This readme.txt file was generated on <20200514> by ------------------- GENERAL INFORMATION ------------------- 1. Title of Dataset Supporting data for 3D Printed Stem-Cell Derived Neural Progenitors Generate Spinal Cord Scaffolds 2. Author Information Principal Investigator Contact Information Name: Michael C. McAlpine Institution: University of Minnesota Address: Department of Mechanical Engineering, University of Minnesota, Minneapolis, MN 55455, USA Email: mcalpine@umn.edu ORCID: 0000-0001-7869-7598 Associate or Co-investigator Contact Information Name: Ann M. Parr Institution: University of Minnesota Address:Department of Neurosurgery, Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA Email: amparr@umn.edu ORCID: 3. Date of data collection (single date, range, approximate date) Approximate date: 20160905-20181005 4. Geographic location of data collection (where was data collected?): The University of Minnesota 5. Information about funding sources that supported the collection of the data: Conquer Paralysis Now Minnesota Spinal Cord Injury and Traumatic Brain Injury Research Grant Program National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health (Award No. 1DP2EB020537) CTSI KL2 Scholar Program of the National Institutes of Health (Award No. NIHCON000000033119-3002) -------------------------- SHARING/ACCESS INFORMATION -------------------------- 1. Licenses/restrictions placed on the data: Material may be reused with appropriate attribution. 2. Links to publications that cite or use the data: Joung, D., Truong, V., Neitzke, C., Guo, S., Walsh, P., Monat, J., . . . McAlpine, M. (2018). 3D Printed Stem‐Cell Derived Neural Progenitors Generate Spinal Cord Scaffolds. Advanced Functional Materials, 28(39). https://doi.org/10.1002/adfm.201801850 3. Links to other publicly accessible locations of the data: N/A 4. Links/relationships to ancillary data sets: N/A 5. Was data derived from another source? No 6. Recommended citation for the data: Joung, Daeha; Truong, Vincent; Neitzke, Colin C; Guo, Shuang-Zhuang; Walsh, Patrick J; Monat, Joseph R; Meng, Fanben; Park, Sung Hyun; Dutton, James R; Parr, Ann M; McAlpine, Michael C. (2020). Supporting data for 3D Printed Stem-Cell Derived Neural Progenitors Generate Spinal Cord Scaffolds. Retrieved from the Data Repository for the University of Minnesota, https://doi.org/10.13020/femp-z102. --------------------- DATA & FILE OVERVIEW --------------------- All the .opj files were generated using OriginPro 9.0.0 (64-bit) b45. All the .nd2 files were generated from a Nikon resource via the University of Minnesota University Imaging Centers (UIC). The files can be opened using NIS-Elements and NIS-Elements Viewer. Code files (Excel) were created to program Fisnar F5200N gantry robot via Smart Robot. 1. File List A. Filename: Figure 1 data.rar Short description: Raw data files for Figure 1c,d,e,f B. Filename: Figure 2 data.rar Short description: Raw data files for Figure 2a,b,c,d,e C. Filename: Figure 3 data.rar Short description: Raw data files for Figure 3a,b,c,d,e,f D. Filename: Figure 4 data.rar Short description: Raw data files for Figure 4a,b,c,d,e,f,g,h,i E. Filename: Figure 5 data.rar Short description: Raw data files for Figure 5a,b,c,d,e,f,g F. Filename: Figure S1 data.rar Short description: Raw data files for Figure S1a,b G. Filename: Figure S2 data.rar Short description: Raw data files for Figure S2 H. Filename: Figure S3 data.rar Short description: Raw data files for Figure S3 I. Filename: Figure S4 data.rar Short description: Raw data files for Figure S4 J. Filename: Figure S5 data.rar Short description: Raw data files for Figure S5a,b K. Filename: Figure S6 data.rar Short description: Raw data files for Figure S6a,b,c,d L. Filename: Figure S7 data.rar Short description: Raw data files for Figure S7a,b M. Filename: Figure S8 data.rar Short description: Raw data files for Figure S8a,b N. Filename: Figure S9 data.rar Short description: Raw data files for Figure S9 O. Filename: Figure S9 data.rar Short description: Raw data files for Figure S9 P. Filename: Figure S9 data.rar Short description: Raw data files for Figure S9 Q. Filename: Figure S10 data.rar Short description: Raw data files for Figure S10a,b,c R. Filename: Figure S11 data.rar Short description: Raw data files for Figure S11a,b,c,d S. Filename: Figure S12 data.rar Short description: Raw data files for Figure S12 T. Filename: Fisnar_printing_code.rar Short description: Code files (excel) for adaptive printing controller (For Fisnar 3D printer). 