EP4179081A1 - Alginate beads and production thereof - Google Patents
Alginate beads and production thereofInfo
- Publication number
- EP4179081A1 EP4179081A1 EP21742832.5A EP21742832A EP4179081A1 EP 4179081 A1 EP4179081 A1 EP 4179081A1 EP 21742832 A EP21742832 A EP 21742832A EP 4179081 A1 EP4179081 A1 EP 4179081A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- alginate
- solution
- beads
- oil
- emulsions
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 235000010443 alginic acid Nutrition 0.000 title claims abstract description 261
- 229920000615 alginic acid Polymers 0.000 title claims abstract description 261
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 title claims abstract description 260
- 229940072056 alginate Drugs 0.000 title claims abstract description 260
- 239000011324 bead Substances 0.000 title claims abstract description 160
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 46
- 238000000034 method Methods 0.000 claims abstract description 44
- 244000005700 microbiome Species 0.000 claims abstract description 44
- 150000002500 ions Chemical class 0.000 claims abstract description 43
- 238000007711 solidification Methods 0.000 claims abstract description 40
- 230000008023 solidification Effects 0.000 claims abstract description 40
- 238000012258 culturing Methods 0.000 claims abstract description 22
- 239000000839 emulsion Substances 0.000 claims description 105
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 23
- 235000010413 sodium alginate Nutrition 0.000 claims description 23
- 239000000661 sodium alginate Substances 0.000 claims description 23
- 229940005550 sodium alginate Drugs 0.000 claims description 23
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 claims description 22
- 239000003945 anionic surfactant Substances 0.000 claims description 22
- 229910001424 calcium ion Inorganic materials 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- 230000001939 inductive effect Effects 0.000 claims description 12
- 230000008859 change Effects 0.000 claims description 10
- 230000000694 effects Effects 0.000 claims description 8
- 239000006143 cell culture medium Substances 0.000 claims description 5
- 239000000725 suspension Substances 0.000 claims description 5
- KBPLFHHGFOOTCA-UHFFFAOYSA-N caprylic alcohol Natural products CCCCCCCCO KBPLFHHGFOOTCA-UHFFFAOYSA-N 0.000 claims 1
- 150000001768 cations Chemical class 0.000 abstract description 10
- 230000035899 viability Effects 0.000 abstract description 10
- 239000002775 capsule Substances 0.000 abstract description 8
- 239000007787 solid Substances 0.000 abstract description 7
- 230000015572 biosynthetic process Effects 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 175
- 210000004027 cell Anatomy 0.000 description 71
- 239000002609 medium Substances 0.000 description 45
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 29
- 239000011575 calcium Substances 0.000 description 29
- 229910052791 calcium Inorganic materials 0.000 description 29
- 239000012071 phase Substances 0.000 description 19
- 238000011534 incubation Methods 0.000 description 18
- 239000004094 surface-active agent Substances 0.000 description 17
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 description 12
- 229920000642 polymer Polymers 0.000 description 12
- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N coumarin Chemical compound C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 description 10
- 238000010586 diagram Methods 0.000 description 10
- 239000004205 dimethyl polysiloxane Substances 0.000 description 10
- 239000011521 glass Substances 0.000 description 10
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 239000007864 aqueous solution Substances 0.000 description 7
- 238000005520 cutting process Methods 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical compound [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 description 6
- 239000002253 acid Substances 0.000 description 6
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 6
- 229910052782 aluminium Inorganic materials 0.000 description 6
- 239000008346 aqueous phase Substances 0.000 description 6
- 238000005538 encapsulation Methods 0.000 description 6
- 239000012530 fluid Substances 0.000 description 6
- 239000004033 plastic Substances 0.000 description 6
- 239000013641 positive control Substances 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 229910000077 silane Inorganic materials 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 5
- 125000000129 anionic group Chemical group 0.000 description 5
- -1 cations ions Chemical class 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 229960000956 coumarin Drugs 0.000 description 5
- 235000001671 coumarin Nutrition 0.000 description 5
- 238000013461 design Methods 0.000 description 5
- 238000009792 diffusion process Methods 0.000 description 5
- 239000000017 hydrogel Substances 0.000 description 5
- 239000013642 negative control Substances 0.000 description 5
- 239000002736 nonionic surfactant Substances 0.000 description 5
- 239000002245 particle Substances 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- UQDUPHDELLQMOV-UHFFFAOYSA-N 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-heptadecafluorooctan-1-ol Chemical compound OC(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)F UQDUPHDELLQMOV-UHFFFAOYSA-N 0.000 description 4
- 230000001464 adherent effect Effects 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 239000002738 chelating agent Substances 0.000 description 4
- 239000000693 micelle Substances 0.000 description 4
- 102000039446 nucleic acids Human genes 0.000 description 4
- 108020004707 nucleic acids Proteins 0.000 description 4
- 150000007523 nucleic acids Chemical class 0.000 description 4
- 238000012805 post-processing Methods 0.000 description 4
- 239000004971 Cross linker Substances 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 239000011258 core-shell material Substances 0.000 description 3
