ALGINATE MICROBEADSPRODUCTIONINTRODUCTION
Microencapsulation has been one of the most interesting fields in the area of pharmaceuticaltechnology since its inception many years ago.
Microencapsulation products (microparticles, microbeads) are small entities that contain an active
agent or core material surrounded by a shell or embedded into a matrix structure.[1]Among all these materials, alginate is a good candidate for many applications. Alginate spheres
are one of the most widely investigated cell encapsulation materials as they are biocompatible,
nontoxic, biodegradable, and relatively inexpensive.[2]Their structural similarity to the extracellular matrices of living tissues allows for applications inwound healing, delivery of bioactive agents such as small chemical drugs and proteins, and celltransplantation.[3]Alginate microbeads are widely used in pharmaceutical research, tissue engineering, andregenerative medicine.[4]Although the production of alginate microbeads appears to be a successful drug delivery system, it
is often hindered by technical challenges, mainly associated with high viscosity of polymer fluids.[3]Many methods are used for encapsulation of agents (bacteria, pancreatic islets, API) in
microbeads, such as extrusion, batch method, spray drying…
The most widespread method for producing alginate microbeads is extrusion, where a solution ofalginate is injected through a needle into a calcium bath to produce ionically crosslinked beads.
This easy to use method allows one to produce alginate
microbeads which have a wide size distribution,
without possible automation and no control over thebead size. This is a big limitation because in microbead
production, particle size is key parameter.
Size is directly correlated to the application, and differs
depending on the product requirements. For instance,
in food, microgels with micrometer sizes are preferredin order to minimize the sensory perception of powdery
or graininess in food such as yoghurt and ice cream [5]1Figure 1: Extrusion method for
Alginate microbeads productionAPPLICATION NOTE
In biomedical applications, drug delivery through submicron nanogels allows drugs to betargeted at specific sites in the body. Nanogels (100–200 nm) used in cancer therapy experimentstend to preferentially accumulate in a number of cancerous tumors [6] [7]As the reproducibility and throughput of such methods is low, there is demand for better
techniques and control of the process to produce alginate microbeads.
Droplet-based microfluidics, offers an efficient method for improvement of this process. It is a
powerful tool which enables the production of micrometric size monodispersed droplets.

Figure 2: Fluigent microfluidic
method for emulsion production

Following is a method for encapsulation of reagent into alginate microbeads with total controlof bead formation.
Droplet-based microfluidic method is used to precisely control the production of microbeads
without the drawbacks of large size distribution that present other methods.
Microfluidics allows one to have better control over the process of production and encapsulation
and reach higher monodispersity than other methods such as extrusion methods, batch...

EXTRUSIONMETHODFLUIGENTMICROFLUIDIC METHOD

Particle size distribution

Up to 50%~2%

Reproducibility

LowHigh

Live particle size control

NoPreciseContinuous (in line)
production

NoYesFigure 3: Comparison between extrusion
method and Fluigent microfluidic method2

ALGINATE MICROBEADS PRODUCTION
MATERIALS AND METHODS
Materials
Reagents:» Dispersed phase: Alginate (Sigma Aldrich) and Calcium EDTA disodium solution (SigmaAldrich). The solution is composed of 1% Alginate, 100mM solution of calcium EDTA
together dissolved in Water and then filtered using syringe filter at 0.2µm.
» Priming and cleaning phase: Distilled water filtered using syringe filter at 0.2µm.
» Continuous phase: 2% dSURF solution. dSurf solution is composed of Fluigent surfactant
at 2% dissolved in HFE 7500 fluorinated oil filtered using syringe filter at 0.2µm.
»

Collection sample: 2% dSURF (Fluigent) with 0.05% v/v acetic acid (Sigma Aldrich)» Emulsion breaker: Perfluoroctanol (PFO) solution (Sigma Aldrich) (20% v/v in HFE 7500
fluorinated oil)
Products/Instrument:» Microfluidic flow controller: The Flow EZ is the most advanced flow controller for pressurebased fluid control. It can be combined with a Flow Unit to control pressure or flow rate.
It can be used without a PC. Two Flow EZ with 2 bar of full scale pressure are used in thesetup presented here.
» Flow sensor: The Flow Unit is a flow sensor that allows real time flow rate measurement.
By combining a Flow Unit with the Flow EZ, it is possible to switch from pressure control
to flow rate control, allowing for the generation of highly monodispersed droplets over along period of time. Two Flow Units M are used here to monitor and control the flow ratesof the dispersed and continuous phase during the run.
» 2-Switch: Easy to use 3-port/2-way microfluidic valve is used to switch between water andalginate solution. This allows to have better reproducibility by priming the system and add
cleaning step during the experiment.
» Droplet generator: The RayDrop droplet and emulsion generator is used to control thegeneration of alginate droplet. The RayDrop is based on the alignment of two capillaries
immersed in a pressurized chamber containing the continuous phase. The dispersed
phase exits one of the capillaries through a 3D-printed nozzle, placed in front of theextraction capillary for collecting the droplets. This non-embedded implementation ofan axisymmetric flow-focusing is referred to co-flow-focusing. The advantage lies in hisgeometry which is leading the droplet formation and then remove all wettability issues that
could appear in other microfluidic chip. This allows one to generate highly monodispersed
emulsions with many fluids, including alginate.