2. Are there multiple versions of the dataset? No. 3. Description of figures 1c: Comparison of a transected rat spinal cord and the design principle for scaffolds consisting of multiple, continuous channels. The number of channels can be scaled according to the size of the scaffold needed. 1d: Top view image of scaffold channels demonstrates a printing resolution of ~150 µm. Channels are continuous throughout the scaffold, allowing for axonal extension. 1e: Side view of a 5 mm long scaffold. 1f: A 2×2×5 mm3 sized scaffold on top of a finger shows the scale of a scaffold. 2a: Cell viability of 3D bioprinted fibroblast-, spinal neuronal progenitor cells (sNPCs), and oligodendrocyte progenitor cells (OPC)-laden GelMa and GEL/FIB bioinks cultured for 3 hours (Day 0), 24 hours (Day 1), and 72 hours (Day 4). 2b: Cell viability of 3D bioprinted sNPC-, and OPC-laden Matrigel suspensions with the use of 25%, 50%, 75%, and 100% Matrigel concentrations. Matrigel was diluted with N2/B27 basal media, and cell viability was measured after 1 day in culture (24 hours after printing). 2c: Cell viability of cultured, bioprinted sNPCs and OPCs in 50% Matrigel suspension cultured at Day 0, Day 1, and Day 4. 2d,e: LIVE/DEAD® sNPC staining after short-term post-printing of (d) ~0.2 μl and (e) ~2 μl volumes, used to determine the maximum printing times before cells needed to be placed under in vitro culture conditions. 3a: Distribution of cell types in specific channels: sNPCs only (left), OPCs only (middle), and sNPCs and OPCs (right). Groups of sNPCs and OPCs are interspersed with a distribution resolution of ~200 μm in a single channel (highlighted on the right channel). sNPCs are detected with human-specific antibody SC121 (red), and OPCs express eGFP (green). The image was taken 24 hours post-printing. 3b: sNPCs printed in a scaffold after 4 days of culture. Antibody to β3III-tubulin detects axonal projections in the channels. 3c: sNPCs and OPCs co-printed in a scaffold after 4 days of culture. β3III-tubulin shows axonal projections down the channels, and the OPCs express mCherry. 3d: Close-up image of eGFP (green) expressing 3D printed OPCs express the OPC marker Sox10 (red) after 1 day of culture. 3e: Close-up image of 3D printed sNPCs detected with antibody to the mature neuron marker NeuN, and axons with antibody to mark β3III-tubulin in a channel after 7 days of culture. 3f: Close-up image of 3D printed sNPCs and OPCs in a channel shows axon projections and OPC processes in close proximity to the axons after 4 days of culture. The OPCs and axons express mCherry (red) and the β3III-tubulin (green), respectively. 4a: Cross-sectional view of the AG/MC scaffold showing ~150 µm channel resolution. 4b-f: Scaffolds were fabricated and cells were alive for 3 days after printing in all three layers. (b),(c) Top-down, (d) Cross-sectional, and (f) Longitudinal side view of fluorescence images of a 3D printed scaffold. 4g-i: Time progression of sNPCs in an alginate scaffold showing processes elongating over a period of 3 days in culture. 5a,b: Video-captured sequence of the calcium imaging of sNPCs bioprinted in a silicone scaffold with long axon projections 14 days after printing. High-resolution confocal imaging shows cell bodies and adjacent axons with calcium transients. 5c-f: Video-captured sequence of the calcium imaging of sNPCs bioprinted in a silicone scaffold. Cells in the scaffold before and after (c,d) 70 mM potassium chloride (KCl), and (e,f) 100 μM glutamate were added. 