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 Ca2+ 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 Ca2+ 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. 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, Wc, Hc, Lc denote the dimensions of cell culture area, and have units of (mm); Wm, Hm, Lm denote the dimensions of microchannels and have units of (µm). n/a=data not available. Author (paper) Cultured cell types Main applications Taylor et al, 20031 rat cortical neurons coupled with microcontact printing technique to study guided axon growth Taylor et al, 2005 rat and mouse cortical and hippocampal neurons, postnatal rat pups oligodendrocytes isolate axonal mRNA, study axonal injury and regeneration Liu et al, 20083 rat sympathetic neurons, kidney epithelial cells, PRV strains analysis of neuron-to-cell transmission and viral transport in axon Park et al, 20084 rat cortical neurons Hengst et al, 20095 rat dorsal root ganglia and dorsal spinal commissural neurons Arundell et al, 20116 PC12, SH-SY5Y, cortical neurons. Kanagasabapathi et al, 20127 rat cortical and thalamic neurons Southam et al, 20138 rat spinal motor neurons, rat hind-limb muscles, rat spinal glial cells 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 Park et al, 20139 Mouse ESCs and mouse myoblasts (C2C12) Co-culture of ESC derived motoneurons and myoblasts 2 Device dimensions WcxHcxLc= 1.5x0.1x8 WmxHmxLm= 10x3x150 WcxHcxLc= 1.5x0.1x7 WmxHmxLm = 10x3x(150, 450, 900) WcxHcxLc= n/a WmxHmxLm = 10xn/ax450 WcxHcxLc= 1.5x0.1x7 WmxHmxLm = 10x3x150 WcxHcxLc= n/a WmxHmxLm = 10x3x450 WcxHcxLc= 10xn/ax10 WmxHmxLm = 8x3x560 WcxHcxLc= 1.5x0.1x8 WmxHmxLm = 10x3x150 WcxHcxLc= n/a WmxHmxLm = 10x3x450 WcxHcxLc= 12.75x4.76x6.35 WmxHmxLm = 10x2.5x500 References 1. 2. 3. 4. 5. 6. 7. 8. 9. A. M. Taylor, S. W. Rhee, C. H. Tu, D. H. Cribbs, C. W. Cotman and N. L. Jeon, Langmuir, 2003, 19, 1551-1556. A. M. Taylor, M. Blurton-Jones, S. W. Rhee, D. H. Cribbs, C. W. Cotman and N. L. Jeon, Nat Methods, 2005, 2, 599-605. W. W. Liu, J. Goodhouse, N. L. Jeon and L. W. Enquist, PLoS One, 2008, 3, e2382. J. W. Park, B. Vahidi, H. J. Kim, S. W. Rhee and N. L. Jeon, Biochip Journal, 2008, 2, 44-51. U. Hengst, A. Deglincerti, H. J. Kim, N. L. Jeon and S. R. Jaffrey, Nat Cell Biol, 2009, 11, 1024-1030. M. Arundell, V. H. Perry and T. A. Newman, Lab on a chip, 2011, 11, 3001-3005. T. T. Kanagasabapathi, P. Massobrio, R. A. Barone, M. Tedesco, S. Martinoia, W. J. Wadman and M. M. Decre, J Neural Eng, 2012, 9, 036010. K. A. Southam, A. E. King, C. A. Blizzard, G. H. McCormack and T. C. Dickson, J Neurosci Methods, 2013, 218, 164-169. 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, 2833-2835. Supplementary Figure 1 Supplementary Figure 2 Supplementary Figure 3