A self-sufficient micro-droplet generation system using highly porous elastomeric sponges: A versatile tool for conducting cellular assays
Introduction
Microfluidic systems facilitate cellular assays using reduced amounts of reagents, buffers and biological samples [1,2]. Such systems are inexpensive, portable, disposable, can be mass produced, and importantly can be customised to meet the requirements of the user [[2], [3], [4]].
In particular, microfluidic droplet generation systems enable encapsulation of cells inside microscale droplets containing desired reagents, chemicals, fluorescent probes, and functionalised beads to enable the stimulation of encapsulated cells and consequent detection of cellular responses [[5], [6], [7], [8]]. The encapsulated cells can undergo various physical and chemical stimuli before they are sorted, cultured, lysed or even released from the droplets for further analysis [[9], [10], [11], [12]]. Conventional droplet generation systems rely on external pumping mechanisms (i.e. syringe or pressure pumps) to drive a pair of immiscible liquids such as cell suspension and oil into microfluidic structures (i.e. T-junction or flow focusing channels) [13,14]. These systems facilitate the high throughput generation of uniformly sized droplets and allow for the modulation of the size of droplets by varying the flow ratio of carrier and discrete fluids [[15], [16], [17], [18], [19]]. Despite these features, the size and cost of existing syringe or pressure pumps might limit the application of microfluidic droplet generation systems to research laboratories. This limitation motivated us to develop a self-sufficient [20], manually-operated droplet generation system to produce droplets directly inside Petri dishes or microwell plates, which are routinely used for cellular assays in biomedical laboratories.
Highly porous sponges facilitate the storage and release of aqueous solutions due to their permeability and high surface area. These sponges can be made from different materials such as cellulose [[21], [22], [23]], hydrogels [24], and polymers [25,26]. In particular, polymeric porous sponges made of polydimethylsiloxane (PDMS) are suitable for microfluidic applications due to their biocompatibility, stability, transparency, and elasticity. PDMS sponges have been utilised for the active storage and release of solutions [27,28]. PDMS sponges are generally made by templating and the subsequent removal of crystal structured materials such as sugar cubes [29]. Despite their simple fabrication procedure, sugar templated sponges consist of an open network of neighbouring pores, which limit their capability for producing microscale droplets. Alternatively, highly porous sponges can be made by injecting microscale droplets of water into uncured polymers [30,31]. Curing of the polymer and subsequent evaporation of the encapsulated water droplets leads to the formation of a highly porous sponge, comprising of large pores that are interconnected by very small holes [32]. The existence of small interconnecting holes reduces the release rate of stored solutions [32], and can facilitate the generation of microscale droplets upon manual compression, as will be explored in the present work.
Here, we present a novel approach for generating microscale droplets by compressing highly porous PDMS sponges loaded with aqueous solutions into a cell culture microwell filled with oil. Upon manual compression of the sponge, hundreds of droplets are generated inside the microwell. We demonstrate the capability of our droplet generation system for encapsulating THP-1 human monocytes inside droplets. The number of encapsulated cells is proportional to the volume of droplets as well as the concentration of cells in the cell suspension. We further apply our droplet generation system for conducting cytotoxicity assays. As a proof-of-concept, we study the response of encapsulated THP-1 cells to hydrogen peroxide using fluorescent microscopy. We also demonstrate the ability to generate droplets with different chemical contents into the same microwell. Our sponge based droplet generation system provides an effective platform for conducting customised cellular assays.
Section snippets
Fabrication of PDMS porous sponge
The PDMS sponge was fabricated using a microfluidic T-junction droplet generation system imprinted into a PDMS slab, as recently reported by us [32], and briefly summarised in Fig. 1. Deionised water mixed with a surfactant (polysorbate 20, Sigma-Aldrich) (19:1 v/v) was injected into a continuous phase consisting of PDMS base, PDMS curing agent (Sylgard 184, Dow Corning) and monohydroxy terminated PDMS (Sigma-Aldrich) (15:2:5 v/v) to create droplets of water in uncured PDMS. The resulting
Droplet generation using the PDMS porous sponge
The process of droplet generation consists of both loading and compression steps, as schematically shown in Fig. 2. During the loading step, the sponge is placed in a cell culture microwell filled with aqueous solutions, and gently compressed two to three times to be filled (Fig. 2a–c). The loaded sponge is then gently placed into a second microwell filled with olive oil, and gently compressed to release the stored solution into the surrounding oil (Fig. 2d–e). This leads to generation of
Conclusion
In summary, we demonstrated a novel mechanism for generating microscale droplets of aqueous solutions in oil using a highly porous PDMS sponge. Upon manual compression, hundreds of droplets are generated inside a cell culture microwell with ∼64% of droplets ranging from 5 to 50 μm in diameter. Sieving of droplets using conventional cell strainers allows for removal of large droplets, increasing the homogeneity of droplet dimensions in a simple and controlled manner. This method was used for the
Conflict of interest
The authors declare no competing financial interest.
Acknowledgements
Authors acknowledge the RMIT Micro Nano Research Facilities (MNRF) for fabrication of the sponge, and the RMIT Microscopy and Microanalysis Facility (RMMF) for scanning electron microscopy characterisation of PDMS sponges. S.B. Acknowledges Australian Research Council for funding under discovery for early career researcher award, DECRA DE170100239. K.K. acknowledges Australian Research Council for funding under discovery projects DP170102138 and DP180102049.
