Browsing by Author "Lin, Tsai-Yu"

Now showing 1 - 7 of 7
  • Thumbnail Image
    Item
    Characterization, Enrichment, and Computational Modeling of Cross-Linked Actin Networks in Transformed Trabecular Meshwork Cells
    (Association for Research in Vision and Ophthalmology, 2025) Li, Haiyan; Harvey, Devon H.; Dai, Jiannong; Swingle, Steven P.; Compton, Anthony M.; Sugali, Chenna Kesavulu; Dhamodaran, Kamesh; Yao, Jing; Lin, Tsai-Yu; Sulchek, Todd; Kim, Taeyoon; Ethier, C. Ross; Mao, Weiming; Ophthalmology, School of Medicine
    Purpose: Cross-linked actin networks (CLANs) are prevalent in the glaucomatous trabecular meshwork (TM). We previously developed the GTM3L cell line, which spontaneously forms fluorescently labeled CLANs, by transducing GTM3, a transformed glaucomatous TM cell line, with a lentivirus expressing the LifeAct-GFP fusion protein. Here, we determined if LifeAct-GFP viral copy numbers are associated with CLANs, developed approaches to increase CLAN incidence, and computationally studied the biomechanical properties of CLAN-containing GTM3L cells. Methods: GTM3L cells were fluorescently sorted for viral copy number analysis to determine whether increased CLAN incidence was associated with copy number. CLAN incidence was increased by combining (1) differential adhesion sorting, (2) cell deswelling, and (3) cell stiffness selection. GTM3L cells were cultured on glass or soft hydrogels for stiffness measurement by atomic force microscopy. Computational models studied the biomechanical properties of CLANs. Results: GTM3L cells had one LifeAct-GFP viral copy/cell on average, and viral copy number or LifeAct-GFP expression level did not associate with CLAN incidence rate. However, CLAN rate was increased from -0.28% to -50% by combining the three enrichment methods noted above. Further, GTM3L cells formed more CLANs on a stiff versus a soft substrate. Computational modeling predicted that CLANs contribute to higher cell stiffness, including increased resistance of the nucleus to tensile stress when CLANs are physically linked to the nucleus. Conclusions: It is possible to greatly enhance CLAN incidence in GTM3L cells. CLANs are mechanosensitive structures that affect cell biomechanical properties. Further research is needed to determine the biomechanics, mechanobiology, and etiology of CLANs in the TM.
  • Thumbnail Image
    Item
    Comparative study of visible light polymerized gelatin hydrogels for 3D culture of hepatic progenitor cells
    (Wiley, 2017-03) Greene, Tanja; Lin, Tsai-Yu; Andrisani, Oaurania M.; Lin, Chien-Chi; Department of Biomedical Engineering, School of Engineering and Technology
    Photopolymerization techniques have been widely used to create hydrogels for biomedical applications. Visible light-based photopolymerizations are commonly initiated by type II (i.e., noncleavage-type) photoinitiator in conjunction with a coinitiator. On the other hand, type I photoinitiators (i.e., cleavage type) are rarely compatible with visible light-based initiation due to their limited molar absorbability in the visible light wavelengths. Here, we report visible light initiated orthogonal photoclick crosslinking to fabricate gelatin-norbornene and poly(ethylene glycol)-tetra-thiol hydrogels using either cleavage-type (i.e., lithium acylphosphinate, LAP) or noncleavage-type photoinitiator (i.e., eosin-Y, EY) without the use of a coinitiator. Regardless of the initiator type, the step-growth gelatin-PEG hybrid hydrogels crosslinked and degraded similarly. While both systems exhibited similar cytocompatibility for hepatic progenitor HepaRG cells, gelation initiated by noncleavage-type initiator EY afforded slightly higher degree of hepatic gene expression.
