(M-484) Laminin-decorated gelatin microporous injectable hydrogel for the encapsulation and differentiation of neural progenitor cells

Introduction: Nervous tissues have limited capacity to regenerate, and damage to these tissues, due to neural injuries or neurological disorders, is often debilitating to the affected patients. The delivery of neural progenitor cells (NPCs), through biomaterials as a delivery vehicle is a promising strategy to promote neural tissue repair. Neural differentiation of NPCs is facilitated by cell spreading and morphological changes, as well as cell-cell interactions. An effective delivery vehicle for the nervous tissue regeneration should promote such cell-matrix and cell-cell interactions. In this report, we investigated the use of an injectable microporous hydrogel, formed by annealing of gelatin microgels, to promote neural differentiation of encapsulated NPCs. NPCs can undergo morphological changes and establish cell-cell interactions through the interconnected pore network between microgels. In addition, we demonstrate a novel method of improving NPC adhesion in the microgel matrix by a facile covalent incorporation of laminin by microbial transglutaminase (mTG) during the microgel annealing process. We found that neuronal differentiation was significantly enhanced in the gelatin microgel-based microporous hydrogel. Covalent incorporation of laminin further enhanced the formation of functional neural tissue.


Materials and Methods: Gelatin microgels were produced by a previously described process, using a water-in-oil emulsion. The temperature-mediated physical crosslinking of gelatin was utilized to crosslink microgels, which were separated via vacuum filtration. Fluorescent labeling and microscopy was used to demonstrate the covalent immobilization of laminin on the surface of gelatin microgels during crosslinking. ReNcell VM, an established NPC line, was encapsulated in the bulk hydrogel by mixing the cells with a gelatin microgel suspension, mTG, and purified laminin. Cells were encapsulated in the interstitial pore space between microgels. In all experiments, these microporous conditions were compared with a nonporous counterpart, made from an equal concentration of gelatin solution, crosslinked by mTG. Proliferation, organization of cells within the 3D space, and morphological changes of encapsulated cells during differentiation were observed by live/dead imaging. Immunostaining for differentiation markers MAP2 and GFAP, change in mitochondrial activity, and calcium flux were observed to measure ReNcell differentiation and functional neural tissue formation.


Results, Conclusions, and Discussions: A schematic of the experimental setup for cell encapsulation is provided by Fig. 1a. Fluorescence imaging shows that simple mixing of laminin with microgels during the mTG crosslinking led to efficient covalent immobilization of laminin to the surface of microgels (Fig. 1b,c) Decoration of microgels with laminin was explored due to its known effects of enhancing NPC adhesion. Without laminin conjugation, encapsulated NPCs aggregate into cell clusters, as observed by live/dead assay (Fig. 1d). When laminin is immobilized on the microgel surface during crosslinking, cell adhesion and spreading around microgels is increased (Fig. 1e). Cell morphology and viability was also seen to depend on the porous architecture, as the increased porosity provided by microporous hydrogel promoted cell spreading and viability of encapsulated cells. Encapsulated NPCs elongated, and made connections with nearby cells in response to incubation in differentiation medium in the microporous hydrogels (Fig. 1f), and cells stained positively for MAP2 (Fig. 1g) and GFAP, markers for neurons and astrocytes respectively, were identified after differentiation for 14 days. The generation of functioning neurons was examined by monitoring calcium flux of encapsulated cells in response to stimulation by neurotransmitter (glutamate). Cells encapsulated in the microporous hydrogels were observed to spontaneously fire when encapsulated in the microporous hydrogel environment, but firing was not observed in the nonporous condition.


In conclusion, the gelatin microgel-based injectable hydrogel provided necessary 3D environment for cell adhesion, rapid spreading, morphological changes and cell-cell interactions, leading to the efficient differentiation of encapsulated cells, and the maturation of differentiated NPCs into electrically active neurons. In summary, these results demonstrate the potential for microporous injectable hydrogels to serve as a scaffold for the encapsulation and delivery of NPCs for repair of nervous tissue.