Dr. Peter Thurgood received his PhD in Biomedical Engineering from RMIT University, Australia in 2018. His research interests include self-sufficient microfluidic systems for various biomedical applications.
References (54)
- et al.
Immunology on chip: promises and opportunities
Biotechnol. Adv.
(2014) - et al.
Droplet-based microfluidic platforms for single T cell secretion analysis of IL-10 cytokine
Biosens. Bioelectron.
(2011) - et al.
An integrated gas-liquid droplet microfluidic platform for digital sampling and detection of airborne targets
Sens. Actuators B Chem.
(2018) - et al.
High-throughput production of satellite-free droplets through a parallelized microfluidic deterministic lateral displacement device
Sens. Actuators B Chem.
(2018) - et al.
A “cleanroom-free” and scalable manufacturing technology for the microfluidic generation of lipid-stabilized droplets and cell-sized multisomes
Sens. Actuators B Chem.
(2018) - et al.
Detecting enzymatic reactions in penicillinase via liquid crystal microdroplet-based pH sensor
Sens. Actuators B Chem.
(2018) - et al.
Generating ultra-small droplets based on a double-orifice technique
Sens. Actuators B Chem.
(2018) - et al.
Surface tension- and buoyancy-driven flows across horizontally propagating chemical fronts
Adv. Colloid Interface Sci.
(2018) Instability of a meniscus due to surface tension gradient-driven flow
J. Colloid Interface Sci.
(1999)- et al.
Gas diffusion and evaporation control using EWOD actuation of ionic liquid microdroplets for gas sensing applications
Sens. Actuators B Chem.
(2018)
Cells on chips
Nature
The present and future role of microfluidics in biomedical research
Nature
Microfluidic Platforms for the Investigation of Intercellular Signalling Mechanisms
Small
Microdroplets: a sea of applications?
Lab Chip
Droplet microfluidic technology for single-cell high-throughput screening
Proc. Natl. Acad. Sci.
Quantitative tracking of the growth of individual algal cells in microdroplet compartments
Integr. Biol.
Fluorescence-activated droplet sorting (FADS): efficient microfluidic cell sorting based on enzymatic activity
Lab Chip
Probing cellular heterogeneity in cytokine-secreting immune cells using droplet-based microfluidics
Lab Chip
Single-cell barcoding and sequencing using droplet microfluidics
Nat. Protoc.
Passive and active droplet generation with microfluidics: a review
Lab Chip
Formation of dispersions using “flow focusing” in microchannels
Appl. Phys. Lett.
Formation of droplets and bubbles in a microfluidic T-junction-scaling and mechanism of break-up
Lab Chip
Emerging droplet microfluidics
Chem. Rev.
Droplet microfluidics
Lab Chip
Self-contained microfluidic systems: a review
Lab Chip
Piezoresistive sensor with high elasticity based on 3D hybrid network of sponge@CNTs@Ag NPs
ACS Appl. Mater. Interfaces
Channel crack-designed Gold@PU sponge for highly elastic piezoresistive sensor with excellent detectability
ACS Appl. Mater. Interfaces
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Dr. Peter Thurgood received his PhD in Biomedical Engineering from RMIT University, Australia in 2018. His research interests include self-sufficient microfluidic systems for various biomedical applications.
Dr. Sara Baratchi received her PhD in Cell Biology from Deakin University, Australia in 2011. She is the named author of 38 journal publications, and the recipient of several awards, including the 2017–2019 Discovery for Early Career Researchers Award (DECRA) from the Australian Research Council. Her research interests include studying the mechanotransduction of blood flow in endothelial cells and leukocytes utilising innovative microfluidic platforms and different imaging techniques.
Mr. Crispin Szydzik is currently a PhD student at RMIT University’s School of Engineering. His research interests include microfluidic systems for biomedical applications.
Mr. Jiu Yang Zhu is currently a PhD student at RMIT University’s School of Engineering. His research interests include microfluidics and liquid metal enabled systems.
Prof. Saeid Nahavandi received his PhD from Durham University, UK. He is an Alfred Deakin Professor, Chair of Engineering, and the Director of the Institute for Intelligent Systems Research & Innovation (IISRI) at Deakin University, Australia. His research interests include modeling of complex systems, robotics and haptics.
Prof. Arnan Mitchell received his PhD from RMIT University, Australia in 1999. He is highly multidisciplinary, spanning integrated photonics, functional and solar materials, microsystems, and lab-on-a-chip technology. He creates technology platforms to enable fundamental scientific and biomedical research, and is committed to industrial translation with patents and industry projects in defence, communications and biomedical diagnostics.
Dr. Khashayar Khoshmanesh received his PhD in Biomechanical Engineering from Deakin University, Australia in 2010. He is the named author of 85 journal papers, and the recipient of several grants, and awards, including the Discovery for Early Career Researcher Award (DECRA) and two Discovery Grants from the Australian Research Council. He is currently a Senior Research Fellow at RMIT University's School of Engineering. His research interests include microfluidic platforms for various cellular assays, self-sufficient microfluidic technologies, and liquid metal enabled miniaturised systems.