High throughput generation and trapping of individual agarose microgel using microfluidic approach

Abstract

Microgel is a kind of biocompatible polymeric material, which has been widely used as micro-carriers in materials synthesis, drug delivery and cell biology applications. However, high-throughput generation of individual microgel for on-site analysis in a microdevice still remains a challenge. Here, we presented a simple and stable droplet microfluidic system to realize high-throughput generation and trapping of individual agarose microgels based on the synergetic effect of surface tension and hydrodynamic forces in microchannels and used it for 3-D cell culture in real-time. The established system was mainly composed of droplet generators with flow focusing T-junction and a series of array individual trap structures. The whole process including the independent agarose microgel formation, immobilization in trapping array and gelation in situ via temperature cooling could be realized on the integrated microdevice completely. The performance of this system was demonstrated by successfully encapsulating and culturing adenoid cystic carcinoma (ACCM) cells in the gelated agarose microgels. This established approach is simple, easy to operate, which can not only generate the micro-carriers with different components in parallel, but also monitor the cell behavior in 3D matrix in real-time. It can also be extended for applications in the area of material synthesis and tissue engineering.

Long‐Term Perfusion Culture of Monoclonal Embryonic Stem Cells in 3D Hydrogel Beads for Continuous Optical Analysis of Differentiation

“…Few solutions have been devised for the spatial immobilization of hydrogel beads that would allow cell culture in a 3D matrix: an order of magnitude fewer cell‐containing beads were trapped in previous work: 28, 70, and 80, respectively, whereas our devices can reach a capacity of 2000 hydrogel beads. In addition, hydrogel beads composed of alginate, agarose, or matrigel were used for cell encapsulation and trapping, yet these studies were not extended to experiments probing molecular function (such as the immunostaining and RT‐qPCR of encapsulated stem cell colonies shown here) and multiple rather than single cells were encapsulated.…”

Multiphase microfluidic synthesis of micro- and nanostructures for pharmaceutical applications

“…Various types of natural and synthetic polymers have been used for making microgels, e.g., alginate (Utech et al, 2015), agarose (Shi et al, 2013), chitosan/dextran , gel-forming peptides (Tsuda et al, 2010), poly(Nisopropyl acrylamide) (PNIPAM) (Seiffert and Weitz, 2010), and poly(2-methacryloyloxyethyl phosphorylcholine) (Park et al, 2014). The polymer-chain chemistry 13 of microgels determines their hydrophilicity/hydrophobicity which, in turn, determines the types of solutions where the microgels can be dispersed (e.g., in aqueous or organic solvents) as well as the types of cargoes that can be loaded.…”

“…Droplets containing a pre-microgel mixture are formed in an immiscible carrier phase, and then turn in microgel particles through various subsequent droplet gelation methods, such as chemically (e.g., by photo-polymerization (Jagadeesan et al, 2011;Lu et al, 2015;Park et al, 2014) and free-radical copolymerization (Seiffert, 2012)) or physically (e.g., by temperature changes (Shi et al, 2013) and ionic cross-linking (Guo et al, 2011;Miyama et al, 2013)). The size of microgels depends on the size of the emulsified droplets, thus is tunable by adjusting the flow rates of the fluids during the microfluidic droplet formation or by controlling the dimensions of the microfluidic devices.…”

“…Various cells have been encapsulated within microgels, such as MCF-7 breast cancer cells (Yu et al, 2015), mesenchymal stem cells (Utech et al, 2015), human umbilical vein endothelial cells , human epithelial carcinoma cells (Miyama et al, 2013), adenoid cystic carcinoma cells (Shi et al, 2013), and Madin Darby canine kidney cells (Eydelnant et al, 2014). 3D cell cultures in microgels offer several advantages, including tunable shear forces imposed on cells, easy visualization (e.g., by conjugating fluorescent agent to the microgel's material (Utech et al, 2015)), easy control over the transport of oxygen, nutrients, growth factors and waste (McGuigan and Sefton, 2007) as well as the mechanical and chemical stability in aqueous media such as buffer or cell culture media.…”

“…Synthetic polymer-based microgels did not gain popularity in cell culture applications due to the harsh conditions involved in the process of microgel preparation, such as strong shear forces, ultraviolet irradiation, and large temperature gradients, which cause severe cell damage or even mortality (Velasco et al, 2012). In contrast, natural polymerbased microgels, such as alginate (Eydelnant et al, 2014;Miyama et al, 2013;Utech et al, 2015;Yu et al, 2015), agarose (Eydelnant et al, 2014;Shi et al, 2013) and chitosan , are widely used for cell encapsulation because of their biocompatibility and mild conditions required to achieve gelation, thus preserving cell viability. Cross-linking methods for gelation affect significantly the network structures of the formed microgels, so the ability to control cross-linking process allows the formation of homogeneous network structure with precise internal structure and tunable stiffness Utech et al, 2015), enabling the stable entrapment of cells in a controlled microenvironment.…”