Application note: cell encapsulation in small double emulsions

Microfluidic droplet generation is a powerful technique for encapsulating biological molecules or cells within precisely controlled nL- to pL-volumes. Microfluidic droplets have been used for a wide variety of applications, including directed evolution of enzymes and proteins, digital PCR, large-scale gene assembly, cell culture, and, recently, single-cell genomic, epigenomic, and transcriptomic analyses.

The need for cell encapsulation methods and sorting devices that are safer, more effective, and simpler to use than current technologies has grown due to the exponential growth of novel techniques of cell analysis.

To perform cell encapsulation, double emulsions are ideally generated because, unlike single emulsions, they provide aqueous compartments as well as an aqueous carrier fluid, which makes the emulsion compatible with most flow cytometry and cell sorting systems.

For successful sorting, DE droplets must be significantly smaller (< 60 µm in diameter) than commercial cell sorters nozzles (typically 70–130 μm in diameter) while simultaneously large enough to encapsulate variants of interest within the inner core volume. Therefore, the possibility of developing a cell encapsulation method in double emulsions small enough to be compatible with commercial cell sorters would be a major breakthrough in the field of biomedical research.

Typically, double emulsions are produced in batches by a two-step emulsification process, resulting in a highly polydisperse population with low encapsulation efficiency. Therefore, droplet generation using microfluidics is an alternative, as it offers maximum control over droplet generation.

With the RayDrop platform, we demonstrate an easy-to-use and robust cell encapsulation method for encapsulating large, complex cells within highly monodisperse DE droplets small enough (∼ 25µm to 60µm) for high-throughput cell screening/sorting. We demonstrate the capabilities of this method by encapsulating Human Adult Peripheral Blood Mononuclear Cells (PBMC) in highly monodisperse double emulsions of 50µm in size.

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Application note: bacteria and yeast encapsulation method in small double emulsions

In recent years, the microencapsulation of Bacteria and Yeast has expanded due to the advantages and novel information provided by these methods. In particular, droplet microfluidics has become a popular technique for bacterial and yeast encapsulation due to its performance, efficiency, and precision. This method has been used in various areas of microbiology, including pathogen detection and identification, antibiotic susceptibility testing, microbial physiology studies, and biotechnological applications.

Bacteria and Yeast encapsulation is a crucial technique in the investigation of microorganisms that play a vital role in the ecosystem and have a potent metabolic capacity for producing biopharmaceuticals and recombinant proteins.

To perform this encapsulation method, double water-in-oil-in-water (W/O/W) emulsions containing the microorganisms of interest are generated. Unlike single water-in-oil (W/O) emulsions, double emulsions provide an aqueous carrier fluid, making them compatible with most flow cytometry and cell sorting setups. They also protect sensitive biological structures from the sheath fluid, preserve the link between microorganisms and the substances they secrete, and allow for in vitro analysis of biomolecules.

Droplet-based microfluidic techniques, particularly for the microencapsulation of Bacteria and Yeast, offer superior control over droplet generation, producing highly stable and monodisperse double emulsions. To be compatible with cell sorting and analysis devices, the droplets must be small (< 60µm) in diameter and large enough to encapsulate the variants of interest.

Therefore, in this application note, we demonstrate an easy-to-use and reliable workflow for encapsulating the yeast strain S. Cerevisiae CEN.PK 113-7D and the bacterial strain L. cremoris MG1363_GFP into highly monodisperse double emulsions with a diameter of 42 μm, suitable for high-throughput screening and sorting. The workflow uses the Cell Encapsulation Platform, consisting of Fluigent’s fluid handling system and Secoya’s emulsification technology, the RayDrop®.

This application note was created in collaboration with Fluigent and TU Delft.

