Production of food-grade microcarriers based on by-products from the food industry to facilitate the expansion of bovine skeletal muscle satellite cells for cultured meat production

publishedVersio

A practical checklist for return of results from genomic research in the European context

An increasing number of European research projects return, or plan to return, individual genomic research results (IRR) to participants. While data access is a data subject’s right under the General Data Protection Regulation (GDPR), and many legal and ethical guidelines allow or require participants to receive personal data generated in research, the practice of returning results is not straightforward and raises several practical and ethical issues. Existing guidelines focusing on return of IRR are mostly project-specific, only discuss which results to return, or were developed outside Europe. To address this gap, we analysed existing normative documents identified online using inductive content analysis. We used this analysis to develop a checklist of steps to assist European researchers considering whether to return IRR to participants. We then sought feedback on the checklist from an interdisciplinary panel of European experts (clinicians, clinical researchers, population-based researchers, biobank managers, ethicists, lawyers and policy makers) to refine the checklist. The checklist outlines seven major components researchers should consider when determining whether, and how, to return results to adult research participants: 1) Decide which results to return; 2) Develop a plan for return of results; 3) Obtain participant informed consent; 4) Collect and analyse data; 5) Confirm results; 6) Disclose research results; 7) Follow-up and monitor. Our checklist provides a clear outline of the steps European researchers can follow to develop ethical and sustainable result return pathways within their own research projects. Further legal analysis is required to ensure this checklist complies with relevant domestic laws

Tuning of 2D cultured human fibroblast behavior using lumichrome photocrosslinked collagen hydrogels

Collagen is extensively used in fabrication of hydrogels for biomedical applications but needs improvement after its isolation from tissues due to slow gelation and poor mechanical properties. Crosslinking could tailor such properties. Collagen has previously been crosslinked by chemical or photochemical methods. Chemical crosslinkers are often toxic, and the crosslinking reaction is difficult to control. Photochemical crosslinkers are usually biocompatible compounds that are activated upon irradiation. Riboflavin (vitamin B2), a photochemical crosslinker of collagen, photodegrades to lumichrome upon irradiation. Cyclodextrins have previously been used to increase the aqueous solubility of lumichrome and regulate collagen self-assembly. In this study, lumichrome dissolved by cyclodextrin complexation was used as a photochemical crosslinker of collagen. Lumichrome photocrosslinking reduced the gelation time to 10 s, compared to 90 min for physical crosslinking. The formed hydrogels exhibited increased elasticity, water absorption properties and water holding capacity compared to physically crosslinked collagen hydrogels and riboflavin photocrosslinked collagen hydrogels. Fibroblasts achieved a myofibroblastic phenotype when cultivated in 2D on lumichrome photocrosslinked gels as observed from histology. These biocompatible photocrosslinked hydrogels could have potential applications in biomedical applications, such as wound healing

Tuning of 2D cultured human fibroblast behavior using lumichrome photocrosslinked collagen hydrogels

Collagen is extensively used in fabrication of hydrogels for biomedical applications but needs improvement after its isolation from tissues due to slow gelation and poor mechanical properties. Crosslinking could tailor such properties. Collagen has previously been crosslinked by chemical or photochemical methods. Chemical crosslinkers are often toxic, and the crosslinking reaction is difficult to control. Photochemical crosslinkers are usually biocompatible compounds that are activated upon irradiation. Riboflavin (vitamin B2), a photochemical crosslinker of collagen, photodegrades to lumichrome upon irradiation. Cyclodextrins have previously been used to increase the aqueous solubility of lumichrome and regulate collagen self-assembly. In this study, lumichrome dissolved by cyclodextrin complexation was used as a photochemical crosslinker of collagen. Lumichrome photocrosslinking reduced the gelation time to 10 s, compared to 90 min for physical crosslinking. The formed hydrogels exhibited increased elasticity, water absorption properties and water holding capacity compared to physically crosslinked collagen hydrogels and riboflavin photocrosslinked collagen hydrogels. Fibroblasts achieved a myofibroblastic phenotype when cultivated in 2D on lumichrome photocrosslinked gels as observed from histology. These biocompatible photocrosslinked hydrogels could have potential applications in biomedical applications, such as wound healing.publishedVersio

5-FU resistant EMT-like pancreatic cancer cells are hypersensitive to photochemical internalization of the novel endoglin-targeting immunotoxin CD105-saporin

