loss during the procedures. The usage of pancreatic islet-like aggregates derived from pluripotent stem cells (SC-islets) could solve the problem of shortage of islets. Incorporating these SC-islets into a supporting scaffold could protect them during handling.FN-silk, a recombinantly produced spider silk protein functionalized with a cell adhesion motif from fibronectin, can generate 3D networks that mimic the extracellular matrix (ECM). We herein demonstrate a reproducible method for integrating SC-islets well-distributed within stable 3D networks of FN-silk. These SC-islets showed high viability and maintained functionality, with increased glucagon expression and improved beta cell maturation as compared to free SC-islets. Additionally, the support of FN-silk networks enables cultivation of SC-islets under conditions needed for scale-up. Moreover, the integration of the SC-islets into sheets of FN-silk networks facilitates handling during transplantation into the anterior chamber of the eye (ACE) of rabbits. Such transplanted FN-silk incorporated SC-islets were found vascularized six weeks post-transplantation, with sustained insulin and glucagon expression.
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| 9. |
- Thatikonda, Naresh, et al.
(författare)
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Genetic fusion of single-chain variable fragments to partial spider silk improves target detection in micro- and nanoarrays
- 2016
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Ingår i: Biotechnology Journal. - : Wiley-VCH Verlagsgesellschaft. - 1860-6768 .- 1860-7314. ; 11:3, s. 437-448
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Tidskriftsartikel (refereegranskat)abstract
- Immobilizing biomolecules with retained functionality and stability on solid supports is crucial for generation of sensitive immunoassays. However, upon use of conventional immobilization strategies, a major portion of the biomolecules (e.g. antibodies) frequently tends to lose their bioactivity. In this study, we describe a procedure to immobilize human single-chain variable fragment (scFv) via genetic fusion to partial spider silk, which have a high tendency to adhere to solid supports. Two scFvs, directed towards serum proteins, were genetically fused to partial spider silk proteins and expressed as silk fusion proteins in E. coli. Antigen binding ability of scFvs attached to a partial silk protein denoted RC was investigated using microarray analysis, whereas scFvs fused to the NC silk variant were examined using nanoarrays. Results from micro- and nanoarrays confirmed the functionality of scFvs attached to both RC and NC silk, and also for binding of targets in crude serum. Furthermore, the same amount of added scFv gives higher signal intensity when immobilized via partial spider silk compared to when immobilized alone. Together, the results suggest that usage of scFv-silk fusion proteins in immunoassays could improve target detection, in the long run enabling novel biomarkers to be detected in crude serum proteomes.
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