Chemical transfection of cells in picoliter aqueous droplets in fluorocarbon oil.
ABSTRACT The manipulation of cells inside water-in-oil droplets is essential for high-throughput screening of cell-based assays using droplet microfluidics. Cell transfection inside droplets is a critical step involved in functional genomics studies that examine in situ functions of genes using the droplet platform. Conventional water-in-hydrocarbon oil droplets are not compatible with chemical transfection due to its damage to cell viability and extraction of organic transfection reagents from the aqueous phase. In this work, we studied chemical transfection of cells encapsulated in picoliter droplets in fluorocarbon oil. The use of fluorocarbon oil permitted high cell viability and little loss of the transfection reagent into the oil phase. We varied the incubation time inside droplets, the DNA concentration, and the droplet size. After optimization, we were able to achieve similar transfection efficiency in droplets to that in the bulk solution. Interestingly, the transfection efficiency increased with smaller droplets, suggesting effects from either the microscale confinement or the surface-to-volume ratio.
- SourceAvailable from: Yi-Ping Ho[Show abstract] [Hide abstract]
ABSTRACT: An attractive option for tissue engineering is to use of multicellular spheroids as microtissues, particularly with stem cell spheroids. Conventional approaches of fabricating spheroids suffer from low throughput and polydispersity in size, and fail to supplement cues from extracellular matrix (ECM) for enhanced differentiation. In this study, we report the application of microfluidics-generated water-in-oil-in-water (w/o/w) double-emulsion (DE) droplets as pico-liter sized bioreactor for rapid cell assembly and well-controlled microenvironment for spheroid culture. Cells aggregated to form size-controllable (30-80 μm) spheroids in DE droplets within 150 min and could be retrieved via a droplet-releasing agent. Moreover, precursor hydrogel solution can be adopted as the inner phase to produce spheroid-encapsulated microgels after spheroid formation. As an example, the encapsulation of human mesenchymal stem cells (hMSC) spheroids in alginate and alginate-arginine-glycine-aspartic acid (-RGD) microgel was demonstrated, with enhanced osteogenic differentiation further exhibited in the latter case.Scientific Reports 01/2013; 3:3462. · 5.08 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: A novel coaxial electrospray technology is developed to generate microcapsules with a hydrogel shell of alginate and an aqueous liquid core of living cells using two aqueous fluids in one step. Approximately 50 murine embryonic stem (ES) cells encapsulated in the core with high viability (92.3 ± 2.9%) can proliferate to form a single ES cell aggregate of 128.9 ± 17.4 μm in each microcapsule within 7 days. Quantitative analyses of gene and protein expression indicate that ES cells cultured in the miniaturized 3D liquid core of the core-shell microcapsules have significantly higher pluripotency on average than the cells cultured on the 2D substrate or in the conventional 3D alginate hydrogel microbeads without a core-shell architecture. The higher pluripotency is further suggested by their significantly higher capability of differentiation into beating cardiomyocytes and higher expression of cardiomyocyte specific gene markers on average after directed differentiation under the same conditions. Considering its wide availability, easiness to set up and operate, reusability, and high production rate, the novel coaxial electrospray technology together with the microcapsule system is of importance for mass production of ES cells with high pluripotency to facilitate translation of the emerging pluripotent stem cell-based regenerative medicine into the clinic.Integrative Biology 07/2014; · 4.32 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: We present a droplet-based microfluidics protocol for high-throughput analysis and sorting of single cells. Compartmentalization of single cells in droplets enables the analysis of proteins released from or secreted by cells, thereby overcoming one of the major limitations of traditional flow cytometry and fluorescence-activated cell sorting. As an example of this approach, we detail a binding assay for detecting antibodies secreted from single mouse hybridoma cells. Secreted antibodies are detected after only 15 min by co-compartmentalizing single mouse hybridoma cells, a fluorescent probe and single beads coated with anti-mouse IgG antibodies in 50-pl droplets. The beads capture the secreted antibodies and, when the captured antibodies bind to the probe, the fluorescence becomes localized on the beads, generating a clearly distinguishable fluorescence signal that enables droplet sorting at ∼200 Hz as well as cell enrichment. The microfluidic system described is easily adapted for screening other intracellular, cell-surface or secreted proteins and for quantifying catalytic or regulatory activities. In order to screen ∼1 million cells, the microfluidic operations require 2-6 h; the entire process, including preparation of microfluidic devices and mammalian cells, requires 5-7 d.Nature Protocol 04/2013; 8(5):870-91. · 8.36 Impact Factor