3Setu fmicrobeads productionAPPLICATION NOTE
Methods
Solution preparation:» Dispersed phase: Make a fresh solution of 1% alginate and 0.1 M Calcium EDTA dissolved
in distilled water:
1. Alginate powder was dissolved in distilled water in order to make a solution of 2%(w/v) alginate.
2. Calcium EDTA was dissolved in distilled water at 0.1M.
3. The two solution were then mixed together to make a fresh solution of 1% alginate
and 0.05M calcium EDTA.
4. To allow for good reproducibility during droplet generation process, the solution
has been filtered with using syringe filter at 0.2µm.
»Continuous phase: Use the 2% dSURF solution. Again, make sure to filter the solution toavoid any issues during droplet generation process.

»Collection solution: The collection solution is composed of 0.05 % (v/v) acetic acid in thecontinuous phase (2% dSURF)»Emulsion breaker: The emulsion breaking solution is composed of Perfluoroctanol (PFO)
solution (Sigma Aldrich) dissolved at 20% v/v in dSURF or HFE 7500 fluorinated oil

Figure 5: Scheme of the setup for Alginate microbeads production

Droplet generation process:
The droplet generation set-up is illustrated in the previous picture. For droplet generation
process we used the RayDrop droplet generator. 2 Flow EZ were connected to alginate solution,
pure water as dispersed phase and dSurf as continuous phase.

4

ALGINATE MICROBEADS PRODUCTION
1. First, water is injected as dispersed phase and dSurf as continuous phase to prime thesystem and generate water in oil droplets. This step allows for the removal of all air bubbles
present in the system (tubing, connectors…) until a steady state is reached.
2. As soon as a steady state of droplet generation is reached, Alginate solution can be easily
injected by switching the first 2-switch to the 2nd position. The pressure may be adjusted
to maintain the proper flow rate with the alginate solution.
3. Alginate droplet should appear in less than 1 min.
4. Pressure and/ or Flow rate can be then adjusted to target the desired droplet size
For more information on droplet generation check the Good practice guide for more detail on
droplet generation process with the Raydrop.
Microbeads Precipitation:
Once droplets are generated, they are recovered and collected into a reservoir.
This step will generate polymerization process and beads will be recovered. By decreasing the pHof the alginate droplet using acetic acid in the collection reservoir, the calcium-EDTA dissociates
and releases CA2+ ions which allows for crosslinking of alginate chains.

Figure 6: Description of alginate crosslinking mechanism

Droplet monitoring and Analysis:
To observe droplet generation an Inverted microscope (Leica DM IRB) coupled with anIDS UI-3070CP camera have been used. For continuous droplet visualization and measurement,
the software uEye Cockpit from IDS has been used. To estimate droplet frequency the following
formula has been used.
Droplet rate (Hz) = Droplet phase flow rate (µL/s) / Droplet volume (µL).
Bead recovery:
After precipitation, the beads are in suspension in the collection vial (dSurf + acetic acid). To
break the emulsion and recover the beads some steps must be followed:» Addition of PFO diluted at 20% in HFE to break the emulsion (approximately the same
volume as the recovered emulsion volume)» After this operation, all the drops collect in a sort of clump which at first gives the impressionthat everything has coalesced into a single large drop. On closer inspection we can seethat it is a clump but that there is no coalescence» Then remove the remaining oil/surfactant with a pipette. There is just the cluster of beads
in the bottom of the Falcon tube.
» Add PBS buffer to resuspend the beads (a dye in the buffer can be used at this step tohave better contrast between these two aqueous phases)

5

APPLICATION NOTERESULTS
Configuration 1: RayDrop: inlet nozzle = 30µm, outlet capillary= 150µm

Dispersed phase/ Droplet phase

6Continuousphase

Droplets
PictureP(mbar)Q(µL/min)Pmbar)

Q(µL/min)

Diameter(µm)

Rate(Hz)

312

0.5

1816

100

95

19319.5

11134.5

100

106

25

776

5309

25155

4213031034425plug

251303101001150

127

166

1699

151001183

1332171699

15160

1810

115320ALGINATE MICROBEADS PRODUCTION
After generation, droplets are recovered in the collection
reservoir where the crosslinking mechanism appears as
described in the method section. Microbeads were thenrecovered and analyzed for monodispersity measurement.
Using this setup, we were able to produce alginate beads
with a dispersion in beads size lower than 5%. (around 50
microbeads have been analysed for a CV of about 2%).
Figure 8: Pictures of Alginate beads in suspension in dSurf after crosslinking

Bead recovery:
To recover only the beads and put them insuspension into aqueous solution, the PFO solution
was added. After breaking the emulsion, beads
are resuspended in PBS solution. To have better
contrast and be able to observe alginate beads, wehave added here blue food dye in the PBS.

