Microfluidic Generation of Monodisperse, Structurally Homogeneous Alginate Microgels for Cell Encapsulation and 3D Cell Culture

Abstract

Recent studies have shown that basic cellular behavior varies significantly between two- and three-dimensional culture systems. To identify the origins of these fundamental differences the design of reliable and precisely controlled environments is essential. While 2D cell culture is a well-established technique, the fabrication of defined three-dimensional culture models is still challenging. We present a new method for the microfluidic generation of a micron-sized three-dimensional cell culture system. We use a triggered ionic crosslink formation to generate highly monodisperse and structurally homogeneous alginate microbeads. Aqueous droplets containing a mixture of alginate and a water-soluble calcium-EDTA complex are formed by droplet-based microfluidics. In their complexed form, the calcium ions are homogenously distributed inside the droplet but not accessible for the crosslinking process. Acid addition is used to trigger the degradation of the complex, releasing calcium ions on demand that can physically crosslink the alginate chains. A homogeneous hydrogel network is thus generated which can be transferred into an aqueous environment without losing its structural integrity. Single cells can be encapsulated into these controlled microenvironments which provide structural support while allowing for continuous nutrient supply. We encapsulate individual mesenchymal stem cells (MSCs) into the microbeads which show the aspired cell growth and proliferation.

Versatile, cell and chip friendly method to gel alginate in microfluidic devices

“…We monitored conditions of pH during gelling using a fluorescent dye (N-(rhodamine 6G)-lactam-ethylenediamine (R6G-EDA)) 28 and photon counting using a confocal microscope and compared the evolution of pH with an alginate containing CaEDTA and gelled by acetic acid contained in the oil phase as described by Utech and coworkers. 29 As expected, using ligand exchange, pH was maintained at a constant pH 6.7 for the duration of the experiment, however upon addition of fluorinated oil containing acetic acid, pH rapidly dropped from pH 6.7 to pH 4.6 as H + diffused from the oil phase to the aqueous alginate phase, a prerequisite to initiate gelling with this method (ESI Fig. 2).…”

“…29,41 Introducing aqueous Ca 2+ directly to the alginate stream in a device will result in immediate gelling and clogging of the channels. Therefore, in addition to having an inlet for Ca 2+ and alginate, an extra inlet is often introduced to the microfluidic geometry whereby sheathing buffer is injected to temporary shield the Ca 2+ stream from the alginate.…”

“…To release the chelated Ca 2+ , an acidified carrier fluid is typically applied to decrease the pH through the diffusion of H + from the continuous to the dispersed phase resulting in subsequent internal gelation of the alginate. 29,38,41,[44][45][46] While effective in achieving a slower and more homogeneous gelation process that does not clog microfluidic channels immediately and results in well dispersed microparticles, unfortunately all of these approaches are reliant on a pH drop well below the physiological range and are therefore detrimental to cell viability. A recent study 29 showed that cell viability can somewhat be enhanced if the cell-loaded gels are rinsed from the acidic environment shortly after gelation, however, even after short time scales (~2min), the survival rate of the encapsulated cells showed a decrease to ~80% and to ~0% after 30 min between encapsulation, gelation and resuspension in cell medium.…”

“…47 However, the use of microcrystals or micron sized agglomerates of nanocrystals of CaCO 3 as a gelling strategy can result in inhomogeneous gelling since the distribution of these particles will vary between droplets and the crystals may also cause clogging of narrow junctions in microfluidic devices. 29,48 Additionally, this gelation strategy is very slow (~hours), which limits the applications for synthesis of more complex structures e.g. core-shell or janus particles since the synthesis of such structures requires rapid gelation.…”

Microfluidic generation of Buddha beads-like microcarriers for cell culture

“…In comparison with other approaches, microfluidic microcarriers are a promising technique for cell encapsulation because of their advantages of excellent monodispersity, precise size control, high throughput, and better microenvironmental control [21][22][23][24][25][26][27][28]. However, because of the lack of effective nutrient exchange pathways, most of the microcarriers are restricted to a small size to avoid the formation of necrotic regions [29][30][31]. In addition, conventional microcarriers could only provide a homogeneous microenvironment for uniform cell distribution, whereas cell distribution in vivo is extremely complex.…”