Purified Human Pancreatic Duct Cell Culture Conditions Defined by Serum-Free High-Content Growth Factor Screening

The proliferation of pancreatic duct-like CK19+ cells has implications for multiple disease states including pancreatic cancer and diabetes mellitus. The in vitro study of this important cell type has been hampered by their limited expansion compared to fibroblast-like vimentin+ cells that overgrow primary cultures. We aimed to develop a screening platform for duct cell mitogens after depletion of the vimentin+ population. The CD90 cell surface marker was used to remove the vimentin+ cells from islet-depleted human pancreas cell cultures by magnetic-activated cell sorting. Cell sorting decreased CD90+ cell contamination of the cultures from 34±20% to 1.3±0.6%, yielding purified CK19+ cultures with epithelial morphology. A full-factorial experimental design was then applied to test the mitogenic effects of bFGF, EGF, HGF, KGF and VEGF. After 6 days in test conditions, the cells were labelled with BrdU, stained and analyzed by high-throughput imaging. This screening assay confirmed the expected mitogenic effects of bFGF, EGF, HGF and KGF on CK19+ cells and additionally revealed interactions between these factors and VEGF. A serum-free medium containing bFGF, EGF, HGF and KGF led to CK19+ cell expansion comparable to the addition of 10% serum. The methods developed in this work should advance pancreatic cancer and diabetes research by providing effective cell culture and high-throughput screening platforms to study purified primary pancreatic CK19+ cells

A fluorophore-tagged RGD peptide to control endothelial cell adhesion to micropatterned surfaces

The long-term patency rates of vascular grafts and stents are limited by the lack of surface endothelialisation of the implanted materials. We have previously reported that GRGDS and WQPPRARI peptide micropatterns increase the endothelialisation of prosthetic materials in vitro. To investigate the mechanisms by which the peptide micropatterns affect endothelial cell adhesion and proliferation, a TAMRA fluorophore-tagged RGD peptide was designed. Live cell imaging revealed that the micropatterned surfaces led to directional cell spreading dependent on the location of the RGD-TAMRA spots. Focal adhesions formed within 3 h on the micropatterned surfaces near RGD-TAMRA spot edges, as expected for cell regions experiencing high tension. Similar levels of focal adhesion kinase phosphorylation were observed after 3 h on the micropatterned surfaces and on surfaces treated with RGD-TAMRA alone, suggesting that partial RGD surface coverage is sufficient to elicit integrin signaling. Lastly, endothelial cell expansion was achieved in serum-free conditions on gelatin-coated, RGD-TAMRA treated or micropatterned surfaces. These results show that these peptide micropatterns mainly impacted cell adhesion kinetics rather than cell proliferation. This insight will be useful for the optimization of micropatterning strategies to improve vascular biomaterials

Multifunctional surface modifications to capture and expand flowing endothelial colony- forming cells

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Design of a 3D printer head for additive manufacturing of sugar glass for tissue engineering applications

Additive manufacturing is now considered as a new paradigm that is foreseen to improve progress in many fields. The field of tissue engineering has been facing the need for tissue vascularization when producing thick tissues. The use of sugar glass as a fugitive ink to produce vascular networks through rapid casting may offer the key to vascularization of thick tissues produced by tissue engineering. Here, a 3D printer head capable of producing complex structures out of sugar glass is presented. This printer head uses a motorized heated syringe fitted with a custom made nozzle. The printer head was adapted to be mounted on a commercially available 3D printer. A mathematical model was derived to predict the diameter of the filaments based on the printer head feed rate and extrusion rate. Using a 1 mm diameter nozzle, the printer accurately produced filaments ranging from 0.3 mm to 3.2 mm in diameter. One of the main advantages of this manufacturing method is the self-supporting behaviour of sugar glass that allows the production of long, horizontal, curved, as well as overhanging filaments needed to produce complex vascular networks. Finally, to establish a proof of concept, polydimethylsiloxane was used as the gel matrix during the rapid casting to produce various “vascularized” constructs that were successfully perfused, which suggests that this new fabrication method can be used in a number of tissue engineering applications, including the vascularization of thick tissues

A Multi-Parameter, High-Content, High-Throughput Screening Platform to Identify Natural Compounds that Modulate Insulin and Pdx1 Expression