Published:October 03, 2011
r2011 American Chemical Society
dx.doi.org/10.1021/ac2022794|Anal. Chem. 2011, 83, 8816–8820
Chemical Transfection of Cells in Picoliter Aqueous Droplets in
Fangyuan Chen,†,‡Yihong Zhan,§Tao Geng,§Hongzhen Lian,†Peisheng Xu,zand Chang Lu*,‡
†Department of Chemistry, Nanjing University, Nanjing 210093, P.R. China
‡Department of Chemical Engineering, Virginia Tech, Blacksburg, Virginia 24061, United States
§Department of Agricultural and Biological Engineering, Purdue University, West Lafayette, Indiana 47907, United States
S Supporting Information
with water-in-oil droplets generated at rates of hundreds to
thousands per second provides a high-throughput platform for
chemical and biological screenings.6,7The tiny volume of these
droplets (in the range of pico- to nanoliters) creates confined
microscale compartments for observation of unique biology,8
screening optimal conditions,9,10and analysis of chemical/bio-
logical molecules of extremely low quantities.11?15The manip-
ulation and analysis of cells in droplets is also an intensively
studied field.16The encapsulation of individual cells is often
low density with the eventual cell occupancy following Poisson
statistics.18Interesting efforts have been made on the use of cell
alignment by hydrodynamics for high-efficiency single cell
encapsulation.19Furthermore, in contrast to mineral oil and
other hydrocarbon oils used in early reports, the newly demon-
strated fluorocarbon oil/surfactant system proved to be highly
compatible with in-droplet cell survival and growth due to its gas
permeability.18,20,21All these works paved the way to wide
application of droplet microfluidics to cell manipulation and
Delivery of genes into cells is an important first step for
studying the functions of genes. Functional genomic studies
often demand systematically expressing or silencing genes
ecent years have witnessed explosion of interests in droplet-
based microfluidic technology.1?5Droplet microfluidics
corresponding to a large fraction of the genome, and such
screening allows the identification of genes that are required
cells guarantee that the proteins are synthesized in situ with
proper post-translational protein folding and glycosylation. Drop-
let microfluidics has been applied to produce high-quality
cationic lipid/DNA complexes used in gene delivery (with cell
transfection conducted in bulk medium).22,23However, there
have been very few reports on gene delivery or cell transfection
inside droplets. Chemical transfection has been demonstrated in
fairly large droplets (∼150 nL) on a digital microfluidic platform
that does not require interface between the oil and aqueous
phases.24We used electroporation to deliver genes into cells
encapsulated in aqueous droplets in hydrocarbon oil.25Cells
were released from the droplets and transferred to bulk media
immediately after electroporation. In comparison, chemical
transfection requires long-time incubation (several hours) in
droplets. Such operation is not practical using hydrocarbon oil
because its low permeability to gases and strong extraction of
organic transfection reagents would lead to massive cell death
and very low transfection.
August 28, 2011
October 3, 2011
ABSTRACT: The manipulation of cells inside water-in-oil droplets is
essential for high-throughput screening of cell-based assays using droplet
microfluidics. Cell transfection inside droplets is a critical step involved in
functional genomics studies that examine in situ functions of genes using the
droplet platform. Conventional water-in-hydrocarbon oil droplets are not
compatible with chemical transfection due to its damage to cell viability and
extraction of organic transfection reagents from the aqueous phase. In this
work, we studied chemical transfection of cells encapsulated in picoliter
droplets in fluorocarbon oil. The use of fluorocarbon oil permitted high cell
the incubation time insidedroplets, the DNA concentration, and the droplet
size. After optimization, we were able to achieve similar transfection efficiency in droplets to that in the bulk solution. Interestingly,
the transfection efficiency increased withsmaller droplets, suggestingeffects fromeither the microscaleconfinementorthe surface-
dx.doi.org/10.1021/ac2022794 |Anal. Chem. 2011, 83, 8816–8820
In this report, we investigated chemical transfection of cells in
picoliter aqueous droplets in fluorocarbon oil. Droplets encapsulat-
at a frequency of several thousand Hz by flow focusing. We have
examined the effects of various parameters such as the incubation
the cell viability and transfection efficiency. The fluorocarbon oil/
in the droplets). Overall, the transfection efficiency in droplets was
similar to that in bulk medium. Interestingly, we found that the
transfection efficiency increased with smaller droplet size.
