Browsing by Author "Raza, Asad"

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    The influence of matrix degradation and functionality on cell survival and morphogenesis in PEG-based hydrogels
    (Wiley, 2013) Raza, Asad; Lin, Chien-Chi; Biomedical Engineering, Purdue School of Engineering and Technology
    Two norbornene-functionalized PEG macromers are synthesized to render hydrogels with different hydrolytic degradability. Dithiol-containing linkers such as dithiothreitol or biscysteine-containing peptides are used to control proteolytic degradability. The influence of thiol-ene gel degradability on cell survival and morphogenesis in 3D is assessed using hMSCs and pancreatic MIN6 β cells. The initial cell viability can be negatively affected in highly crosslinked thiol-ene hydrogels. When cells are encapsulated in thiol-ene gels lacking cell-adhesive motifs, their survival and proliferation are promoted in more hydrolytically labile hydrogels. The degree of 3D cell spreading in encapsulated hMSCs is enhanced when the matrices are immobilized with cell-adhesive motifs.
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    The influence of matrix properties on growth and morphogenesis of human pancreatic ductal epithelial cells in 3D
    (Elsevier, 2013) Raza, Asad; Ki, Chang Seok; Lin, Chien-Chi; Biomedical Engineering, Purdue School of Engineering and Technology
    A highly tunable synthetic biomimetic hydrogel platform was developed to study the growth and morphogenesis of pancreatic ductal epithelial cells (PDEC) under the influence of a myriad of instructive cues. A PDEC line, PANC-1, was used as a model system to illustrate the importance of matrix compositions on cell fate determination. PANC-1 is an immortalized ductal epithelial cell line widely used in the study of pancreatic tumor cell behaviors. PANC-1 cells are also increasingly explored as a potential cell source for endocrine differentiation. Thus far, most studies related to PANC-1, among other PDEC lines, are performed on 2D culture surfaces. Here, we evaluated the effect of matrix compositions on PANC-1 cell growth and morphogenesis in 3D. Specifically, PANC-1 cells were encapsulated in PEG-based hydrogels prepared by step-growth thiol-ene photopolymerization. It was found that thiol-ene hydrogels provided a cytocompatible environment for encapsulation and 3D culture of PANC-1 cells. In contrast to a monolayer morphology on 2D culture surfaces, PANC-1 cells formed clusters in 3D thiol-ene hydrogels within 4 days of culture. After culturing for 10 days, however, the growth and structures of these clusters were significantly impacted by gel matrix properties, including sensitivity of the matrix to proteases, stiffness of the matrix, and ECM-mimetic motifs. The use of matrix metalloproteinase (MMP) sensitive linker or the immobilization of fibronectin-derived RGDS ligand in the matrix promoted PANC-1 cell growth and encouraged them to adopt ductal cyst-like structures. On the other hand, the encapsulated cells formed smaller and more compact aggregates in non-MMP responsive gels. The incorporation of laminin-derived YIGSR peptide did not enhance cell growth and caused the cells to form compact aggregates. Immobilized YIGSR also enhanced the expression of epithelial cell markers including β-catenin and E-cadherin. These studies have established PEG-peptide hydrogels formed by thiol-ene photo-click reaction as a suitable platform for studying and manipulating pancreatic epithelial cell growth and morphogenesis in 3D.
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    Tunable hydrogels for pancreatic tissue engineering
    (2014-01-03) Raza, Asad; Lin, Chien-Chi; Xie, Dong; Mirmira, Raghavendra G.
    Type I diabetes is an autoimmune disorder characterized by the loss of insulin producing islet cell mass. While daily insulin injection provides an easy means of glycemic control, it does not prevent long-term complications associated with diabetes. Islet transplantation has been suggested as a permanent cure for type 1 diabetes. However, the recurrence of host immunity and shortage of donor islets hinder the prevalence of islet transplantation. Biomaterial strategies provide an alternative route to solving the problems associated with host immune response and shortage of donor islets. One highly recognized platform for achieving these goals are hydrogels, which are hydrophilic crosslinked polymers with tissue-like elasticity and high permeability. Hydrogels prepared from poly(ethylene glycol) (PEG) derivatives are increasingly used for a variety of tissue engineering applications, including encapsulation of pancreatic islets and serving as a material platform for pseudo-islet differentiation. PEG hydrogels formed by mild and rapid thiol-ene photo-click reactions are particularly useful for studying cell behaviors in three-dimension (3D). Thiol-ene PEG-based hydrogels can be rendered biodegradable if appropriate macromer and cross-linker chemistry is employed. However, the influence of hydrogel matrix properties on the survival, growth, and morphogenesis of cells in 3D has not been fully evaluated. This thesis aims at using norbornene-functionalized PEG macromers to prepare thiol-ene hydrogels with various stiffness and degradability, from which to study the influence of hydrogel properties on pancreatic cell fate processes in 3D. Toward establishing an adaptable hydrogel platform for pancreatic tissue engineering, this thesis systematically studies the influence of hydrogel properties on encapsulated endocrine cells (e.g., MIN6 beta-cells) and exocrine cells (PANC-1 cells), as well as human mesenchymal stem cells (hMSC). It was found that thiol-ene photo-click hydrogels provide a cytocompatible environment for 3D culture of these cells. However, cell viability was negatively affected in hydrogels with higher cross-linking density. In contrast to a monolayer when cultured on a 2D surface, cells with epithelial characteristic formed clusters and cells with mesenchymal features retained single cell morphology in 3D. Although cells survived in all hydrogel formulations studied, the degree of proliferation, and the size and morphology of cell clusters formed in 3D were significantly influenced by hydrogel matrix compositions. For example: encapsulating cells in hydrogels formed by hydrolytically degradable macromer positively influenced cell survival indicated by increased proliferation. In addition, when cells were encapsulated in thiol-ene gels lacking cell-adhesive motifs, hydrolytic gel degradation promoted their survival and proliferation. Further, adjusting peptide crosslinker type and immobilized ECM-mimetic bioactive cues provide control over cell fate by determining whether observed cellular morphogenesis is cell-mediated or matrix-controlled. These fundamental studies have established PEG-peptide hydrogels formed by thiol-ene photo-click reaction as a suitable platform for pancreatic tissue engineering