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Application note : A quick and efficient double emulsion generation method for flow cytometry droplet sorting

In this paper, we describe the encapsulation of a fluorescent strain of Escherichia coli bacteria in w/o/w double emulsions and flow cytometry droplet sorting using a flow cytometry cell sorter (also commonly known as FACS). The Secoya’s RayDrop platform (including the RayDrop^® device and distributed by Fluigent) allows the bacteria to be compartmentalized within a protective shell and ensures compatibility with the sorting device. The platform generates double emulsions in one step, scaling the production of common two-step double emulsion generation methods by up to ten times. Several hundred microliters of emulsions can be produced in 30 minutes, and multiple conditions can be tested within a single day, com- pared to a week with traditional methods. In addition, the platform integrates fluidic, pneumatic and optic solutions, and does not require complex fabrication steps nor surface treatment. This application note was written in collaboration with Delphine Lestrade (head of the flow cytometry platform at Toulouse White Biotechnology, TWB) and Sophie Lajus (researcher at the Toulouse Biotechnology Institute, TBI)

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White Paper : 5 use cases of controlled double emulsification process for encapsulation

Emulsions are usually manufactured in batch processes on a large scale. These produced emulsions require large amounts of energy and have wide-size distributions with low reproducibility. Moreover, when it comes to encapsulating active pharmaceutical ingredients (APIs) in these drops, the process is complex, and losses are high. Indeed, the APIs must be encapsulated in a material that can then deliver APIs in a delayed and controlled manner. In high-value-added areas such as the pharmaceutical industry, the use of microfluidic emulsion systems allows for users to obtain monodisperse emulsions and improve the quality of the product, with notably reduced API losses due to higher encapsulation efficiency than batch processes.

In this context, Secoya has developed a device named Raydrop^®, which is a microfluidic droplet generator that facilitates the production of emulsions. Raydrop^® technology aims for a more robust production with less wear than most current microfluidic emulsification devices. Depending on the configuration of the Raydrop^®, it is possible to create either simple or double emulsions.

By presenting five various possibilities for microcapsules production (Chitosan, PLGA, Polymethacrylate resin, Alginate, Agarose) using the double emulsion Raydrop^®  integrative platform,  this white paper outlines the versatility of Secoya encapsulation technology.

 

POLYMETHACRYLATE MICROCAPSULES WITH AN OILY CORE GENERATION

This Application Note is complementary to the application note entitled Polymethacrylate resin microcapsules synthesis, available on the website of Secoya Technologies (https://secoya-tech.com/documents/application-note-polymethacrylate-resin-microcapsules-synthesis/). The main difference is the composition of the core phase. In this document, the capsules contain an oily core, which is essential to encapsulate oily-soluble molecules. The capsule formation is, here again, made by the cross-linking of the polymeric shell of the double emulsion. This reaction consolidates the shell phase and makes it solid.  Thus, the oil is encapsulated in polymeric microcapsules (PMCs) with tunable sizes.

QUICK START GUIDE: PLGA capsules

Application note: Multiple emulsions

Traditionally, batch methods are used to produce emulsions in industry. The use of bulk mixing allows to produce huge quantities of emulsions but to the detriment of quality. Indeed, the shear distribution in a bulk mixer is various, leading to numerous particles sizes and a low encapsulation rate in the case of API encapsulation or double emulsion production (core-shell particles). [I] A fortiori, batch method makes the control of multiple emulsions more complex, i.e. the encapsulation of a precise number of droplets of liquid A in a droplet of liquid B.

In general, the use of microfluidics helps to reach low size dispersity and so monodispersed emulsions with a high control over both the size and structure can be obtained. [II] Microfluidic tools are also used to create emulsions of varying compositions. With this technology, it is possible to produce water–in-oil–in-water (W/O/W) emulsions or oil–in-water–in-oil (O/W/O) emulsions. A microfluidic device developed by Secoya Technologies – called the RayDrop® – allows to easily produce such highly controlled emulsions. Examples of applications in double emulsion can be found in the white paper entitled Generation of microcapsules, available on our website https://secoya-tech.com/documents/.

Inspired by the publication of LI, Er Qiang and al. [III], we wanted to demonstrate the possibility to produce multiple emulsions using the RayDrop®. For instance, these multiple emulsions are precursors in the creation of solid microcapsules used for triggered release. [I] Furthermore, these multicompartmental microspheres are interesting to co-encapsulate incompatible solutions (which would react if they were in contact). [IV]

In this Application Note, aqueous droplets (called “core”) in an oily droplet (called “shell”) are obtained using the combination of two RayDrop® devices placed in series. The influence of the fluidic parameters on the number of cores contained in the oily shell is underlined in this application note.

