Cell Microencapsulation in Polyethylene Glycol Hydrogel Microspheres Using Electrohydrodynamic Spraying.

Microencapsulation of cells is beneficial for various biomedical applications, such as tissue regeneration and cell delivery. While a variety of techniques can be used to produce microspheres, electrohydrodynamic spraying (EHS) has shown promising results for the fabrication of cell-laden hydrogel microspheres in a wide range of sizes and in a relatively high-throughput manner. Here we describe an EHS technique for the fabrication of cell-laden polyethylene glycol (PEG) microspheres. We utilize mild hydrogel gelation chemistry and a combination of EHS parameters to allow for cell microencapsulation with high efficiency and viability. We also give examples on the effect of different EHS parameters such as inner diameter of the needle, voltage and flow rate on microsphere size and encapsulated cell viability.

Templated Macroporous Polyethylene Glycol Hydrogels for Spheroid and Aggregate Cell Culture.

Macroporous cell-laden hydrogels have recently gained recognition for a wide range of biomedical and bioengineering applications. There are various approaches to create porosity in hydrogels, including lyophilization or foam formation. However, many do not allow a precise control over pore size or are not compatible with in situ cell encapsulation. Here, we developed novel templated macroporous hydrogels by encapsulating uniform degradable hydrogel microspheres produced via microfluidics into a hydrogel slab. The microspheres degraded completely leaving macropores behind. Microsphere degradation was dependent on the incubation medium, microsphere size, microsphere confinement in the hydrogel as well as cell encapsulation. Uniquely, the degradable microspheres were biocompatible and when laden with cells, the cells were deposited in the macropores upon microsphere degradation and formed multicellular aggregates. The hydrogel-encapsulated cell aggregates were used in a small drug screen to demonstrate the relevance of cell-matrix interactions for multicellular spheroid drug responsiveness. Hydrogel-grown spheroid cultures are increasingly important in applications such as in vitro tumor, hepatocellular, and neurosphere cultures and drug screening; hence, the templated cell aggregate-laden hydrogels described here would find utility in various applications.

Cell Attachment and Spreading on Carbon Nanotubes Is Facilitated by Integrin Binding.

Owing to their exceptional physical, chemical, and mechanical properties, carbon nanotubes (CNTs) have been extensively studied for their effect on cellular behaviors. However, little is known about the process by which cells attach and spread on CNTs and the process for cell attachment and spreading on individual single-walled CNTs has not been studied. Cell adhesion and spreading is essential for cell communication and regulation and the mechanical interaction between cells and the underlying substrate can influence and control cell behavior and function. A limited number of studies have described different adhesion mechanisms, such as cellular process entanglements with multi-walled CNT aggregates or adhesion due to adsorption of serum proteins onto the nanotubes. Here, we hypothesized that cell attachment and spreading to both individual single-walled CNTs and multi-walled CNT aggregates is governed by the same mechanism. Specifically, we suggest that cell attachment and spreading on nanotubes is integrin-dependent and is facilitated by the adsorption of serum and cell-secreted adhesive proteins to the nanotubes.

Directed and enhanced neurite outgrowth following exogenous electrical stimulation on carbon nanotube-hydrogel composites.

OBJECTIVE: The objective of this work was to test the synergistic effects of substrate stiffness, electro-conductivity, composition and electrical stimulation on the morphology, alignment and directional neurite outgrowth of neuron-like PC12 cells. The use of exogenous electrical stimulation has emerged as a promising new intervention to promote neural regeneration following injury. For critical gap size nerve injuries, a permissive biomaterial coupled to electrical stimulation may be needed to provide guidance and support for neurite outgrowth. Thus, the combinatorial effects of biomaterial composition and properties and exogenous electrical stimulation need interrogation to develop successful therapeutic interventions. Carefully designed in vitro models are ideally suited to perform such multidimensional detailed studies. APPROACH: We assembled a simple electrical stimulation device to deliver uniform electrical current with minimum voltage field variation through a hydrogel. We used polyacrylamide (PA), polyethylene glycol (PEG), and multi-walled carbon nanotubes (MWCNT)-PEG nanocomposite hydrogels of varying stiffness, resistivity and MWCNT concentration. Cells were seeded on the substrates for 24 h, stimulated for 1 h at 30 V m-1 DC, and then cultured for additional 24 h. Non-stimulated cells were used as controls. To induce neurite outgrowth, cells were primed with nerve growth factor (100 ng ml-1). MAIN RESULTS: For all substrates tested, electrical stimulation induced neurite alignment at 60-90° angle to the applied current. It also increased total neurite outgrowth by 18%-49% and mean neurite length by 20%-46% (increase dependent on the underlying substrate) compared to non-stimulated cells. The nanocomposite composed of 20% w/v PEG and 0.1% w/v MWCNTs resulted in the highest total neurite outgrowth and mean neurite length, which were further significantly enhanced by electrical stimulation by 2-fold and 1.8-fold, respectively. SIGNIFICANCE: Our results indicate that nanocomposites, where carbon nanotubes have been added to hydrogel substrates, in combination with electrical stimulation provided improved conditions for neural growth and regeneration.

A Two-Step Method for Transferring Single-Walled Carbon Nanotubes onto a Hydrogel Substrate.

Carbon nanotube (CNT)-hydrogel nanocomposites are beneficial for various biomedical applications, such as nerve regeneration, tissue engineering, sensing, or implant coatings. Still, there are impediments to developing nanocomposites, including attaining a homogeneous CNT-polymer dispersion or patterning CNTs on hydrogels. While few approaches have been reported for patterning CNTs on polymeric substrates, these methods include high temperature, high vacuum or utilize a sacrificial layer and, hence, are incompatible with hydrogels as they lead to irreversible collapse in hydrogel structure. In this study, a novel two-step method is designed to transfer CNTs onto hydrogels. First, dense CNTs are grown on quartz substrates. Subsequently, hydrogel solutions are deposited on the quartz-grown CNTs. Upon gelation, the hydrogel with transferred CNTs is peeled from the quartz. Successful transfer is confirmed by scanning electron microscopy and indirectly by cell attachment. The efficient transfer is attributed to π-interactions pregelation between the polymers in solution and the CNTs.