Engineering a functional neuro-muscular junction model in a chip

Transcription

1 Electronic Supplementary Material (ESI) for RSC Advances. This journal is The Royal Society of Chemistry 2014 Engineering a functional neuro-muscular junction model in a chip Ziqiu Tong, Oscar Seira, Cristina Casas, Diego Reginensi, Antoni Homs-Corbera, Josep Samitier, José Antonio Del Río Electronic supplemental information Figure S1. Comparison of motoneurons growing on 2 µm and 10 µm wide microchannel chips. Characterization of 2 µm wide (a) and 10 µm wide (c) microchannels via SEM imaging. Neuronal Class III β-tubulin (TUJ1) staining of axons exiting in 2 µm wide (b) and 10 µm wide (d) microchannels. Scale bars: (b) 20 µm, (d) 100 µm. Figure S2. Effect of neurotropic factors and Semaphorin 3A on motoneuron axon growth. Representative micrographs of axonal growth in: normal motoneuron growth media which contains growth factors of CNTF and GDNF (a), additional neurotropic factors of BDNF and HGF (b), and the addition of Sem3A to the growth media (c). Pictures were taken 7 days after the initial cell seeding. Fractions of channels having axons across the entire channel under each condition are calculated and plotted. Asterisk denotes statistically significant by Student t-test ( * P < 0.01) comparison to control. Scale bars: 50 µm. Figure S3. Comparison of mitochondria imaging on petridish and on chip. MitoTracker Deep Red FM was used for live cell staining of mitochondria in petridish (a) and on chip with 10 µm wide microchannels (c). Figure b shows bright field image of axons entering the 10 µm wide microchannels (b). Scale bars (a): 200 µm; (b, c): 50 µm. Video S1. Time-lapse movie illustrating differentiated myotubes (arrows) exhibiting spontaneous beating activity in a myotube compartment (see Figure 1 for details of fabrication). Video S2. Time-lapse movie illustrating Ca 2+ transients in C2C12 myotubes cultured in a compartimentalized chip in the absence of motoneurons. KCl (100 mm) was added in the somal compartment (without motoneurons) at 50 sec. C2C12 Ca 2+ changes was measured for 8 min. Notice the asynchronous transients in C2C12 myotubes. The addition of KCl does not modify the asynchronous activity and the number of activated cells.

2 Table S1. Summary of recently published studies using microfluidic chip for neuron cultures, especially targeting for axon isolations. Note: W=width, H=height, L=length, c=cell culture area, m=microchannels, W c, H c, L c denote the dimensions of cell culture area, and have units of (mm); W m, H m, L m denote the dimensions of microchannels and have units of (µm). n/a=data not available. Author (paper) Cultured cell types Main applications Device dimensions W coupled with microcontact printing c xh c xl c = 1.5x0.1x8 Taylor et al, rat cortical neurons W technique to study guided axon growth m xh m xl m = 10x3x150 Taylor et al, Liu et al, Park et al, Hengst et al, Arundell et al, Kanagasabapathi et al, Southam et al, Park et al, rat and mouse cortical and hippocampal neurons, postnatal rat pups oligodendrocytes rat sympathetic neurons, kidney epithelial cells, PRV strains rat cortical neurons rat dorsal root ganglia and dorsal spinal commissural neurons PC12, SH-SY5Y, cortical neurons. rat cortical and thalamic neurons rat spinal motor neurons, rat hind-limb muscles, rat spinal glial cells Mouse ESCs and mouse myoblasts (C2C12) isolate axonal mrna, study axonal injury and regeneration analysis of neuron-to-cell transmission and viral transport in axon analysis of axon regeneration focusing on the inhibitory protein NOGO-66 and MAG elucidating the mechanism for local protein translation in axonal outgrowth (PAR complex) Demonstration of new method of creating macro/micro cocultured platform coupled with microelectrode arrays (MEA) for spontaneous eletrical activity recording co-culture of motoneuron and muscle cells to establish neural muscular junction formation Co-culture of ESC derived motoneurons and myoblasts W c xh c xl c = 1.5x0.1x7 W m xh m xl m = 10x3x(150, 450, 900) W m xh m xl m = 10xn/ax450 W c xh c xl c = 1.5x0.1x7 W m xh m xl m = 10x3x150 W m xh m xl m = 10x3x450 W c xh c xl c = 10xn/ax10 W m xh m xl m = 8x3x560 W c xh c xl c = 1.5x0.1x8 W m xh m xl m = 10x3x150 W m xh m xl m = 10x3x450 W c xh c xl c = 12.75x4.76x6.35 W m xh m xl m = 10x2.5x500 References 1. A. M. Taylor, S. W. Rhee, C. H. Tu, D. H. Cribbs, C. W. Cotman and N. L. Jeon, Langmuir, 2003, 19, A. M. Taylor, M. Blurton-Jones, S. W. Rhee, D. H. Cribbs, C. W. Cotman and N. L. Jeon, Nat Methods, 2005, 2, W. W. Liu, J. Goodhouse, N. L. Jeon and L. W. Enquist, PLoS One, 2008, 3, e J. W. Park, B. Vahidi, H. J. Kim, S. W. Rhee and N. L. Jeon, Biochip Journal, 2008, 2, U. Hengst, A. Deglincerti, H. J. Kim, N. L. Jeon and S. R. Jaffrey, Nat Cell Biol, 2009, 11, M. Arundell, V. H. Perry and T. A. Newman, Lab on a chip, 2011, 11, T. T. Kanagasabapathi, P. Massobrio, R. A. Barone, M. Tedesco, S. Martinoia, W. J. Wadman and M. M. Decre, J Neural Eng, 2012, 9, K. A. Southam, A. E. King, C. A. Blizzard, G. H. McCormack and T. C. Dickson, J Neurosci Methods, 2013, 218, H. S. Park, S. Liu, J. McDonald, N. Thakor and I. H. Yang, Conference proceedings :... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference, 2013, 2013,

3 Supplementary Figure 1

4 Supplementary Figure 2

5 Supplementary Figure 3