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Application note: CHITOSAN MICROCAPSULES CREATION

Over the past few decades, core-shell microcapsules have been extensively used for the delivery and release of materials in the pharmaceutical, cosmetic, and food industries. The encapsulation of Active Pharmaceutical Compounds in core-shell microcapsules is of great interest for several purposes: taste and odor masking, controlled release of drugs… In pharmaceutics the possibility to encapsulate drugs, nutrients, and living cells that can be protected by a solid biocompatible shell in order to target a specific site is an intense field of research.

However, classical methods of microencapsulation like coacervation, spray drying, solvent evaporation, etc, require complex process and equipment and make difficult to control the size and load of the microcapsules.

In contrast, microfluidics allows to produce monodisperse double emulsions which lead to monodispersed microcapsules with a high control over both size and structure. Microfluidic tools are also used in order to create capsules of varying compositions. With this technology, it is possible to encapsulate aqueous or oily phases. The encapsulation of aqueous phases allows the capsule to contain proteins or active pharmaceutical ingredients (APIs). On the other hand, oily phases containing lipophilic or poorly water-soluble drugs can also be encapsulated. Moreover, capsules can be used for drug delivery or acid-triggered gastric delivery depending on the composition of the shell.

In this Application Note, chitosan-shell/oily-core microcapsules are obtained using the Raydrop® double emulsion generator, a capillary based microfluidic device. Indeed, microcapsules consisting of a chitosan shell and an oily core have been extensively studied as chitosan – a cationic polysaccharide – exhibits numerous benefits: excellent biological activity, good biocompatibility [1] and biodegradability and pH sensitivity [2] for acid-triggered gastric delivery. The method of production as well as the influence of the fluidic parameters on the size and the release from the oil across the shell are studied and presented.

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Application note: POLYMETHACRYLATE RESIN MICROCAPSULES SYNTHESIS

Over the past few decades, core-shell microcapsules have been extensively used for the delivery and release of materials in the pharmaceutical, cosmetic, and food industries [1]. The encapsulation of Active Pharmaceutical Compounds in core-shell microcapsule is of great interest for several purposes: taste and odor masking, controlled release of drugs… In pharmaceutics the possibility to encapsulate drugs, nutrients, and living cells that can be protected by a solid biocompatible shell in order to target a specific site is an intense field of research.

However, classical methods of microencapsulation, like coacervation, spray drying, solvent evaporation, etc, require complex process and equipment and make difficult to control the size and load of the microcapsules.

In contrast, microfluidics allows to produce monodisperse double emulsions which lead to monodispersed microcapsules with a high control over both the size and the structure. Microfluidics tools are also used in order to create capsules of varying compositions. With this technology, it is possible to encapsulate aqueous or oily phases. The encapsulation of aqueous phases allows the capsule to contain proteins or active pharmaceutical ingredients (APIs). On the other hand, oily phases containing lipophilic or poorly water-soluble drugs can also be encapsulated. Moreover, capsules can be used for drug delivery or acid-triggered gastric delivery depending on the composition of the shell.

In this Application Note, capsules are formed by consolidating shell phase of the resulting double emulsions by UV-crosslinking of polymers [2] and photoinitiator used as shell phase. Core and continuous phases are aqueous phases non-miscible with the shell. Fine control of the fluid flows leads to defined capsule and shell dimensions. In-situ polymerization is achieved, meaning that the droplets are exposed to UV-light while still being moving forward in the output tubing connected to the Raydrop®. Hard shell microcapsules are thus directly collected in the collection vial. The in-situ process allows to avoid coalescence and deformation of the droplets that can arise in an ex-situ process where the droplets are polymerized after collection.

