Diels–Alder Cross-Linked, Washing-Free Hydrogel Films with Ordered Wrinkling Patterns for Multicellular Spheroid Generation

Abstract

Three-dimensional (3D) multicellular spheroids are a new generation in vitro cell model, however, their applications are severely limited by difficulties in their generation. Here patterned poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogel films were synthesized for their generation. Instead of polymerization of HEMA monomers in the presence of a cross-linker, here the PHEMA films were synthesized by cross-linking furan-functionalized linear PHEMA, PHEMA-furan, and maleimide-functionalized linear PHEMA, PHEMA-mal, via Diels−Alder (DA) reaction between furan and maleimide groups. A thermal treatment temperature of 75 °C was chosen for the cross-linking reaction. The occurrence of DA reaction was confirmed by IR spectra. Using this method, cross-linked PHEMA films with smooth surface were successfully synthesized in situ in the well of cell culture plates. The films were then patterned by simply adding water to swell them. Highly ordered, honeycomb-like wrinkling patterns were successfully obtained by adjusting the furan and maleimide contents in the precursor linear polymers. The patterned hydrogel films were used to generate multicellular spheroids. Guided by the patterns, 3D spheroids with narrow size distribution, tunable size, and high cell viability were successfully obtained. The patterned PHEMA films reported here exhibited a lot of advantages. The patterning method was quite simple and required no template or special equipment. They were synthesized in situ in commercial cell culture plates. Particularly, thanks to the clean nature of the DA reaction, no low molecular weight monomer, cross-linker, initiator, or catalyst, which were potentially cytotoxic, was involved in the film synthesis, and no byproduct was produced and left in the film. The resulting films presented a high biocompatibility, allowing the avoidance of the tedious washing step. The films synthesized here were expected to have high potential for massive production of well-defined multicellular spheroids.

Evaluation of the Usefulness of Human Adipose-Derived Stem Cell Spheroids Formed Using SphereRing® and the Lethal Damage Sensitivity to Synovial Fluid In Vitro

“…Genes related to cell-cell interaction, cell assembly, and organization also showed significant changes, suggesting that the formation of dense cell-cell aggregates led to changes in genes related to active cell-cell signaling and interaction. The mechanism of action of ADSCs in joints may be explained by angiogenic factors, immunosuppressive factors, and tissue regeneration [56], and the effect may be enhanced by spheroid formation. IPA revealed a gene network including Akt, MAPK, and PI3K, and MSC-derived spheroids reportedly induce PI3K-Akt signaling pathway activation, which might be similar to what occurred in the present study [57].…”

Ultrasound-Responsive Peptide Nanogels to Balance Conflicting Requirements for Deep Tumor Penetration and Prolonged Blood Circulation

“…Three-dimension (3D) TNBC cell mass was built for investigating the penetrating capacity of nanogels according to previous report. 54 Briefly, poly(2-hydroxyethyl methacrylate) solution at 15 mg/mL was spread on the bottom of cell culture flask, and then air-dried to prepare strongly hydrophobic coating under sterile environment. Individual suspended TNBC cells at a density of 1.0 × 10 5 were slowly added dropwise into the above flask, and the cell mass with uniform diameter were screened during culture.…”

“…Three-dimension (3D) TNBC cell mass was built for investigating the penetrating capacity of nanogels according to previous report . Briefly, poly­(2-hydroxyethyl methacrylate) solution at 15 mg/mL was spread on the bottom of cell culture flask, and then air-dried to prepare strongly hydrophobic coating under sterile environment.…”

Uniformly Sized Stem Cell Spheroids for Treatment of Hind Limb Ischemia: Size Effect

“…The PHEMA films were synthesized in situ in the wells of commercial cell culture plates and spontaneously patterned by swelling in water without using any template, mask, or special facilities. [20][21][22][23] Guided by these uniform microstructures, uniform-sized multicellular spheroids of adipose-derived stromal cells (ADSCs) were generated successfully. In addition, this method allows for high-throughput production of the spheroids and fine control on the spheroid sizes.…”