Bioamine-crosslinked gellan gum hydrogel for neural tissue engineering

Abstract

Neural tissue engineering and three-dimensional in vitro tissue modeling require the development of biomaterials that take into account the specified requirements of human neural cells and tissue. In this study, an alternative method of producing biomimetic hydrogels based on gellan gum (GG) was developed by replacing traditional crosslinking methods with the bioamines spermidine and spermine. These bioamines were proven to function as crosslinkers for GG hydrogel at +37 °C, allowing for the encapsulation of human neurons. We studied the mechanical and rheological properties of the formed hydrogels, which showed biomimicking properties comparable to naïve rabbit brain tissue under physiologically relevant stress and strain. Human pluripotent stem cell-derived neuronal cells demonstrated good cytocompatibility in the GG-based hydrogels. Moreover, functionalization of GG hydrogels with laminin resulted in cell type-specific behavior: neuronal cell maturation and neurite migration.

Screening of Hydrogels for Human Pluripotent Stem Cell–Derived Neural Cells: Hyaluronan‐Polyvinyl Alcohol‐Collagen‐Based Interpenetrating Polymer Network Provides an Improved Hydrogel Scaffold

“…A 3D hydrogel staining protocol optimized previously was used after 2 or 4 weeks of cell culture, and the same protocol was also used for 2D controls. Briefly, cells were fixed in 4% paraformaldehyde for 30 min, and unspecific staining was blocked by 10% normal donkey serum, 0.1% Triton‐X 100, and 1% bovine serum albumin (all from Sigma‐Aldrich) for 1 h at room temperature.…”

“…Compression testing was performed as previously described, using a BOSE Electroforce Biodynamic 5100 machine equipped with a 225 N load sensor and Wintest 4.1 software (Bose Corporation, Eden Prairie, Minnesota, USA). Briefly, HA1‐PVA‐Col and HA2‐PVA hydrogel samples were cast into a homemade cylindrical mold with an approximate height of 6 mm and a diameter of 12 mm and stored overnight before compression testing to ensure complete gelation.…”

“…A total of seven different hydrogels were prepared and used in 3D culture experiments: PuraMatrix (PM; BD Biosciences, San Jose, CA, USA [Catalog No. 354250]/3DM Inc., Cambridge, MA, USA), gellan gum (GG; Gelzan, Sigma‐Aldrich;), collagen hydrogel (Col1), hyaluronan–polyvinyl alcohol based hydrogels (HA1‐PVA, HA2‐PVA, HA1‐PVA‐Col), and hyaluronan–collagen type I–poly(ethylene glycol) ether tetrasuccinimidyl glutarate hydrogel (HA‐Col1‐4SPEG).…”

“…Based on previous studies and in‐house laboratory standards, both positive and negative 3D hydrogel controls were used. The controls were as follows: positive control PM and negative control nonfunctionalized GG . In addition, in‐house 2D laboratory standard coating control mouse laminin (Sigma‐Aldrich) was used .…”

“…For PM, cells were mixed with 0.25% PM diluted in 10% sucrose in phosphate‐buffered saline (PBS). GG solution (5 mg mL −1 ) was prepared as previously described, and 1.25% spermidine trihydrochloride (SPD; Sigma‐Aldrich) was used as a cross‐linker, both dissolved in 10% sucrose.…”