Device Fabrication and Operation. The microfluidic chip
was fabricated in polydimethylsiloxane (PDMS) (GE Silicones
RTV 615, MG Chemicals) using soft lithography.26SU-8 2025
for 30 s to create a 52 μm thick layer on a 3 in. silicon wafer. The
photoresist was then exposed to UV light and developed to
produce the SU-8/silicon wafer master. Prepolymer PDMS was
poured onto the master at a 10:1 ratio, degassed, and cured in an
oven at 80 ?C for 1 h. The PDMS piece was then cut and peeled
from the master, and access holes for inlets and outlets were
punched with a flat needle. Glass slides were cleaned in a basic
solution (H2O/30% NH4OH/27% H2O2= 5:1:1, v/v) at 75 ?C
for 3 h before they were rinsed by DI water and blown dry.
Precleaned glass slide and PDMS were treated in a plasma
cleaner (Harrick) and then brought into contact to form
irreversible bonding. The device was baked at 80 ?C for 1 h for
further strengthening of the bonding. Before use, Aquapel (PPG
Industries) was used to coat the channel before it was blown out
of the channel by air. Aquapel treatment was important for
generating a continuous oil phase that wet the surface. Water-in-
FC-40 (3M) containing 5.0% (w/w) perfluorinated polyether
polyethylene glycol (PFPE-PEG) block-copolymer surfactant
(synthesis described below) together with culture medium con-
taining cells, DNA, and transfection reagent (PolyFect). The oil
was prefiltered by a 0.2 μm filter (VWR). Flows were driven at
constant volumetric flow rates by two syringe pumps (Fusion
acid functionality) was purchased from Dupont. HFE-7100
(>99.5% methoxy-nonafluorobutane) was purchased from 3M.
further purification. The triblock copolymer PEG-PFPE-PEG
was synthesized according to a method (Scheme 1) modified
from the literature.27Briefly, Krytox 157FSH was reacted with
thionyl chloride at the molar ratio of 1:10 under reflux overnight
with nitrogen purge. The excess thionyl chloride and solvent
were removed by rotary evaporation. The resulting intermediate
was dissolved in the mixed solvent of HFE-7100 and benzotri-
MW: 400 Da) solution under stirring with the existence of
triethylamine (TEA). The reaction was kept under reflux for
24 h. The reaction product was purified by filtration and washed
with chloroform and DI water twice. The final product, PEG-
PFPE-PEG, was dried under high vacuum.
Plasmid Preparation. We used pEGFP-C1 plasmid (4.7 Kb,
a model vector for observing transfection.28The plasmid was
propagated in E.coli cells and purifiedusing QIAfilter Plasmid Giga
plasmid DNA was dissolved in Tris-EDTA buffer and stored at
?20 ?C. The DNA concentration was detected by UV absorbance
at 260 nm. The OD 260/280 nm ratio was between 1.8 and 2.0.