Figure 9: Alginate beads in suspension into PBS

In this configuration beads from 90µm to 150µm can be made.
However, RayDrop versatility and flexibility allows one to easily change configuration and change
capillary size. This allow to target other droplet/beads size using the RayDrop.
Configuration 2: RayDrop: Inlet nozzle = 30µm, outlet nozzle= 60µm
In this case we have changed the outlet capillary
to another nozzle of about 60µm ID. This increases
the shear stress to the dispersed phase and thenproduce smaller alginate droplet with higher
generation frequency.
With this specific configuration 70µm OD andesmaller alginate droplets were easily andsuccessfully generated. Flexibility in term of
droplet size is however limited (dripping regime
range is reduced and jetting comes quickly withthis specific configuration).
Figure 10: Alginate beads formation with nozzle of 60µm in outlet

7

APPLICATION NOTECONCLUSION
In this application note we have demonstrated that alginate beads
can be successfully produced using the RayDrop with precise controlof droplet size.
Microbeads of 95-160µm diameter were generated with standard
RayDrop configuration (Nozzle of 30µm and outlet capillary 150µm)
using alginate solution at 1% in water. Other concentrations of alginate such as 2%, which is widely used in biological applications,have also successfully tested by following the same process. In thislast case, higher internal diameter tubing has to be used to deal
with a more viscous solution.
As a consequence, this setup and protocol can be used for encapsulation of mammalian cells, bacteria and other reagents into alginate
beads.
We have successfully demonstrated that using Fluigent product allow to control precisely alginate beads formation with high monodispersity.
As presented, using the RayDrop with different nozzle size allow tohave better flexibility. Changing capillary size can be easily done by
the user to target different droplet sizes.

8ALGINATE MICROBEADS PRODUCTIONREFERENCES[1] Obeidat, W. M. (2009). Recent Patents Review in Microencapsulation of Pharmaceuticals
Using the Emulsion Solvent Removal Methods, 178–192.
[2] Andersen, Therese & Strand, Berit & Formo, K. & Alsberg, Eben & Christensen, B.E.. (2011).
Alginates as biomaterials in tissue engineering. Carbohydrate Chemistry. 37. 227-258.
10.1039/9781849732765-00227.
[3] Yong, K., & Mooney, D. J. (2012). Progress in Polymer Science Alginate : Properties andbiomedical applications. Progress in Polymer Science, 37(1), 106–126. https://doi.org/10.1016/j.
progpolymsci.2011.06.003[4] Applications, M. (2013). Alginate-Based Biomaterials for Regenerative Medicine
Applications, 1285–1297. https://doi.org/10.3390/ma6041285[5] Taylor, P., Heidebach, T., Först, P., Kulozik, U., Heidebach, T., & Orst, P. F. (2012).
Microencapsulation of Probiotic Cells for Food Applications Microencapsulation ofProbiotic Cells, (June 2013), 37–41. https://doi.org/10.1080/10408398.2010.499801[6] Yih, T. C. (2006). Engineered Nanoparticles as Precise Drug Delivery Systems, 1190, 1184–1190. https://doi.org/10.1002/jcb.20796[7] Ching, S. H., Bansal, N., & Bhandari, B. (2017). Alginate gel particles – A review of production
techniques and physical properties. Critical Reviews in Food Science and Nutrition, 57(6),1133–1152. https://doi.org/10.1080/10408398.2014.965773

9For additional information, contact us by email : contact@fluigent.com or consult our website : www.fluigent.com

APPLICATION NOTEAPPENDIX
Alginate 1% flow sensor calibration

For better precision one flow unit M has been calibrated to have the exact flow rate of Alginate
solution during all experiment.
We calibrated the Alginate by weighting the volµme during a specific time. This experimentgave us the following equation:
Qreal= 1,1019*QFU-1,0461
The Flow unit have been placed in water calibration mode and thanks to the previous equation
we enter this equation in the AIO software to have the real Alginate flow rate.

Full Alginate phase diagram

APPLICATION NOTECONTACT⽇本代理店: 株式会社ASICON
Local distributor in Japan: ASICON Tokyo Ltd.
asicon-tokyo@asicon-tokyo.com
www.asicon-tokyo.com