A Balance between Inter‐ and Intra‐Microgel Mechanics Governs Stem Cell Viability in Injectable Dynamic Granular Hydrogels

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Abstract

Injectable hydrogels are increasingly explored for the delivery of cells to tissue. These materials exhibit both liquid‐like properties, protecting cells from mechanical stress during injection, and solid‐like properties, providing a stable 3D engraftment niche. Many strategies for modulating injectable hydrogels tune liquid‐ and solid‐like material properties simultaneously, such that formulation changes designed to improve injectability can reduce stability at the delivery site. The ability to independently tune liquid‐ and solid‐like properties would greatly facilitate formulation development. Here, such a strategy is presented in which cells are ensconced in the pores between microscopic granular hyaluronic acid (HA) hydrogels (microgels), where elasticity is tuned with static covalent intra‐microgel crosslinks and flowability with mechanosensitive adamantane‐cyclodextrin (AC) inter‐microgel crosslinks. Using the same AC‐free microgels as a 3D printing support bath, the location of each cell is preserved as it exits the needle, allowing identification of the mechanism driving mechanical trauma‐induced cell death. The microgel AC concentration is varied to find the threshold from microgel yielding‐ to AC interaction‐dominated injectability, and this threshold is exploited to fabricate a microgel with better injection‐protecting performance. This delivery strategy, and the balance between intra‐ and inter‐microgel properties it reveals, may facilitate the development of new cell injection formulations.

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Floor plate-derived dopamine neurons from hESCs efficiently engraft in animal models of PD

Sonja Kriks, Jae Won Shim, Jinghua Piao … (2011)

SUMMARY Human pluripotent stem cells (hPSCs) are a promising source of cells for applications in regenerative medicine. Directed differentiation of hPSCs into specialized cells such as spinal motoneurons 1 or midbrain dopamine (DA) neurons 2 has been achieved. However, the effective use of hPSCs for cell therapy has lagged behind. While mouse PSC-derived DA neurons have shown efficacy in models of Parkinson’s disease (PD) 3, 4 , DA neurons from human PSCs generally display poor in vivo performance 5 . There are also considerable safety concerns for hPSCs related to their potential for teratoma formation or neural overgrowth 6, 7 Here we present a novel floor plate-based strategy for the derivation of human DA neurons that efficiently engraft in vivo, suggesting that past failures were due to incomplete specification rather than a specific vulnerability of the cells. Midbrain floor plate precursors are derived from hPSCs in 11 days following exposure to small molecule activators of sonic hedgehog (SHH) and canonical WNT signaling. Engraftable midbrain DA neurons are obtained by day 25 and can be maintained in vitro for several months. Extensive molecular profiling, biochemical and electrophysiological data define developmental progression and confirm identity of hPSC-derived midbrain DA neurons. In vivo survival and function is demonstrated in PD models using three host species. Long-term engraftment in 6-OHDA-lesioned mice and rats demonstrates robust survival of midbrain DA neurons, complete restoration of amphetamine-induced rotation behavior and improvements in tests of forelimb use and akinesia. Finally, scalability is demonstrated by transplantation into Parkinsonian monkeys. Excellent DA neuron survival, function and lack of neural overgrowth in the three animal models indicate promise for the development of cell based therapies in PD.

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Cytocompatible Click-based Hydrogels with Dynamically-Tunable Properties Through Orthogonal Photoconjugation and Photocleavage Reactions

Cole DeForest, Kristi S. Anseth (2011)

To provide insight as to how cells receive information from their external surroundings, synthetic hydrogels have emerged as systems for assaying cell function in well-defined microenvironments where single cues can be introduced and subsequent effects individually elucidated. However, as the field seeks to answer more complex biological questions, advanced material systems are needed that allow dynamic alteration of the 3D cellular environment with orthogonal reactions that enable multiple levels of control of biochemical and biomechanical signals. Here, we sought to synthesize one such 3D culture system using cytocompatible and wavelength-specific photochemical reactions to create hydrogels that allow orthogonal and dynamic control of the material properties through independent spatiotemporally-regulated photocleavage of crosslinks and photoconjugation of pendant functionalities. Results demonstrate the versatile nature of the chemistry to create programmable niches to study and direct cell function by modifying the local hydrogel environment.

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Sequential Click Reactions for Synthesizing and Patterning 3D Cell Microenvironments

Cole DeForest, Brian D. Polizzotti, Kristi S. Anseth (2009)

Click chemistry provides extremely selective and orthogonal reactions that proceed with high efficiency and under a variety of mild conditions, the most common example being the copper(I)-catalyzed reaction of azides with alkynes1,2. While the versatility of click reactions has been broadly exploited3–5, a major limitation is the intrinsic toxicity of the synthetic schemes and the inability to translate these approaches to biological applications. This manuscript introduces a robust synthetic strategy where macromolecular precursors react via a copper-free click chemistry6, allowing for the direct encapsulation of cells within click hydrogels for the first time. Subsequently, an orthogonal thiol-ene photocoupling chemistry is introduced that enables patterning of biological functionalities within the gel in real-time and with micron-scale resolution. This material system allows one to tailor independently the biophysical and biochemical properties of the cell culture microenvironments in situ. This synthetic approach uniquely allows for the direct fabrication of biologically functionalized gels with ideal structures that can be photopatterned and all in the presence of cells.