cells were cultured in F-12 Nutrient medium (Fisher Scientific)
supplemented with 10% (v/v) fetal bovine serum (Sigma) and
100 mg/mL streptomycin (Sigma) at 37 ?C with 5% CO2. Cells
were subcultured every 2 days at a ratio of 1:10. PolyFect
(a cationic dendrimer from Qiagen) was used to deliver plasmid
DNA into cells. PolyFect dissolved in the culture medium with
no serum was mixed with DNA solution at a ratio suggested by
the manufacturer’s protocol. The PolyFect/DNA mixture was
incubated for 5 min at room temperature to allow PolyFect/DNA
complex to form. In the droplet transfection experiments, we mixed
50 μL of newly formed PolyFect/DNA complexes with 150 μL of
CHO cell sample (107cells/mL). Such a mixture was immediately
Scheme 1. Synthesis of PEG-PFPE-PEG Triblock Copolymer Surfactant
dx.doi.org/10.1021/ac2022794 |Anal. Chem. 2011, 83, 8816–8820
loaded into a syringe and used in the droplet production by flow
the droplet layer to prevent evaporation and coalescence during
incubation in a cell incubator (37 ?C, 5% CO2). After incubation of
a certain time, 400 μL of culture medium was added to the emulsion
a CCD camera (ORCA-R2, Hamamatsu) for examination of the
transfection. Cell viability after droplet transfection was measured
upper solution was aspirated, and PBS was added to rinse cells. Cells
we examined the PI exclusion by fluorescence and phase contrast
imaging. The transfection efficiency (defined by the percentage of
transfection, 150 μL of cell sample (107cells/mL) was mixed with
50 μLof PolyFect/DNA complexes and culturedinthe 96wellplate
’RESULTS AND DISCUSSION
Figure 1 shows the device design for generating aqueous
droplets in fluorocarbon oil by flow focusing.29The aqueous
phase (containing DNA, the transfection reagent PolyFect, and
cells) came into the device from the center stream while
fluorocarbon oil (FC-40) containing a perfluorinated polyether
polyethylene glycol (PFPE-PEG) block-copolymer surfactant20
(5%, w/w) came in from the two side streams. PolyFect (from
Qiagen) is a cationic activated dendrimer that forms a complex
mixing of DNA, PolyFect, and cells was conducted immediately
before the droplet production in order to make sure that no
transfection occurredinthebulk solution. The PFPE-PEG block
copolymer was synthesized following a published protocol with
minor modifications(detailed in the Experimental Section).27In
our experiments, the droplet size was varied in the range of 44 to
26 μm in the diameter by maintaining the flow rate of the aqueous
phase at 16 μL/min and varying the oil phase flow between 64
and 150 μL/min (shown in Supporting Information Figure S1).
Figure 1. Layout of the microfluidic device for generating microscale
droplets that encapsulate cells, DNA, and transfection reagent. The
droplets of 44 μm diameter were generated at a frequency of ∼6000 Hz
by having a flow rate of 16 μL/min for the aqueous phase from the
central channel anda total flow rate of64 μL/min forthe oilphase from
the two side channels.
for various times up to 6 h.
dx.doi.org/10.1021/ac2022794 |Anal. Chem. 2011, 83, 8816–8820
The cell concentration in the aqueous phase was ∼7.5 ? 106
cells/mL. When the droplet diameter was ∼44 μm, this cell
the droplets and double occupancy in ∼4% of the droplets, with
fixed cell concentration, when the droplet size decreased, the
droplet occupancy of cells became lower.
Figure 2 shows that the droplets were very stable during the
first3 hof incubation (37 ?C, 5% CO2in acell incubator). Some
level of coalesce was observed around 4 h. More unevenness in
the droplet size was exhibited at 5 and 6 h (11 and 22% RSD in
liquid among droplets. The evaporation from the droplets
diameter after 6 h (from 44 to 42 μm). We extracted cells from
droplets at various times during incubation to examine their
viability and transfection. Encapsulated cells were transferred
from droplets into bulk culture medium for viability examination
(tested immediately after the incubation in the droplets) and
transfection efficiency determination (conducted after 40 h of
bulk culture medium effectively terminated transfection when
and permits high cell viability after incubation in the droplets.
longer incubation. The transfection efficiency increased with
longer incubation time in the droplets but such increase pla-
teaued after 3 h (Figure 3B). We also varied the DNA concen-
tration between 1.1 and 3.0 μg/mL (while keeping PolyFect
amount proportional to that of DNA) (Figure 4). We tested the
transfection both in droplets (Figure 4A) and in bulk culture
medium (Figure 4B). In both cases, higher DNA concentration
improved the transfection efficiency until it reached a plateau.