Diabetes is a devastating disease that is ultimately caused by the malfunction or loss of insulin-producing pancreatic beta-cells. Drugs capable of inducing the development of new beta-cells or improving the function or survival of existing beta-cells could conceivably cure this disease. We report a novel high-throughput screening platform that exploits multi-parameter high-content analysis to determine the effect of compounds on beta-cell survival, as well as the promoter activity of two key beta-cell genes, insulin and pdx1. Dispersed human pancreatic islets and MIN6 beta-cells were infected with a dual reporter lentivirus containing both eGFP driven by the insulin promoter and mRFP driven by the pdx1 promoter. B-score statistical transformation was used to correct systemic row and column biases. Using this approach and 5 replicate screens, we identified 7 extracts that reproducibly changed insulin and/or pdx1 promoter activity from a library of 1319 marine invertebrate extracts. The ability of compounds purified from these extracts to significantly modulate insulin mRNA levels was confirmed with real-time PCR. Insulin secretion was analyzed by RIA. Follow-up studies focused on two lead compounds, one that stimulates insulin gene expression and one that inhibits insulin gene expression. Thus, we demonstrate that multi-parameter, high-content screening can identify novel regulators of beta-cell gene expression, such as bivittoside D. This work represents an important step towards the development of drugs to increase insulin expression in diabetes and during in vitro differentiation of beta-cell replacements

Surface grafting of Fc-binding peptides as a simple platformto immobilize and identify antibodies that selectively capture circulating endothelial progenitor cells

Antibody surface immobilization is a promising strategy to capture cells of interest from circulating fluids in vitro and in vivo. An application of particular interest in vascular interventions is to capture endothelial progenitor cells (EPCs) on the surface of stents to accelerate endothelialization. The clinical impact of EPC capture stents has been limited by the lack of efficient selective cell capture. Here, we describe a simple method to immobilize a variety of immunoglobulin G antibodies through their fragment crystallizable (Fc) regions via surface-conjugated RRGW peptides for cell capture applications. As an EPC capture model, peripheral blood endothelial colony-forming cells suspended in cell culture medium with up to 70% serum were captured by immobilized anti-CD144, anti-CD34 or anti-CD309 antibodies under laminar flow. The endothelial colony-forming cells were successfully enriched from a mixture with peripheral blood mononuclear cells using surfaces with anti-CD309 but not anti-CD45. This antibody immobilization approach holds great promise to engineer vascular biomaterials with improved EPC capture potential. The ease of immobilizing different antibodies using the same Fc-binding peptide surface grafting chemistry renders this platform suitable to screen antibodies that maximize cell capture efficiency and selectivity

Supporting information: Magnetic resonance imaging of alginate beads containing pancreatic islets and paramagnetic nanoparticles

Microencapsulation is being investigated as a means to avoid rejection of transplanted pancreatic islets. Monitoring bead distribution and stability in vivo is an important step toward improving microencapsulated islet transplantation strategies. Islet co-encapsulation with gadolinium-labeled mesoporous silica nanoparticles (Gd-MSNs) could allow bead visualization while immobilizing and limiting the potential internalization of the contrast agent. [...] This study paves the way for microencapsulated islet tracking via MRI using co-encapsulated paramagnetic nanomaterials

Atmospheric pressure plasma polymer of ethyl lactate: In vitro degradation and cell viability studies

Ethyl lactate is injected into a dielectric barrier discharge (DBD) to build up a degradable plasma polymer (PP) to be used as a drug delivery system. Plasma power, deposition time, and type of carrier gas (Ar, N2) are correlated to the coating in vitro degradation rate. PPs are characterized by AFM, SEM, IR spectroscopy, XPS, and SEC, while surface profilometry is used to monitor the degradation kinetics. PPs deposited under N2 are mainly composed of hydrophilic functionalities, which explain their fast degradation upon exposure to an aqueous environment. In contrast, PPs synthesized under Ar lead to a slower degradation rate due to their hydrocarbon structure containing some hydrolyzable moieties. The potential of the PPs for vascular applications is verifie

CK19+ cell expansion in medium containing bFGF, EGF, HGF and KGF compared to control media.

<p>A) Growth curve based on the number of CK19+ cells enumerated at different time points in the presence of 20 ng/mL each of bFGF, EGF, HGF and KGF or control media (*p<0.05 for two-way comparisons with the 10% FBS condition). B) Phase contrast images of the cultures taken on day 6. N = 1 pancreas with 6 replicate cultures.</p

Validation of a screening platform to identify CK19+ cell mitogens.

<p>A) Number of CK19+ cells at the end of the culture normalized to the number obtained in basal serum-free medium culture. B) Fraction of CK19+ cells that incorporated BrdU during the last day of culture, normalized to BrdU incorporation in basal medium culture. Unsorted cells were seeded on day 0 while CD90-depleted cells were sorted and seeded on day 1. The data were pooled from cultures ending on day 8 (N = 5 pancreata) or day 6 (N = 3 pancreata) and normalized for each pancreas prior to calculating averages and errors. The non-normalized results obtained at the end of the cultures in basal medium were as follows: the unsorted populations contained 0.05±0.02×10⁵ CK19+ cells/cm² of which 6±3% were BrdU+; the CD90-depleted populations contained 0.07±0.02×10⁵ CK19+ cells/cm² of which 1.0±0.6% were BrdU+. *p<0.05 compared to basal medium.</p