  • Thumbnail Image
    Item
    Designing Visible Light-Cured Thiol-Acrylate Hydrogels for Studying the HIPPO Pathway Activation in Hepatocellular Carcinoma Cells
    (Wiley Blackwell (John Wiley & Sons), 2016-04) Lin, Tsai-Yu; Bragg, John C.; Lin, Chien-Chi; Department of Biomedical Engineering, School of Engineering and Technology
    Various polymerization mechanisms have been developed to prepare peptide-immobilized poly(ethylene glycol) (PEG) hydrogels, a class of biomaterials suitable for studying cell biology in vitro. Here, a visible light mediated thiol-acrylate photopolymerization scheme is reported to synthesize dually degradable PEG-peptide hydrogels with controllable crosslinking and degradability. The influence of immobilized monothiol pendant peptide is systematically evaluated on the crosslinking of these hydrogels. Further, methods are proposed to modulate hydrogel crosslinking, including adjusting concentration of comonomer or altering the design of multifunctional peptide crosslinker. Due to the formation of thioether ester bonds, these hydrogels are hydrolytically degradable. If the dithiol peptide linkers used are susceptible to protease cleavage, these thiol-acrylate hydrogels can be designed to undergo partial proteolysis. The differences between linear and multiarm PEG-acrylate (i.e., PEGDA vs PEG4A) are also evaluated. Finally, the use of the mixed-mode thiol-acrylate PEG4A-peptide hydrogels is explored for in situ encapsulation of hepatocellular carcinoma cells (Huh7). The effects of matrix stiffness and integrin binding motif (e.g., RGDS) on Huh7 cell growth and HIPPO pathway activation are studied using PEG4A-peptide hydrogels. This visible light poly-merized thiol-acrylate hydrogel system represents an alternative to existing light-cured hydrogel platforms and shall be useful in many biomedical applications.
  • Thumbnail Image
    Item
    Enzyme-mediated stiffening hydrogels for probing activation of pancreatic stellate cells
    (Elsevier, 2017-01-15) Liu, Hung-Yi; Greene, Tanja; Lin, Tsai-Yu; Dawes, Camron S.; Korc, Murray; Lin, Chien- Chi; Biomedical Engineering, School of Engineering and Technology
    The complex network of biochemical and biophysical cues in the pancreatic desmoplasia not only presents challenges to the fundamental understanding of tumor progression, but also hinders the development of therapeutic strategies against pancreatic cancer. Residing in the desmoplasia, pancreatic stellate cells (PSCs) are the major stromal cells affecting the growth and metastasis of pancreatic cancer cells by means of paracrine effects and extracellular matrix protein deposition. PSCs remain in a quiescent/dormant state until they are 'activated' by various environmental cues. While the mechanisms of PSC activation are increasingly being described in literature, the influence of matrix stiffness on PSC activation is largely unexplored. To test the hypothesis that matrix stiffness affects myofibroblastic activation of PSCs, we have prepared cell-laden hydrogels capable of being dynamically stiffened through an enzymatic reaction. The stiffening of the microenvironment was created by using a peptide linker with additional tyrosine residues, which were susceptible to tyrosinase-mediated crosslinking. Tyrosinase catalyzes the oxidation of tyrosine into dihydroxyphenylalanine (DOPA), DOPA quinone, and finally into DOPA dimer. The formation of DOPA dimer led to additional crosslinks and thus stiffening the cell-laden hydrogel. In addition to systematically studying the various parameters relevant to the enzymatic reaction and hydrogel stiffening, we also designed experiments to probe the influence of dynamic matrix stiffening on cell fate. Protease-sensitive peptides were used to crosslink hydrogels, whereas integrin-binding ligands (e.g., RGD motif) were immobilized in the network to afford cell-matrix interaction. PSC-laden hydrogels were placed in media containing tyrosinase for 6h to achieve in situ gel stiffening. We found that PSCs encapsulated and cultured in a stiffened matrix expressed higher levels of αSMA and hypoxia-inducible factor 1α (HIF-1α), suggestive of a myofibroblastic phenotype. This hydrogel platform offers a facile means of in situ stiffening of cell-laden matrices and should be valuable for probing cell fate process dictated by dynamic matrix stiffness. STATEMENT OF SIGNIFICANCE: Hydrogels with spatial-temporal controls over crosslinking kinetics (i.e., dynamic hydrogel) are increasingly being developed for studying mechanobiology in 3D. The general principle of designing dynamic hydrogel is to perform cell encapsulation within a hydrogel network that allows for postgelation modification in gel crosslinking density. The enzyme-mediated in situ gel stiffening is innovative because of the specificity and efficiency of enzymatic reaction. Although tyrosinase has been used for hydrogel crosslinking and in situ cell encapsulation, to the best of our knowledge tyrosinase-mediated DOPA formation has not been explored for in situ stiffening of cell-laden hydrogels. Furthermore, the current work provides a gradual matrix stiffening strategy that may more closely mimic the process of tumor development.