 

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White paper: Generation of microcapsules

Emulsions are usually manufactured in batch process in industry on a large scale. These produced emulsions require large amounts of energy and have wide size distributions with low reproducibility. Moreover, when it comes to encapsulating active pharmaceutical ingredients (APIs) in these drops, the processes are complex and losses are high. Indeed, the APIs must be encapsulated in a material that can then deliver APIs in a delayed and controlled manner. In high value-added areas such as the pharmaceutical industry, the use of microfluidic emulsion systems allows to obtain monodisperse emulsions and to improve the quality of the product, with notably reduced APIs losses thanks to a higher encapsulation efficiency as opposed to batch processes.

In this context, Secoya has developed a device named Raydrop® which is a microfluidic droplet generator that facilitates the production of emulsions. The Raydrop® technology aims for a more robust production with less wear as most current microfluidic emulsification devices. Depending on the configuration of the Raydrop®, it is possible to create either simple or double emulsions. In this white paper, this device and the multiple possibilities that it offers concerning the production of double emulsions are presented.

Furthermore, the Raydrop® is now part of a platform that make the utilization of microfluidic easier. This platform already contains all elements needed to produce a reproducible and high-quality emulsion, such as the fluidic elements, the mechanic compounds and optical material. The capacities of this platform are underlined in this white paper as well as a performance illustration using different application cases.

 

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Application note: PLGA MICROCAPSULES CREATION

Over the past few decades, core-shell microcapsules have been extensively used for the delivery and release of materials in the pharmaceutical, cosmetic, and food industries. The encapsulation of Active Pharmaceutical Compounds in core-shell microcapsule is of great interest for several purposes: taste and odor masking, controlled release of drugs… In pharmaceutics the possibility to encapsulate drugs, nutrients, and living cells that can be protected by a solid biocompatible shell in order to target a specific site is an intense field of research [1].

However, classical methods of microencapsulation, like coacervation, spray drying, solvent evaporation, etc, require complex process and equipment and make it difficult to control the size and load of the microcapsules.

In contrast, microfluidics allows to produce monodisperse double emulsions which lead to monodispersed microcapsules with a high control over both the size and the structure [2,3]. Microfluidics tools are also used in order to create capsules of varying compositions. With this technology, it is possible to encapsulate aqueous or oily phases. The encapsulation of aqueous phases allows the capsule to contain proteins or active pharmaceutical ingredients (APIs). On the other hand, oily phases containing lipophilic or poorly water-soluble drugs can also be encapsulated. Moreover, capsules can be used for drug delivery or acid-triggered gastric delivery depending on the composition of the shell.

In this Application Note, PLGA shell/aqueous core microcapsules are obtained using the Raydrop® Double emulsion device, a capillary-based microfluidic device equipped with a 3D printed injection nozzle making the generation of double emulsion easy, in combination with pressure-based flow controllers. The influence of the fluidic parameters on the microcapsule size and the release from the oil across the shell are explored in this application note.

 

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Leaflet – Monodispersed micro-particles for encapsulated drugs

New White paper: 5 USE CASES OF CONTROLLED DOUBLE EMULSIFICATION PROCESS FOR ENCAPSULATION

Droplet-Based Microfluidics: Applications in Pharmaceuticals

Publication on Raydrop

Adrien Dewandre and Youen Vitry, Technology leads at Secoya, and Benoit Scheid, Chairman of the Board of Secoya and Maître de Recherche FNRS at ULB, were involved in an experimental and numerical study of the Secoya droplet generator, the Raydrop. Their work on “Microfluidic droplet generation in a non-embedded co-flow-focusing using 3D printed nozzle” is published in the journal Scientific Reports.

Secoya-Fluigent Webinar on double emulsion

28/10/2020

On November 17rd at 5 pm, Youen Vitry, co-founder and Encapsulation Technology Lead, will give a Webinar in collaboration with Fluigent on the Raydrop technology for the generation of double emulsion.

Register to the webinar : https://www.fluigent.com/resources/webinars/

Secoya-Fluigent Webinar on the Raydrop technology – Replay

28/10/2020

On June 23rd, Adrien Dewandre, co-founder and Encapsulation Technology Lead, has given a Webinar in collaboration with Fluigent on the Raydrop technology for the droplet generation.

Access now to the replay : https://webikeo.com/webinar/raydrop-a-universal-droplet-generator-based-on-a-non-embedded-co-flow-focusing/replay