5g: Time-dependent fluorescent intensity of 10 cells marked in (c-f). Fluorescent intensity was measured after the addition of high KCl and the neurotransmitter glutamate. S1a,b: Fluorescence images (LIVE/DEAD® staining) of 3D printed sNPC-laden gelatin admixed with fibrin (GEL/FIB) matrix, with the use of (a) 10% N2B27 and (b) 30% DMEM culture media for 0, 1, and 4 days. Green and red indicate live and dead cells, respectively. The morphologies of the cells did not show axon propagation. S2:Fluorescence images of 3D printed human fibroblast-laden gelatin methacrylate (GelMa) matrix in a poly(ethylene glycol) diacrylate (PEGDA) scaffold (channel width ~ 150 µm) cultured for 0, 1, and 4 days. The images show live printed fibroblasts within the scaffold channel. S3: Matrigel titration for cell-laden matrices. Bright field microscopy images of varying Matrigel concentrations (a,b) for sNPC-laden matrices, and (c,d) for OPC-laden matrices for culture periods of 0 and 4 days. The cells in Matrigel suspension were tested at four different concentrations: 100% (undiluted), 75%, 50%, and 25% of Matrigel in growth media. S4: Optical microscope images of Matrigel at different time points, showing the evaporation of water. S5: Fluorescence images of 3D printed cell-laden Matrigel (50%) matrices cultured for 0 (3 hours), 1, and 4 days. Timescale images show (a) sNPCs extending axons, and (b) OPCs exhibiting bi-polar processes. S6: Spinal cord scaffold assembly process: 3D bioprinting cells on silicone scaffolds allows for in vitro culture of sNPCs and OPCs. (a) Silicone scaffolds are printed with channels, and (b) cells are dispensed inside the channels. (c) A layer of silicone covers the channels, and (d) scaffolds are placed inside a dish and cultured for 7 days. S7: Mechanical characterization of various scaffolds. (a) Static compression fidelity via stress-strain curves of printed scaffolds and a transected rat spinal cord in the wet (W) and dry (D) states. The 3D printed scaffolds are comprised of PEGDA (PEG), and AG/MC blends. The AG/MC blend hydrogels were named AG1, AG2, and AG3, corresponding to the lower molecular weight of alginate admixed with MC (crosslinking with Ca2+ ion), the higher molecular weight of alginate admixed with MC (crosslinking with Ca2+ ion), and the higher molecular weight of alginate admixed with MC (crosslinking with Ba2+ ion), respectively. (b) Elastic modulus of printed scaffolds. Inset shows photographs of static mechanical tests for representative dry and wet hydrogel scaffolds and a transected rat spinal cord. The compression tests were performed longitudinally. *P < 0.05 was considered statistically significant from spinal cord tissue. S8: Fluorescence images of 3D bioprinted (a) sNPCs and (b) OPCs on PEGDA scaffolds cultured for 0 (3 hours), 1, and 4 days. Although some cells survive after printing on PEGDA (live cells are green), no axons nor processes were observed. S9: Dynamic modulus of the alginate-based scaffolds for both dry and wet states. Plots show Storage Modulus (Eˊ) and Loss Modulus (E˝) versus frequency at 30% strain. S10: Degradation characterization of alginate-based scaffolds. (a)-(c) Evaluation of the degradation of printed alginate-based scaffolds. Representative images of the degradation process of printed alginate-based scaffolds kept in PBS over 4 days: (a) AG1, (b) AG2, and (c) AG3, respectively. S11: Fluorescence images (LIVE/DEAD® staining) of 3D printed alginate-based scaffolds containing OPCs. (a) Cross-sectional and (b) top-down view of fluorescence images of a 3D printed scaffold 3 days after printing show live OPCs within the scaffold. (c),(d) 3 day time progression of OPCs in an alginate scaffold show elongating processes typical of healthy OPC morphology. S12: Photograph of the customized extrusion-based 3D printer set-up, including the controller, the dispensing system, the cooling and heating jackets, a 3-axis gantry robot, and a vision system.