Microfluidic fabrication of bioactive microgels for rapid formation and enhanced differentiation of stem cell spheroids

Abstract

A major challenge in tissue engineering is to develop robust protocols for differentiating ES and iPS cells to functional adult tissues at a clinically relevant scale. The goal of this study is to develop a high throughput platform for generating bioactive, stem cell-laden microgels to direct differentiation in a well-defined microenvironment. We describe a droplet microfluidics system for fabricating microgels composed of polyethylene glycol and heparin, with tunable geometric, mechanical, and chemical properties, at kHz rates. Heparin-containing hydrogel particles sequestered growth factors Nodal and FGF-2, which are implicated in specifying pluripotent cells to definitive endoderm. Mouse ESCs were encapsulated into heparin microgels with a single dose of Nodal and FGF-2, and expressed high levels of endoderm markers Sox17 and FoxA2 after 5 days. These results highlight the use of microencapsulation for tailoring the stem cell microenvironment to promote directed differentiation, and may provide a straightforward path to large scale bioprocessing in the future.

A microfluidic-based cell encapsulation platform to achieve high long-term cell viability in photopolymerized PEGNB hydrogel microspheres

“…The polydispersity of microgel particle size distributions was calculated as the standard deviation of the diameter of particles divided by the mean diameter for both microfluidic and vortex fabrication methods. The microfluidic technique generated microgels with a 6.22% polydispersity while vortexing produced particle samples with an average polydispersity of 23.62%, Monodispersity of microgels generated by microfluidics has been shown to be increased by further reducing channel and particle size 26 . Well-controlled size distributions holds enabling consequences for precision cell encapsulation 61 and high throughput cell screening 37 .…”

“…Miniaturizing PEG hydrogels through microfabrication or microfluidics has emerged as a promising and versatile platform for biosensing 23 , tissue engineering 24 , drug delivery 25,26 , and high throughput screening 27 . The generation of structured hydrogels through bioprinting 28 , photolithography 29 , and liquid bridging 30 have been demonstrated to effectively generate functional platforms for cell studies.…”

“…However, the throughput of SFL is ultimately limited by exposure area and polymerization kinetics 33,34 . Microfluidic emulsification, a two-phase microfluidic technique capable of generating monodisperse aqueous droplets in continuous oil phase has improved microgel fabrication frequency to kHz 26 , thus providing a potential tool for high throughput single cell encapsulation or screening 35-37 . Sustained cell viability following photoencapsulation is a critical parameter for therapeutic cell delivery applications.…”

Parallel droplet microfluidics for high throughput cell encapsulation and synthetic microgel generation

“…Crosslinking PEG-4MAL macromers can be accomplished with dithiol molecules and does not require free radical initiators, which are detrimental to encapsulated cell health 13 . Similar microfluidic schemes for producing microgels [14][15][16] and encapsulating cells [17][18][19] have recently been reported for widely varying applications, including wound healing, stem cell culture and fundamental studies of cell biology. The versatility of microfluidic cell encapsulation extends to other polymers, including Matrigel 20 , agarose 21 and even multilayer core-shell composites such as collagen cores with alginate shells 22 .…”

Microfluidic generation of alginate microgels for the controlled delivery of lentivectors

“…17,18 In contrast to macroscopic hydrogels, microgels exhibit reduced characteristic lengths and increased surface area, which provides potential for manipulation as a delivery platform. These strategies have been used to promote cellular encapsulation, 19,20 control drug release, 21,22 and have formed a platform for responsive materials. [23][24][25][26] Microgel encapsulation may hold advantage for the delivery of lentivectors, [27][28][29] but adapting these microfluidic techniques for producing lentivector-compatible microgels represents an engineering challenge.…”