Controlled embryoid body formation via surface modification and avidin-biotin cross-linking

Abstract

Cell-cell interaction is an integral part of embryoid body (EB) formation controlling 3D aggregation. Manipulation of embryonic stem (ES) cell interactions could provide control over EB formation. Studies have shown a direct relationship between EB formation and ES cell differentiation. We have previously described a cell surface modification and cross-linking method for influencing cell-cell interaction and formation of multicellular constructs. Here we show further characterisation of this engineered aggregation. We demonstrate that engineering accelerates ES cell aggregation, forming larger, denser and more stable EBs than control samples, with no significant decrease in constituent ES cell viability. However, extended culture >/=5 days reveals significant core necrosis creating a layered EB structure. Accelerated aggregation through engineering circumvents this problem as EB formation time is reduced. We conclude that the proposed engineering method influences initial ES cell-ES cell interactions and EB formation. This methodology could be employed to further our understanding of intrinsic EB properties and their effect on ES cell differentiation.

Fig. 1

Optimization of experimental parameters and effect on aggregation. Engineered and control ES cells were seeded at 5 × 10⁴ cells/mL into SCM supplemented with varying concentrations of avidin, incubated for 24 h and average EB diameters measured (a). Engineered and non-engineered ES cells with (control 2) and without (control 1) 10 μg/mL avidin supplementation were seeded at 5 × 10⁴ cells/mL into SCM and agitated at varying rotation speeds for the first 6 h of a 24 h incubation and analyzed by PSS (b). ES cells were seeded at 5 × 10⁴ and 1 × 10⁶ cells/mL into SCM with avidin supplementation, incubated for 3 days and average EB diameters measured (c). *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05. Error bars = SEM

Fig. 2

ES cell aggregation and EB formation. ES cells were seeded into suspension at 5 × 10⁴ cells/mL, rotated at 15 rpm for the first 6 h of a 24 h static incubation and photographed at 10× magnification every 4 h until visible EB formation (a). Surface morphologies of EBs were analyzed by SEM after 1, 3 and 5 days incubation (b)

Fig. 3

Analysis of aggregation dynamics. Engineered and control ES cells were seeded at 5 × 10⁴ cells/mL into AM supplemented with 10 μg/mL avidin and rotated at 15 rpm for 6 h. Suspensions were then incubated in SCM under static conditions for a maximum of 9 days. Average EB diameters (a) and numbers (b) were measured every other day. The numbers of non-aggregated ES cells in suspension were analyzed by Hoechst assay of flow through cells following suspension filtration with a 40 μm sieve to remove EBs (c). Whole samples without filtration were also analyzed by Hoechst assay. The numbers of ES cells constituting EBs were calculated by deducting flow through cell numbers from whole sample cell numbers (d). *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05. Error bars = SEM

Fig. 4

EB structure and constituent ES cell viability. Both engineered (a) and control EBs (b) were incubated with Live/Dead™ solution for 30–60 min to assess ES cell viability. The numbers of dead ES cells within engineered and control EBs seeded at 5 × 10⁴ cells/mL (c) were analysed by cell counts. d shows an H&E stained cross-section from an engineered EB after 9 days static culture and diagrammatic representation of the internal structure. *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05. Error bars = SEM

Fig. 5

Effect of engineering on ES cell density within the EB. Engineered and control EBs were fixed and sectioned after 9 days static culture in SCM. Sections were H&E stained and ES cell densities calculated via nuclei counts within a known area. ES cell densities were measured between engineered and control EBs at both the surface (a) and core (b). ES cell surface densities were compared to ES cell core densities in both engineered (c) and control 1 (d) EBs. *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05. Error bars = SEM