efficient than in droplets (∼44 μm diameter) at low concentra-
tions (<1.9 μg/mL) but such a difference largely diminished in
the high DNA concentration range. This suggests that there was
minor loss in either the DNA or PolyFect amount due to the
interaction with oil phase in the case of droplet transfection. In
practice, this apparently can be compensated by having high
initial DNA and PolyFect concentrations. The use of fluorocarbon
Figure 3. Variation of the cell viability (A) and transfection efficiency
contained DNA of 2.3 μg/mL, associated PolyFect and cells. The
viability was measured immediately after cell release from droplets by
PI exclusion. The transfection efficiency was determined by the percen-
tage of fluorescent cells among live cells, examined at ∼40 h after cells
were released from the droplets by vortex.
Figure 4. Comparison of transfection efficiency in droplets and in well
tion after a 6 h incubation in droplets of 44 μm diameter. Cells were
medium for a 40 h additional culture before the transfection efficiency
was determined. (B) Transfection in 96 well plates by incubating cells
with DNA and PolyFect for 6 h. The cells were then washed to remove
uninternalized DNA and cultured for an additional 40 h before the
transfection efficiency was determined.
dx.doi.org/10.1021/ac2022794 |Anal. Chem. 2011, 83, 8816–8820
oil is important here because it has much lower solubility for
organic molecules such as PolyFect than hydrocarbon oils.
Overall, the cell transfection in droplets yielded very comparable
results as in bulk solution. Images of transfected cells are shown
in Supporting Information Figure S2.
Interestingly, we observed a clear trend that the transfection
efficiency in droplets was higher in smaller droplets. As shown in
Figure 5, there was a consistent increase from 21% to 25% when
the droplet diameter decreased from 44 to 26 μm. The reasons
for such a trend are not entirely clear. Each droplet contained at
least several thousands of plasmid DNA molecules that formed
DNA/PolyFect complexes with their sizes in the range of tens to
hundreds of nanometers.30It is possible that the transfection
benefits from the microscale confinement provided by the
droplets via having higher probability for cell/complexes inter-
action in smaller droplets. Alternatively, surface-to-volume ratio
may play a role in the transfection so that a higher surface-to-
volume ratio in small droplets enhances transfection. The exact
mechanism requires further investigation to illustrate.
in text. This material is available free of charge via the Internet at
This work was supported by National Science Foundation
grants CBET 1016547 and CBET 0967069 and United States
(to C.L.), the Promising Investigator Research Award (PIRA)
from the Office of Research and Graduate Education of USC (to
P.X.), National Natural Science Foundation of China (90913012),
National Basic Research Program of China (973 program,
2011CB911003), and National Natural Science Funds for Crea-
tive Research Groups (20821063) (to H.L.).
(2) Huebner, A.; Sharma, S.; Srisa-Art, M.; Hollfelder, F.; Edel, J. B.;
Demello, A. J. Lab Chip 2008, 8, 1244.
(3) Chiu, D. T.; Lorenz, R. M. Acc. Chem. Res. 2009, 42, 649.
(4) Vyawahare, S.; Griffiths, A. D.; Merten, C. A. Chem. Biol. 2010,
(5) Kintses, B.; van Vliet, L. D.; Devenish, S. R.; Hollfelder, F. Curr.
Opin. Chem. Biol. 2010, 14, 548.
(6) Agresti, J. J.; Antipov, E.; Abate, A. R.; Ahn, K.; Rowat, A. C.;
Baret, J. C.; Marquez, M.; Klibanov, A. M.; Griffiths, A. D.; Weitz, D. A.
Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 4004.
(7) Li, L.; Mustafi, D.; Fu, Q.; Tereshko, V.; Chen, D. L.; Tice, J. D.;
Ismagilov, R. F. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 19243.
(8) Boedicker, J. Q.; Vincent, M. E.; Ismagilov, R. F. Angew. Chem.,
Int. Ed. 2009, 48, 5908.