Abstract Background Development of resistance to 5-fluorouracil (5-FU) is a major problem in treatment of various cancers including pancreatic cancer. In this study, we reveal important resistance mechanisms and photochemical strategies to overcome 5-FU resistance in pancreatic adenocarcinoma. Methods 5-FU resistant (5-FUR), epithelial-to-mesenchymal-like sub-clones of the wild type pancreatic cancer cell line Panc03.27 were previously generated in our lab. We investigated the cytotoxic effect of the endosomal/lysosomal-localizing photosensitizer TPCS2a (fimaporfin) combined with light (photochemical treatment, PCT) using MTS viability assay, and used fluorescence microscopy to show localization of TPCS2a and to investigate the effect of photodamage of lysosomes. Flow cytometric analysis was performed to investigate uptake of photosensitizer and to assess intracellular ROS levels. Expression and localization of LAMP1 was assessed using RT-qPCR, western blotting, and structured illumination microscopy. MTS viability assay was used to assess the effect of combinations of 5-FU, chloroquine (CQ), and photochemical treatment. Expression of CD105 was investigated using RT-qPCR, western blotting, flow cytometry, and fluorescence microscopy, and co-localization of TPCS2a and anti-CD105-saporin was assessed using microscopy. Lastly, the MTS assay was used to investigate cytotoxic effects of photochemical internalization (PCI) of the anti-CD105-immunotoxin. Results The 5-FUR cell lines display hypersensitivity to PCT, which was linked to increased uptake of TPCS2a, altered lysosomal distribution, lysosomal photodamage and increased expression of the lysosomal marker LAMP-1 in the 5-FUR cells. We show that inhibition of autophagy induced by either chloroquine or lysosomal photodamage increases the sensitivity to 5-FU in the resistant cells. The three 5-FUR sub-clones overexpress Endoglin (CD105). Treatment with the immunotoxin anti-CD105-saporin alone significantly reduced the viability of the CD105-expressing 5-FUR cells, whereas little effect was seen in the CD105-negative non-resistant parental cancer cell lines. Strikingly, using the intracellular drug delivery method photochemical internalization (PCI) by combining light-controlled activation of the TPCS2a with nanomolar levels of CD105-saporin resulted in strong cytotoxic effects in the 5-FUR cell population. Conclusion Our findings suggested that autophagy is an important resistance mechanism against the chemotherapeutic drug 5-FU in pancreatic cancer cells, and that inhibition of the autophagy process, either by CQ or lysosomal photodamage, can contribute to increased sensitivity to 5-FU. For the first time, we demonstrate the promise of PCI-based targeting of CD105 in site-specific elimination of 5-FU resistant pancreatic cancer cells in vitro. In conclusion, PCI-based targeting of CD105 may represent a potent anticancer strategy and should be further evaluated in pre-clinical models

Implications of Targeted Genomic Disruption of β-Catenin in BxPC-3 Pancreatic Adenocarcinoma Cells

<div><p>Pancreatic adenocarcinoma (PA) is among the most aggressive human tumors with an overall 5-year survival rate of <5% and available treatments are only minimal effective. WNT/β-catenin signaling has been identified as one of 12 core signaling pathways that are commonly mutated in PA. To obtain more insight into the role of WNT/β-catenin signaling in PA we established human PA cell lines that are deficient of the central canonical WNT signaling protein β-catenin by using zinc-finger nuclease (ZFN) mediated targeted genomic disruption in the β-catenin gene (<i>CTNNB1</i>). Five individual <i>CTNNB1</i> gene disrupted clones (BxPC3ΔCTNNB1) were established from a BxPC-3 founder cell line. Despite the complete absence of β-catenin, all clones displayed normal cell cycle distribution profiles, overall normal morphology and no elevated levels of apoptosis although increased doubling times were observed in three of the five BxPC3ΔCTNNB1 clones. This confirms that WNT/β-catenin signaling is not mandatory for long term cell growth and survival in BxPC-3 cells. Despite a normal morphology of the β-catenin deficient cell lines, quantitative proteomic analysis combined with pathway analysis showed a significant down regulation of proteins implied in cell adhesion combined with an up-regulation of plakoglobin. Treatment of BxPC3ΔCTNNB1 cell lines with siRNA for plakoglobin induced morphological changes compatible with a deficiency in the formation of functional cell to cell contacts. In addition, a re-localization of E-cadherin from membranous in untreated to accumulation in cytoplasmatic puncta in plakoglobin siRNA treated BxPC3ΔCTNNB1 cells was observed. In conclusion we describe in β-catenin deficient BxPC-3 cells a rescue function for plakoglobin on cell to cell contacts and maintaining the localization of E-cadherin at the cellular surface, but not on canonical WNT signaling as measured by TFC/LEF mediated transcription.</p></div

<i>CTNNB1</i> gene disrupted cells displays increased levels of plakoglobin protein.

<p>A) Immunostaining of WT and gene disrupted clones with plakoglobin Ab (green) and DAPI nuclear counter stain (blue). B) WB analysis of total cell lysates from wild type and gene disrupted clones. Position and molecular weight of plakoglobin and actin specific bands are indicated. Quantification of the intensities of the plakoglobin bands relative to the actin bands are shown below. C) Relative quantification of plakoglobin mRNA levels using qRT-PCR in WT and gene disrupted clones. Error bars represent SD of the mean RQ value. D) Co-immunoprecipitation of total cell lysates from WT or gene disrupted cells using anti plakoglobin Ab for immunoprecipitation and anti plakoglobin and anti E-cadherin Abs for detection as indicated.</p