Cell density overrides the effect of substrate stiffness on human mesenchymal stem cells’ morphology and proliferation

Cell–cell interaction via substrate deformation in turn modifies cellular response to substrate rigidity.

shows the range of substrate deformation (at least 2μm of bead displacement obtained from TFM) of a single cell is around 75 μm which is in accordance with. 1 As the cell number increased from one to two, this range increased to 125 μm meaning deformation caused by two closely placed neighbours will reach the third cell which is within that range. As shown in the figure S1A, for seeding density more than 4K, average distance between two cells falls within this range leading to initiation of network formation.

Figure S2: Seeding density effect is independent of cell type: Fluorescent images (A-C) C2C12 cells and (D-F) NIH 3T3 cells on stiffness of 500Pa with seeding density of 1K(A), 4K(B) and 16K (C).
Although we were able to observe the formation of the cellular networks with hMSCs, and literature shows the network formation with endothelial cells, 2 we wanted to check if the observation is cell type specific. To address this question, NIH 3T3 and C2C12 were cultured on the different substrates (500Pa, 1 kPa, 2 kPa, and glass) with different seeding densities. It was observed that both cell types formed the networks from seeding 4K seeding density onwards as shown in the figure S2. Figure S3. Network formation is independent of global growth factor/cytokine concentrations.

Cells were cultured at two different seeding densities 1K cells/cm 2 (A-B) and 4K cells/cm 2 (C-D) with media volume of 3 ml (A and C) and 25 ml (B and D). From A to D, although cell density was
increased by 4 times, the media volume was increased by more than 8 times, making an increase in the global concentration of cytokines unlikely. In figure (E-G), total cell number was increased from 10K to 50K keeping seeding density constant by changing seeding area. We observe that formation of networks is independent of total number of cells thus total cytokine concentration.
With increased seeding density, not only does intercellular separation distance decreased, but the concentration of secreted molecules increased, which might had an effect on individual cell spreading and the formation of global patterns. To test that possibility, we performed two experiments. First, we

strings (C), open (D and E) and eventually create closed networks (E and F) and monolayers (H and I).
With the increase in the seeding density, the average distance between the two cells decreases. At sparse seeding density, the cells tend to remain round as shown in the image S4 A. As the distance between the two cells decreases (approximately 100-200 μm) they start to form the protrusion towards       During the initial hours of seeding the cells interact with the surrounding, and forms protrusions towards its neighbour in the vicinity as explained earlier. AFM measurements done during the initial hours of seeding showed that within 45 minutes of seeding the cells starts to orient towards the neighboring cells. Due to this there is a small increase in the stiffness of the gel in between the two cells as measured for figure S9 A and shown by black solid line in figure S9 C. The stiffness showed an increase around two fold compared to the base stiffness at 45 minutes of seeding. As the time lapsed, the stiffness between the two cells increased as measured for figure S9 B and shown by red dotted line of figure S9 C. After 4 hours of seeding the stiffness changed from 2 fold to around 3.7 times the base value. As mentioned earlier, the formation of network begins within 4 hours of seeding.

The scatter plot shows the ratio of BrdU+ cells to BrdU-cells with respect to the number of neighbours that a particular cell had in its vicinity. Graphs A), B), and C) , obtained from three independent experiments, show that ratio of BrdU+ cells over BrdU-cells increases with
Image S9 D and S9 E shows the phase image of the cells at 50 minutes and 4 hours after seeding.
During the initial hours of seeding the cells remained round (figure S9 D) but as the time progressed they started to make protrusions towards the neighboring cell (figure S9 E).