Hydrogel design of experiments methodology to optimize hydrogel for iPSC-NPC culture

Abstract

Bioactive signals can be incorporated in hydrogels to direct encapsulated cell behavior. Design of experiments methodology methodically varies the signals systematically to determine the individual and combinatorial effects of each factor on cell activity. Using this approach enables the optimization of three ligands concentrations (RGD, YIGSR, IKVAV) for the survival and differentiation of neural progenitor cells.

Keywords: design of experiments; hyaluronic acid; hydrogel; induced pluripotent stem cells; neurons.

Figure 1

Hyaluronic acid was modified to contain an acrylate functional group and (A) degree of modification (16%) was confirmed via NMR spectroscopy. Hydrogel formulations with different hyaluronic acid concentrations (3.5–4.5%) were made and (B–C) the mechanical properties characterized. (D) F-actin of iPS-NPCs encapsulated in these three gel conditions were stained at days 1 and 7 and (E) cell spreading was greatest in the softest gel (3.5%). The symbol ** represents statistical significance to the level of p < 0.01. Statistical significance was determined using multiple comparisons with a Tukey post hoc test.

Figure 2

Statistical designs of experiments approach was used to (A) investigate the interactions between RGD, YIGSR and IKVAV adhesion peptides (0–100 μM each) and their ability to promote cell survival. Experiments give a (B) predictive max peptide mixture and (C) the effect magnitude of all factors and two-factor interactions. Confirmation of (D) max and min predictive peptide mixture. (E–G) A second series of optimization experiments to test higher peptide concentrations for RGD and IKVAV (100–200 μM each). (H–J) A third series of optimization experiments to test higher peptide concentration for IKVAV (200–300 μM). (K) Validation of the three optimized conditions compared to RDG, RGD and 100 μM of each peptide. Phase images of the (L–N) three optimized conditions with spread cells outlined. Scale bar = 100 μm.

Figure 3

iPS-NPCs cultured for one week in (A) 2-D, and (B–D) hydrogels were stained for DOUBLECORTIN (DCX) and DAPI. Number of (E) DCX positive cells and (F) length were quantified. Cells were also stained for (G–J) tuj1 and DAPI with (K) tuj1 positive cells and (L) length quantified. Flow cytometry data for (M–N) SOX2 and (O–P) DCX indicate that the 3D hydrogels promote differentiation of the iPS-NPCs.