Cells in Hyaluronic Acid Gels Stephanie Seidlits Schmidt Lab Group Meeting April 9, 2008

Guiding cells with protein structures in GMHA gels Motivation: create 3D tissue engineering constructs that can be patterned for cell guidance and/or co-cultures

Microcontact printed bIKVAV on 1:1 PA:PEGDA- streptavidin labeled with Sigma L9393 (Hynd, J. Neurosci Methods, 2007); scale bar = 50 um

IKVAV immobilization on protein structures BSA-FLbBSA+NAv+bIKVAV fluorescein filter cube BSA-FLbBSA+NAv+bIKVAV TRITC filter cube

Crosslinking of IKVAV-Acryloyl 400 mg/mL BSA, 5 mM MB: w/ 50 ug/mL Ac-IKVAV:IKVAV alone: p<0.05, but lots of background…

E18 hippocampal neurons on bIKVAV- PA-PEGDA after 4 weeks (Hynd, J. Neurosci Methods, 2007); scale bar = 100 um

My structures for cells

IKVAV I made IKVAV from NE peptide IKVAV I made bBSA alone bBSA + NAv alone bBSA alone - cells attached to 37.5% of structures (n=8); of these 33.3% spread and aligned NAv alone - cells attached to 57.1% of structures (n=7); of these 25% spread and aligned IKVAV - cells attached to 70% of structures (n=40); of these 71.4% spread and aligned

beta III tubulin (green) DAPI (blue) S100 (red)

Pins, JBMR, 2006

top left: fabricated at gel surface; bottom left: fabricated ~20 um into the gel; top right: fabricated ~37 um into the gel Grasshopper stack

Proposed DMD Shape/Slices

Plans Paper to submit by summer: –Repeat DRGs again for higher “n” for quantification –Higher resolution imaging of DRGs –IKVAV immobilization - see if varying crosslink power varies signal –stability of IKVAV immobilization in culture conditions –Ac-IKVAV? Try again w/ less MB, more IKVAV?

Plans Next paper: –DRGs with DMD structures –Quantification of peptide binding with fluorescein-labeled peptide –Effect of concentration, feature size, angle on cells - focal adhesion staining? –Creation and testing of gradients for guidance –Spatially defined co-cultures –Tests with more progenitors? Look at synapse formation?

Thanks! Christine Schmidt Jason Shear Rex Nielson Zin Khaing Undergraduates who helped with this project - Rebecca Rosenberger, Nathan Thompson

Midbrain progenitor encapsulation in MAHA gels Motivation: Investigate effects of stiffness in 3D culture on MAHA; encourage higher percentage of neuronal differentiation/viability in vitro

MAHA 2x~102 5x~36N/A 10x~ x~15418 Need to keep collecting more data and optimizing consistency…

Viability of midbrain progenitors in MAHA gels Same trend in results between assays, but quantification very different May be due to protocol variations - such as days in culture before passaging

MAHA degradation Repeat all together with shorter gelling times and 5 uL PBS added to mimic cell encapulation

Mechanical Data Variability due to MAHA batches?

Fluoreporter Assay R 2 =0.972 d.o.m. (200 mg/mL bBSA) =

Immunostaining On PLL coverslips (1 week in culture) - green = GFP, blue = DAPI, red = nestin(left)/beta-III tubulin (right)

Plans Continue MAHA NMR, mechanical testing, and degradation studies Repeat cell titer assay for quantification Compare differentiation of MB progenitors on 2D and 3D and between different stiffness gels by counting immunostained cells –Need to optimize immunostaining protocol for this