Additional file 2 of Simultaneous induction of vasculature and neuronal network formation on a chip reveals a dynamic interrelationship between cell types
Creators: name: Isosaari, Lotta
name: Vuorenpää, Hanna
name: Yrjänäinen, Alma
name: Kapucu, Fikret Emre
name: Kelloniemi, Minna
name: Pakarinen, Toni-Karri
name: Miettinen, Susanna
name: Narkilahti, Susanna
Publisher: name: figshare
Year: 2023
Subjects: subject: Medicine
subject: Cell Biology
subject: Physiology
subject: FOS: Biological sciences
subject: 69999 Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer

description: Additional file 2: Supplementary Material. Supplementary Fig. 1. Neurons in collagen I-fibrin hydrogel in different medium without aprotinin supplementation on day 3 of culturing. Degradation and shrinkage can be seen rapidly after plating the cells to hydrogel. Scale bar is 500 μm. Supplementary Fig. 2. Angiotool was used to analyze the vessel area percentage in the BMSC and ASC multicultures in NMM, 50:50 and EGM-2 media. Supplementary Fig. 3. Hydrogel integrity is affected by the cell culture media. a Hydrogel integrity test with fluorescent beads for multicultures in the microfluidic chip in different cell culture media at day 8 timepoint. Interstitial flow through hydrogel carries fluorescent beads across the hydrogel region. Beads flow more freely through hydrogel in NMM and 50:50 media, whereas in EGM-2 beads follow the vascular structures. White arrows indicate the flow of the beads. Scale bar is 1000 μm. b Phase-contrast images of multicultures in different cell culture media at day 9 timepoint. Images showed shrinkage of hydrogel in microfluidic chips in BMSC and ASC multicultures in NMM and 50:50 media. Scale bar is 500 μm. Supplementary Fig. 4. Neurons stained with neuronal markers βtubIII+MAP-2 in different medium in neuronal monocultures and in multicultures of neurons, ECs, and BMSCs/ASCs on day 14. a Tilescan images of neurons in microfluidic chips. In EGM-2 neuronal monoculture, neurons grow to medium channels and form aggregates in the hydrogel. In EGM-2 multicultures or NMM neuronal monoculture these effects cannot be seen, as only few cells grow in the channels compared to EGM-2 monocultures. Scale bar is 500 μm. b Close ups of the tilescan images of neurons in different media in EGM monoculture, NMM monoculture and multicultures with BMSCs or ASCs as the mural cell type. Supplementary Fig. 5. Formation of vascularization, neuronal networks and supporting mural cells in the multicultures. a Live image timelapse of the formation of vascular structures indicated by the GFP tag of HUVECs trough the 14-day culturing period in BMSC and ASC multicultures. Scale bar is 1000 μm. b ICC staining of multicultures with neuronal markers showed the similar formation of neuronal networks in both multiculture formats. c ICC staining of multicultures with mural cell marker showed the pericytic characteristics of BMSCs and ASCs to cells in multicultures at day 14 timepoint. Both BMSCs and ASCs expressed the mural cell marker and spread throughout the multicultures. However, the morphology of the differentiated mural cells was different when using cells of different origin. Scale bar is 500 μm. Supplementary Fig. 6. Reproducibly established vasculature in multicultures in different experiments. a Representative live images of the vasculature formation in BMSC and ASC multicultures indicated by the GFP or RFP tag of HUVECs on days 13-14 of culturing. Scale bar is 1000 μm. b Angiotool was used to analyze the vessel area in the BMSC and ASC multicultures in different experiments. BMSC and ASC cell lines used in experiments are color coded with different shades of blue and green. In experiments 4 and 5 ASCs were not utilized. More detailed information of the cell lines is listed in supplementary tables 1,2 and 3. Supplementary Fig. 7. Angiotool was used to analyze the vessel area percentage in the BMSC and ASC multi- and cocultures in EGM-2. Supplementary Fig. 8. Proteome Profiler human angiogenesis array analysis. a Protein secretion profile of BMSC and ASC multicultures normalized to BMSC and ASC cocultures, respectively. Most of the angiogenesis-related proteins are overexpressed in multicultures compared to cocultures. b Proteome Profiler membranes from BMSC and ASC multi- and cocultures used for spot intensity quantification. Medium for analysis was collected from experiment 6. Details of the BMSC and ASC cell lines used in the experiment are listed in Supplementary Tables 1, 2 and 3. Supplementary Table 1. Donor information for the used primary mesenchymal stem/stromal cell lines BMSC 1-2 and ASC 1-2. For donor cell line BMSC 1, Body Mass Indexwas not available. Supplementary Table 2. Surface marker expression of the studied donor BMSCs and ASCs. Individual donor cell line passage during analysis is denoted in the column ‘’P’’. Positive &gt; 80%; low &lt; 10%; negative &lt; 2%. The cells were characterized as MSCs due to positive expression of CD73, CD90, and CD105, and low or negative expression of CD14, CD19, CD45 [67]. The expression of CD34 and HLA-DR was present at variable levels. The heterogeneity in surface marker expression if compared to the ISCT requirements could be explained by changes in cell culturing conditions [68]. Supplementary Table 3. Experimental information of the used primary mesenchymal stem/stromal cell lines BMSC 1-2 and ASC 1-2. Experiments were repeated 7 and 5 times for BMSCs and ASCs, respectively. Supplementary Table 4. Proteins analyzed with the Proteome Profiler array.