Translate this page to:
Choose Language…
In JoVE (1)
Other Publications (5)
Articles by Sandeep Koshy in JoVE
JoVE General
Culture of myeloid dendritic cells from bone marrow precursors
1Medical Sciences Program, McMaster University, 2Centre for Gene Therapeutics, McMaster University, 3Department of Chemical Engineering, University of Waterloo
This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.
Other articles by Sandeep Koshy on PubMed
Vesicular Stomatitis Virus As a Novel Cancer Vaccine Vector to Prime Antitumor Immunity Amenable to Rapid Boosting with Adenovirus
Vesicular stomatitis virus (VSV) has proven to be an effective vaccine vector for immunization against viral infection, but its potential to induce an immune response to a self-tumor antigen has not been investigated. We constructed a recombinant VSV expressing human dopachrome tautomerase (hDCT) and evaluated its immunogenicity in a murine melanoma model. Intranasal delivery of VSV-hDCT activated both CD4(+) and CD8(+) DCT-specific T-cell responses. The magnitude of these responses could be significantly increased by booster immunization with recombinant adenovirus (Ad)-hDCT, which led to enhanced efficacy against B16-F10 melanoma in both prophylactic and therapeutic settings. Notably, the interval of VSV/Ad heterologous vaccination could be shortened to as few as 4 days, making it a potential regimen to rapidly expand antigen-specific effector cells. Furthermore, VSV-hDCT could increase DCT-specific T-cell responses primed by Ad-hDCT, suggesting VSV is efficient for both priming and boosting of the immune response against a self-tumor antigen.
Controlling the Porosity and Microarchitecture of Hydrogels for Tissue Engineering
Tissue engineering holds great promise for regeneration and repair of diseased tissues, making the development of tissue engineering scaffolds a topic of great interest in biomedical research. Because of their biocompatibility and similarities to native extracellular matrix, hydrogels have emerged as leading candidates for engineered tissue scaffolds. However, precise control of hydrogel properties, such as porosity, remains a challenge. Traditional techniques for creating bulk porosity in polymers have demonstrated success in hydrogels for tissue engineering; however, often the conditions are incompatible with direct cell encapsulation. Emerging technologies have demonstrated the ability to control porosity and the microarchitectural features in hydrogels, creating engineered tissues with structure and function similar to native tissues. In this review, we explore the various technologies for controlling the porosity and microarchitecture within hydrogels, and demonstrate successful applications of combining these techniques.
Cell-laden Microengineered Gelatin Methacrylate Hydrogels
The cellular microenvironment plays an integral role in improving the function of microengineered tissues. Control of the microarchitecture in engineered tissues can be achieved through photopatterning of cell-laden hydrogels. However, despite high pattern fidelity of photopolymerizable hydrogels, many such materials are not cell-responsive and have limited biodegradability. Here, we demonstrate gelatin methacrylate (GelMA) as an inexpensive, cell-responsive hydrogel platform for creating cell-laden microtissues and microfluidic devices. Cells readily bound to, proliferated, elongated, and migrated both when seeded on micropatterned GelMA substrates as well as when encapsulated in microfabricated GelMA hydrogels. The hydration and mechanical properties of GelMA were demonstrated to be tunable for various applications through modification of the methacrylation degree and gel concentration. The pattern fidelity and resolution of GelMA were high and it could be patterned to create perfusable microfluidic channels. Furthermore, GelMA micropatterns could be used to create cellular micropatterns for in vitro cell studies or 3D microtissue fabrication. These data suggest that GelMA hydrogels could be useful for creating complex, cell-responsive microtissues, such as endothelialized microvasculature, or for other applications that require cell-responsive microengineered hydrogels.
Potentiating Cancer Immunotherapy Using an Oncolytic Virus
Oncolytic viruses (OVs) are highly immunogenic and this limits their use in immune-competent hosts. Although immunosuppression may improve viral oncolysis, this gain is likely achieved at the cost of antitumoral immunity. We have developed a strategy wherein the immune response against the OV leads to enhanced therapeutic outcomes. We demonstrate that immunization with an adenoviral (Ad) vaccine before treatment with an oncolytic vesicular stomatitis virus (VSV) expressing the same tumor antigen (Ag) leads to significantly enhanced antitumoral immunity. Intratumoral replication of VSV was minimally attenuated in Ad-immunized hosts but extending the interval between treatments reduced the attenuating effect and further increased antitumoral immunity. More importantly, our combination approach shifted the immune response from viral Ags to tumor Ags and further reduced OV replication in normal tissues, leading to enhancements in both efficacy and safety. These studies also highlight the benefits of using a replicating, OV to boost a pre-existing antitumoral immune response as this approach generated larger responses versus tumor Ag in tumor-bearing hosts than could be achieved in tumor-free hosts. This strategy should be applicable to other vector combinations, tumor Ags, and tumor targets.
Synthesis and Characterization of Tunable Poly(ethylene Glycol): Gelatin Methacrylate Composite Hydrogels
Poly(ethylene glycol) (PEG) hydrogels are popular for cell culture and tissue-engineering applications because they are nontoxic and exhibit favorable hydration and nutrient transport properties. However, cells cannot adhere to, remodel, proliferate within, or degrade PEG hydrogels. Methacrylated gelatin (GelMA), derived from denatured collagen, yields an enzymatically degradable, photocrosslinkable hydrogel that cells can degrade, adhere to and spread within. To combine the desirable features of each of these materials we synthesized PEG-GelMA composite hydrogels, hypothesizing that copolymerization would enable adjustable cell binding, mechanical, and degradation properties. The addition of GelMA to PEG resulted in a composite hydrogel that exhibited tunable mechanical and biological profiles. Adding GelMA (5%-15% w/v) to PEG (5% and 10% w/v) proportionally increased fibroblast surface binding and spreading as compared to PEG hydrogels (p<0.05). Encapsulated fibroblasts were also able to form 3D cellular networks 7 days after photoencapsulation only within composite hydrogels as compared to PEG alone. Additionally, PEG-GelMA hydrogels displayed tunable enzymatic degradation and stiffness profiles. PEG-GelMA composite hydrogels show great promise as tunable, cell-responsive hydrogels for 3D cell culture and regenerative medicine applications.