(9) Zheng, B.; Roach, L. S.; Ismagilov, R. F. J. Am. Chem. Soc. 2003,
(10) Jambovane, S.; Kim, D. J.; Duin, E. C.; Kim, S. K.; Hong, J. W.
Anal. Chem. 2011, 83, 3358.
(11) He, M.Y.; Edgar, J.S.; Jeffries, G.D. M.; Lorenz, R. M.;Shelby,
J. P.; Chiu, D. T. Anal. Chem. 2005, 77, 1539.
David, P. H.; Kotsopoulos, S. K.; Samuels, M. L.; Hutchison, J. B.;
Larson, J. W.; Topol, E. J.; Weiner, M. P.; Harismendy, O.; Olson, J.;
Link, D. R.; Frazer, K. A. Nat. Biotechnol. 2009, 27, 1025.
(13) Han, Z.; Li, W.; Huang, Y.; Zheng, B. Anal. Chem.2009, 81, 5840.
Chem. 2010, 82, 3183.
(16) Zagnoni, M.; Cooper, J. M. Methods Cell Biol. 2011, 102, 23.
(17) Vijayakumar, K.; Gulati, S.; deMello, A. J.; Edel, J. B. Chem. Sci.
2010, 1, 447.
(18) Koster, S.; Angile, F. E.; Duan, H.; Agresti, J. J.; Wintner, A.;
Schmitz, C.; Rowat, A. C.; Merten, C. A.; Pisignano, D.; Griffiths, A. D.;
Weitz, D. A. Lab Chip 2008, 8, 1110.
(19) Edd, J. F.; Di Carlo, D.; Humphry, K. J.; Koster, S.; Irimia, D.;
Weitz, D. A.; Toner, M. Lab Chip 2008, 8, 1262.
(20) Holtze, C.; Rowat, A. C.; Agresti, J. J.; Hutchison, J. B.; Angile,
Johnson, J. S.; Pisignano, D.; Weitz, D. A. Lab Chip 2008, 8, 1632.
(21) Clausell-Tormos, J.; Lieber, D.; Baret, J. C.; El-Harrak, A.;
Miller, O. J.; Frenz, L.; Blouwolff, J.; Humphry, K. J.; Koster, S.; Duan,
H.; Holtze, C.; Weitz, D. A.; Griffiths, A. D.; Merten, C. A. Chem. Biol.
2008, 15, 427.
(22) Hsieh, A. T.; Hori, N.; Massoudi, R.; Pan, P. J.; Sasaki, H.; Lin,
Y. A.; Lee, A. P. Lab Chip 2009, 9, 2638.
(23) Lee, G. B.; Wu, H. W.; Huang, Y. C.; Wu, C. L. Microfluid.
Nanofluid. 2009, 7, 45.
(24) Barbulovic-Nad, I.; Au, S. H.; Wheeler, A. R. Lab Chip 2010,
(25) Zhan, Y.; Wang, J.; Bao, N.; Lu, C. Anal. Chem. 2009, 81, 2027.
(26) Duffy, D. C.; McDonald, J. C.; Schueller, O. J. A.; Whitesides,
G. M. Anal. Chem. 1998, 70, 4974.
(27) Fabiilli, M. L.; Lee, J. A.; Kripfgans, O. D.; Carson, P. L.;
Fowlkes, J. B. Pharm. Res. 2010, 27, 2753.
(28) Geng, T.; Zhan, Y. H.; Wang, H. Y.; Witting, S. R.; Cornetta,
K. G.; Lu, C. J. Controlled Release 2010, 144, 91.
(29) Tan, Y. C.; Hettiarachchi, K.; Siu, M.; Pan, Y. R.; Lee, A. P.
J. Am. Chem. Soc. 2006, 128, 5656.
1996, 7, 703.
Figure 5. Transfection efficiency in droplets of various sizes (26?49 μm
in the diameter). Droplets of different sizes were generated by varying
the flow rates of the oil and aqueous streams. In all cases, the droplets
contained DNA of 2.3 μg/mL and the incubation time in these droplets
was 6 h. The cells were released from droplets after the incubation and
the transfection efficiency was determined after 40 h of additional