Optimization of nanocellulose hydrogel for mouse intestinal organoid cultivation
Karvonen, Rosa (2022)
Karvonen, Rosa
2022
Master's Programme in Biomedical Technology
Lääketieteen ja terveysteknologian tiedekunta - Faculty of Medicine and Health Technology
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Hyväksymispäivämäärä
2022-05-30
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tuni-202204263660
https://urn.fi/URN:NBN:fi:tuni-202204263660
Tiivistelmä
Background and objectives of the study: Nowadays, biomedical research aims to build three-dimensional (3D) organ models to mimic better complex tissues and their functions. Organoids are self-organizing organ-like structures with near-native microanatomy. Organoids are a potential platform for drug screening and disease modelling. Conventionally in cell culturing animal-derived basement membrane extract mimics the extracellular matrix (ECM), which surrounds the cells and provides them with essential biochemical and mechanical cues. However, animal-derived materials suffer from reproducibility problems that limit their use in medical applications. Engineered matrices are promising alternatives to animal-derived materials. Nanocellulose hydrogels have shown their potential to mimic the native ECM with their high biocompatibility and tunable properties. In this thesis avidin-conjugated nanofibrillar cellulose hydrogel, GrowDex-A (GDxA), was studied as a 3D culturing platform for murine intestinal organoids. This thesis aimed to functionalize the GDxA with selected biotinylated proteins or peptides, and to optimize the culturing conditions for intestinal organoid cultivation.
Materials and methods: For GDxA functionalization, selected ECM proteins, vitronectin (VN), and laminin (LN) were biotinylated to bind them to GDxA via avidin-biotin interaction. In addition, biotinylated cyclic RGD (BcRGD) peptide and non-biotinylated laminin were used to functionalize GDxA. Intestinal crypts were cultured in GDxA functionalized with several protein combinations and concentrations. Functionalized GDxA was also supplemented with collagen (COL). Intestinal crypts were cultured in hydrogels with varying GDxA-COL ratios, and delayed crypt addition to premade GDxA-BcRGD-COL hydrogels was also studied. Several variables such as pH of COL, use of inhibitor, and incubation times in different steps of hydrogel preparation were investigated to find the optimal culture conditions. Bright-field microscopy and Live/Dead staining were used to characterize the organoid viability.
Results: Biotinylation was done successfully for VN and LN. A hydrogel containing 0.18 % (w/v) GDxA functionalized with 1.54 µg/ml BcRGD and supplemented with 1.4 mg/ml COL supported the intestinal organoid formation and was used in further experiments. When other GDxA-COL ratios were studied, cystic organoids formed only if hydrogel rolled into a thick mattress-like structure. When delayed crypt addition to premade GDxA-BcRGD-COL hydrogels was studied, cell growth was observed in every culture condition with differing COL pH levels and incubation times. The Live/Dead assay showed that during the first three culture days organoid growth in the GDxA-BcRGD-COL hydrogel seemed to be slower but otherwise similar to organoid growth in the animal-derived reference hydrogel.
Conclusions: Organoid formation was observed under many culturing conditions in GDxA-BcRGD-COL hydrogel, but more optimization is needed to produce GDxA-BcRGD-COL hydrogel reproducibly for intestinal organoid cultivation. More in-depth information on the genetics of the formed organoids is needed to confirm the similarity in growth between GDxA-BcRGD-COL and reference hydrogels. GDxA may have the potential as an alternative for animal-derived matrices for organoid cultivation when it is functionalized with BcRGD and supplemented with COL.
Materials and methods: For GDxA functionalization, selected ECM proteins, vitronectin (VN), and laminin (LN) were biotinylated to bind them to GDxA via avidin-biotin interaction. In addition, biotinylated cyclic RGD (BcRGD) peptide and non-biotinylated laminin were used to functionalize GDxA. Intestinal crypts were cultured in GDxA functionalized with several protein combinations and concentrations. Functionalized GDxA was also supplemented with collagen (COL). Intestinal crypts were cultured in hydrogels with varying GDxA-COL ratios, and delayed crypt addition to premade GDxA-BcRGD-COL hydrogels was also studied. Several variables such as pH of COL, use of inhibitor, and incubation times in different steps of hydrogel preparation were investigated to find the optimal culture conditions. Bright-field microscopy and Live/Dead staining were used to characterize the organoid viability.
Results: Biotinylation was done successfully for VN and LN. A hydrogel containing 0.18 % (w/v) GDxA functionalized with 1.54 µg/ml BcRGD and supplemented with 1.4 mg/ml COL supported the intestinal organoid formation and was used in further experiments. When other GDxA-COL ratios were studied, cystic organoids formed only if hydrogel rolled into a thick mattress-like structure. When delayed crypt addition to premade GDxA-BcRGD-COL hydrogels was studied, cell growth was observed in every culture condition with differing COL pH levels and incubation times. The Live/Dead assay showed that during the first three culture days organoid growth in the GDxA-BcRGD-COL hydrogel seemed to be slower but otherwise similar to organoid growth in the animal-derived reference hydrogel.
Conclusions: Organoid formation was observed under many culturing conditions in GDxA-BcRGD-COL hydrogel, but more optimization is needed to produce GDxA-BcRGD-COL hydrogel reproducibly for intestinal organoid cultivation. More in-depth information on the genetics of the formed organoids is needed to confirm the similarity in growth between GDxA-BcRGD-COL and reference hydrogels. GDxA may have the potential as an alternative for animal-derived matrices for organoid cultivation when it is functionalized with BcRGD and supplemented with COL.