Dynamic Covalent Hydrogels for Triggered Cell Capture and Release

A dual-responsive, cell capture and release surface was prepared through the incorporation of phenylboronic acid (PBA) groups into an oxime-based polyethylene glycol (PEG) hydrogel. Owing to its PEG-like properties, the unfunctionalized hydrogel was nonfouling. The use of highly efficient oxime chemistry allows the incorporation of commercially available 3,5-diformylphenyl boronic acid into the hydrogel matrix. Thus, the surface properties of the hydrogel were modified to enable reversible cell capture and release. Boronic ester formation between PBA groups and cell surface carbohydrates enabled efficient cell capture at pH 6.8. An increase to pH 7.8 resulted in cell detachment. This capture-and-release procedure was performed on MCF-7 human breast cancer cells, NIH-3T3 fibroblast cells, and primary human umbilical vein endothelial cells (HUVECs) and could be cycled with negligible loss in activity. The facile preparation of PBA-functionalized surfaces presented here has applications in biomedical fields such as cell diagnostics and cell culture

Decellularized extracellular matrices produced from immortal cell lines derived from different parts of the placenta support primary mesenchymal stem cell expansion

<div><p>Mesenchymal stem/stromal cells (MSCs) exhibit undesired phenotypic changes during <i>ex vivo</i> expansion, limiting production of the large quantities of high quality primary MSCs needed for both basic research and cell therapies. Primary MSCs retain many desired MSC properties including proliferative capacity and differentiation potential when expanded on decellularized extracellular matrix (dECM) prepared from primary MSCs. However, the need to use low passage number primary MSCs (passage 3 or lower) to produce the dECM drastically limits the utility and impact of this technology. Here, we report that primary MSCs expanded on dECM prepared from high passage number (passage 25) human telomerase reverse transcriptase (hTERT) transduced immortal MSC cell lines also exhibit increased proliferation and osteogenic differentiation. Two hTERT-transduced placenta-derived MSC cell lines, CMSC29 and DMSC23 [derived from placental chorionic villi (CMSCs) and <i>decidua basalis</i> (DMSCs), respectively], were used to prepare dECM-coated substrates. These dECM substrates showed structural and biochemical differences. Primary DMSCs cultured on dECM-DMSC23 showed a three-fold increase in cell number after 14 days expansion in culture and increased osteogenic differentiation compared with controls. Primary CMSCs cultured on the dECM-DMSC23 exhibited a two-fold increase in cell number and increased osteogenic differentiation. We conclude that immortal MSC cell lines derived from different parts of the placenta produce dECM with varying abilities for supporting increased primary MSC expansion while maintaining important primary MSC properties. Additionally, this is the first demonstration of using high passage number cells to produce dECM that can promote primary MSC expansion, and this advancement greatly increases the feasibility and applicability of dECM-based technologies.</p></div

Fluorescence micrographs of DMSC seeded on dECM and control surfaces after 72 h of incubation.

<p>(A) dECM-DMSC23, (B) dECM-CMSC29, (C) fibronectin, and (D) tissue culture plastic. Scale bar is 100 μm for all images. (E) Average DMSC cell spread area. (F) Average CMSC cell spread area. All values are mean ± SD, *p < 0.05.</p

Representative images showing decellularization of DMSC23 cultures.

<p>phase contrast (A) before and (D) after decellularization, fluorescence micrographs of DAPI staining (B) before and (E) after decellularization, and scanning electron micrographs (C) before and (F) after decellularization. Scale bar on phase contrast and fluorescence micrographs is 100 μm. Scale bar on SEM images is 5 μm.</p

Osteogenic differentiation of DMSC and CMSC on dECM substrates.

<p>Representative images of DMSC stained with Alizarin Red S dye after 14 days of osteogenic induction: (A) dECM-DMSC23, (B) dECM-CMSC29, (C) Fibronectin, and (D) TCP. Inset shows control uninduced DMSC. Scalebar is 100 μm. Osteoimage staining in (E) primary DMSC and (F) primary CMSC cultured on dECM and control surfaces after 14 days of osteogenic differentiation. All values are mean ± SD; n = 3; *p < 0.05, **p < 0.01, ***p < 0.001.</p

Initial characterization of dECM prepared from DMSC23 and CMSC29 cell lines.

<p>Immunofluorescence labelling of collagen type 1 (FITC) in dECM-DMSC23 and dECM-CMSC29 (A and B, respectively). Immunofluorescence labelling of fibronectin (Texas Red) in dECM-DMSC23 and dECM-CMSC29 (C and D, respectively). dECM-DMSC23 and dECM-CMSC29 proteoglycan visualized by Alcian blue (E and F, respectively). Scalebars are 100 μm. (G) SDS-PAGE protein profile of dECM-DMSC23 (lane 1), dECM-CMSC29 (lane 2), collagen I (lane 3), fibronectin (lane 4) and protein standards (lane 5).</p

Primary DMSC and CMSC proliferation on dECM substrates.

<p>(A) numbers of primary DMSC and (B) numbers of population doublings after 14 days of culture, and (C) representative photomicrographs showing primary DMSC cultured on dECM substrates and control surfaces on day 7. (D) Numbers of primary CMSC and (E) the numbers of population doublings after 14 days of culture, and (F) representative photomicrographs showing primary CMSC cultured on various dECM substrates and control surfaces on day 7. Significant increases in cell proliferation and population doubling levels were observed using one-way ANOVA with Tukey’s multiple comparison test. All values are mean ± SD; n = 3; *p < 0.05, **p < 0.01. Scalebar is 100 μm.</p

Macroporous Hydrogels Composed Entirely of Synthetic Polypeptides: Biocompatible and Enzyme Biodegradable 3D Cellular Scaffolds

Synthetic polypeptides are a class of bioinspired polymers with well demonstrated biocompatibility, enzyme biodegradability, and cell adhesive properties, making them promising materials for the preparation of macroporous hydrogels as 3D cellular scaffolds. Three-dimensional macroporous hydrogels composed entirely of biocompatible and enzyme biodegradable synthetic polypeptides have thus been prepared. Under cryoconditions, macroporous hydrogels in the form of macroporous cryogels were prepared using a single copolymer component through direct EDC/sulfo-NHS zero-length cross-linking between poly­(l-glutamic acid) (PLG) and poly­(l-lysine) (PLL) residues on a PLG-<i>r</i>-PLL random copolypeptide chain. The resulting macroporous cryogels were found to contain large interconnected pores (≥100 μm) highly suitable for tissue engineering applications. Tuning the relative ratios of the amino acid components could result in cryogels with very different pore structures, swelling, and mechanical properties, suitable for developing gels for a range of possible soft tissue engineering applications. These cryogels were shown to be enzymatically biodegradable and demonstrated excellent biocompatibility, cell attachment and cell proliferation profiles with mammalian fibroblast (NIH-3T3) cells, demonstrating the appeal of these novel cryogels as highly suitable cellular scaffolds