  • Thumbnail Image
    Item
    Meeting FDA Guidance recommendations for replication-competent virus and insertional oncogenesis testing
    (Elsevier, 2022-12-02) Cornetta, Kenneth; Lin, Tsai-Yu; Pellin, Danilo; Kohn, Donald B.; Medical and Molecular Genetics, School of Medicine
    Integrating vectors are associated with alterations in cellular function related to disruption of normal gene function. This has been associated with clonal expansion of cells and, in some instances, cancer. These events have been associated with replication-defective vectors and suggest that the inadvertent exposure to a replication-competent virus arising during vector manufacture would significantly increase the risk of treatment-related adverse events. These risks have led regulatory agencies to require specific monitoring for replication-competent viruses, both prior to and after treatment of patients with gene therapy products. Monitoring the risk of cell expansion and malignancy is also required. In this review, we discuss the rational potential approaches and challenges to meeting the US FDA expectations listed in current guidance documents.
  • Thumbnail Image
    Item
    Replication competent retrovirus testing (RCR) in the National Gene Vector Biorepository: No evidence of RCR in 1,595 post-treatment peripheral blood samples obtained from 60 clinical trials
    (Elsevier, 2023) Cornetta, Kenneth; Yao, Jing; House, Kimberley; Duffy, Lisa; Adusumilli, Prasad S.; Beyer, Rachel; Booth, Claire; Brenner, Malcolm; Curran, Kevin; Grilley, Bambi; Heslop, Helen; Hinrichs, Christian S.; Kaplan, Rosandra N.; Kiem, Hans-Peter; Kochenderfer, James; Kohn, Donald B.; Mailankody, Sham; Norberg, Scott M.; O’Cearbhaill, Roisin E.; Pappas, Jennifer; Park, Jae; Ramos, Carlos; Ribas, Antonio; Rivière, Isabelle; Rosenberg, Steven A.; Sauter, Craig; Shah, Nirali N.; Slovin, Susan F.; Thrasher, Adrian; Williams, David A.; Lin, Tsai-Yu; Medical and Molecular Genetics, School of Medicine
    The clinical impact of any therapy requires the product be safe and effective. Gammaretroviral vectors pose several unique risks, including inadvertent exposure to replication competent retrovirus (RCR) that can arise during vector manufacture. The US FDA has required patient monitoring for RCR, and the National Gene Vector Biorepository is an NIH resource that has assisted eligible investigators in meeting this requirement. To date, we have found no evidence of RCR in 338 pre-treatment and 1,595 post-treatment blood samples from 737 patients associated with 60 clinical trials. Most samples (75%) were obtained within 1 year of treatment, and samples as far out as 9 years after treatment were analyzed. The majority of trials (93%) were cancer immunotherapy, and 90% of the trials used vector products produced with the PG13 packaging cell line. The data presented here provide further evidence that current manufacturing methods generate RCR-free products and support the overall safety profile of retroviral gene therapy.
  • Thumbnail Image
    Item
    Thiol-ene hydrogels as desmoplasia-mimetic matrices for modeling pancreatic cancer cell growth, invasion, and drug resistance
    (Elsevier, 2014-12) Ki, Chang Seok; Lin, Tsai-Yu; Korc, Murray; Lin, Chien-Chi; Department of Engineering Technology, School of Engineering and Technology
    The development of pancreatic ductal adenocarcinoma (PDAC) is heavily influenced by local stromal tissues, or desmoplasia. Biomimetic hydrogels capable of mimicking tumor niches are particularly useful for discovering the role of independent matrix cues on cancer cell development. Here, we report a photo-curable and bio-orthogonal thiol-ene (i.e., cross-linked by mutually reactive norbornene and thiol groups via photoinitiation) hydrogel platform for studying the growth, morphogenesis, drug resistance, and cancer stem cell marker expression in PDAC cells cultured in 3D. The hydrogels were prepared from multi-arm poly(ethylene glycol)-norbornene cross-linked with protease-sensitive peptide to permit cell-mediated matrix remodeling. Collagen 1 fibrils were incorporated into the covalent network while cytokines (e.g., EGF and TGF-β1) were supplemented in the culture media for controlling cell fate. We found that the presence of collagen 1 enhanced cell proliferation and Yes-associated protein (YAP) translocation to cell nuclei. Cytokines and collagen 1 synergistically up-regulated MT1-MMP expression and induced cell spreading, suggestive of epithelial-mesenchymal transition (EMT) in the encapsulated cells. Furthermore, PDAC cells cultured in 3D developed chemo-resistance even in the absence of collagen 1 and cytokines. This phenotype is likely a consequence of the enrichment of pancreatic cancer stem cells that expressed high levels of CD24, sonic hedgehog (SHH), and vascular endothelial growth factor (VEGF).