Title page for ETD etd-050510-114009

Document Type thesis

Author Name Parekh, Darshan P

URN etd-050510-114009

Title Rapid and Uniform Cell Seeding on Fibrin Microthreads to Generate Tissue Engineered Microvessels

Degree MS

Department Biomedical Engineering

Advisors
Marsha Rolle, Advisor
Glenn Gaudette, Committee Member
George Pins, Committee Member

Keywords
Cell seeding
v well seeding
fibrin microthreads
mesencymal stem cells
UV crosslinking

Date of Presentation/Defense 2010-04-14

Availability unrestricted

Abstract
A wide variety of techniques have been explored to synthesize small diameter tissue engineered blood vessels. Toward this end, we are exploring direct cell seeding and culture on tubular mandrels to create engineered vascular tissues. In the present study, v-shaped channels cast from polydimethyl siloxane (PDMS) were used as cell seeding wells. Fibrin microthreads placed in the chamber were used as model tubular seeding mandrels. Human mesenchymal stem cells (hMSCs) were seeded onto fibrin microthreads in v-shaped channels for 4 hours. Cell attachment to the microthreads was confirmed visually by Hoechst nuclear staining and a cell quantification assay showed that 5,114 ± 339 cells attached per 1 cm fibrin microthread sample (n = 6). Fibrin microthreads were completely degraded by hMSCs within 5 days of culture, therefore UV crosslinking was used to increase their mechanical strength and prolong the amount of time cells could be cultured on fibrin microthreads and generate tubular tissue constructs. Cell attachment was unaffected on UV-crosslinked microthreads compared to uncrosslinked microthreads, resulting in a count of 4,944 ± 210 cells per 1 cm of fibrin microthread sample (n = 3). Long term culture of the hMSCs on the UV-crosslinked fibrin microthreads showed an increase in cell number over time to 11,198 ± 582 cells per cm of microthread after 7 days with 92% cell viability (CYQUANT NF/DEAD staining) and evidence of cell proliferation. The results show that the v-well cell seeding technique was effective in promoting rapid hMSC attachment on UV-crosslinked fibrin microthreads and encouraged their growth, maintained viability and also promoted their proliferation over the culture period. In conclusion, the technique could serve as an efficient model system for rapid formation of tissue engineered vascular grafts.

Files
DParekh.pdf

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Document Type	thesis
Author Name	Parekh, Darshan P
URN	etd-050510-114009
Title	Rapid and Uniform Cell Seeding on Fibrin Microthreads to Generate Tissue Engineered Microvessels
Degree	MS
Department	Biomedical Engineering
Advisors	Marsha Rolle, Advisor Glenn Gaudette, Committee Member George Pins, Committee Member
Keywords	Cell seeding v well seeding fibrin microthreads mesencymal stem cells UV crosslinking
Date of Presentation/Defense	2010-04-14
Availability	unrestricted
Abstract A wide variety of techniques have been explored to synthesize small diameter tissue engineered blood vessels. Toward this end, we are exploring direct cell seeding and culture on tubular mandrels to create engineered vascular tissues. In the present study, v-shaped channels cast from polydimethyl siloxane (PDMS) were used as cell seeding wells. Fibrin microthreads placed in the chamber were used as model tubular seeding mandrels. Human mesenchymal stem cells (hMSCs) were seeded onto fibrin microthreads in v-shaped channels for 4 hours. Cell attachment to the microthreads was confirmed visually by Hoechst nuclear staining and a cell quantification assay showed that 5,114 ± 339 cells attached per 1 cm fibrin microthread sample (n = 6). Fibrin microthreads were completely degraded by hMSCs within 5 days of culture, therefore UV crosslinking was used to increase their mechanical strength and prolong the amount of time cells could be cultured on fibrin microthreads and generate tubular tissue constructs. Cell attachment was unaffected on UV-crosslinked microthreads compared to uncrosslinked microthreads, resulting in a count of 4,944 ± 210 cells per 1 cm of fibrin microthread sample (n = 3). Long term culture of the hMSCs on the UV-crosslinked fibrin microthreads showed an increase in cell number over time to 11,198 ± 582 cells per cm of microthread after 7 days with 92% cell viability (CYQUANT NF/DEAD staining) and evidence of cell proliferation. The results show that the v-well cell seeding technique was effective in promoting rapid hMSC attachment on UV-crosslinked fibrin microthreads and encouraged their growth, maintained viability and also promoted their proliferation over the culture period. In conclusion, the technique could serve as an efficient model system for rapid formation of tissue engineered vascular grafts.
Files	DParekh.pdf