- 238000004132 cross linking Methods 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 229920001343 polytetrafluoroethylene Polymers 0.000 description 3
- 239000004810 polytetrafluoroethylene Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 235000012976 tarts Nutrition 0.000 description 3
- BGWLYQZDNFIFRX-UHFFFAOYSA-N 5-[3-[2-[3-(3,8-diamino-6-phenylphenanthridin-5-ium-5-yl)propylamino]ethylamino]propyl]-6-phenylphenanthridin-5-ium-3,8-diamine;dichloride Chemical compound [Cl-].[Cl-].C=1C(N)=CC=C(C2=CC=C(N)C=C2[N+]=2CCCNCCNCCC[N+]=3C4=CC(N)=CC=C4C4=CC=C(N)C=C4C=3C=3C=CC=CC=3)C=1C=2C1=CC=CC=C1 BGWLYQZDNFIFRX-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 239000004904 UV filter Substances 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 238000004581 coalescence Methods 0.000 description 2
- 230000001351 cycling effect Effects 0.000 description 2
- 230000008021 deposition Effects 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 238000000206 photolithography Methods 0.000 description 2
- 239000006041 probiotic Substances 0.000 description 2
- 235000018291 probiotics Nutrition 0.000 description 2
- LLHKCFNBLRBOGN-UHFFFAOYSA-N propylene glycol methyl ether acetate Chemical compound COCC(C)OC(C)=O LLHKCFNBLRBOGN-UHFFFAOYSA-N 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 231100000747 viability assay Toxicity 0.000 description 2
- 238000003026 viability measurement method Methods 0.000 description 2
- 241000203069 Archaea Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 238000012773 Laboratory assay Methods 0.000 description 1
- 241000199919 Phaeophyceae Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000007900 aqueous suspension Substances 0.000 description 1
- 230000008236 biological pathway Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000012864 cross contamination Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000013537 high throughput screening Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 229920005684 linear copolymer Polymers 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 239000010453 quartz Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 239000012798 spherical particle Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 239000007762 w/o emulsion Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
- A61K9/1605—Excipients; Inactive ingredients
- A61K9/1629—Organic macromolecular compounds
- A61K9/1652—Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
- A61K9/1682—Processes
- A61K9/1694—Processes resulting in granules or microspheres of the matrix type containing more than 5% of excipient
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5005—Wall or coating material
- A61K9/5021—Organic macromolecular compounds
- A61K9/5036—Polysaccharides, e.g. gums, alginate; Cyclodextrin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5089—Processes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0012—Cell encapsulation
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/70—Polysaccharides
- C12N2533/74—Alginate
Definitions
- the present invention is in the field of polymer chemistry and relates to methods for producing alginate beads.
- the present application is in the field of mammalian and microbial cell culture analysis and also, in the field of microfluidics, particularly in the field of microfluidic analysis and devices.
- Microfluidic devices are powerful tools that allow to miniaturise and to perform a large number of assays in parallel. As a consequence, microfluidic devices are ideal tools to vastly increase the throughput of many types of laboratory assays, such as screenings analyses or in vitro evolution.
- Microfluidic devices are essentially networks of small channels used for the precise manipulation of small amounts of fluids.
- Miniaturised reaction vessels which are in facts droplets of fluid, flow through the channels of the microfluidic system. Along their flow path, the droplets can be manipulated.
- a reagent can for example be added to at least a subset of the droplets by various methods known in the art, such as nanoinjection. This technique allows the production and precise manipulation of emulsion droplets revealing an enormous potential for high-throughput screening applications.
- microfluidic is also used to produce beads of various compositions and sizes that have applications such as in ELISA assays, in attachment of nucleic acids for molecular biology assays or cell and microbial encapsulation. It has the advantage of producing extremely monodisperse emulsions with a controlled size that can then be solidified post emulsification via cross-linking of a polymer for example.
- Alginate is a polymer extracted from brown algae that has been extensively used in biomedical applications, due to its non-toxicity.
- alginate is used in its hydrogel form. Gelling is obtained via cross-linking of the [0006] alginate polymers. This can be done via UV exposure or heating. However, these methods are harmful to the cells.
- Alginate molecules are also cross-linked in the presence of divalent cations ions (ie. Ca 2 +, Ba 2 +). Therefore, when encapsulating cells or microorganisms this is preferably used to increase their viability.
- divalent cationic cross-linking two main techniques are used: 1) dripping of alginate containing the cells or microorganisms into a calcium bath or 2 slow release of calcium from a chelating agent such as EDTA.
- the first technique can lead to inhomogeneous particles particularly in the production of core-shell capsules smaller than 150 pm, due to a quick solidification process. These particles cannot be analyzed via standard flow cytometry instruments.
- the second technique leads to homogenous beads or core-shell capsules.
- the divalent cation is released either via a decrease in pH, or via competition with another ion.
- the chelating agents render nutrients from the surrounding environment unexploitable for the cells and microorganisms.
- the present invention provides a method for producing alginate beads, wherein the alginate solidifies via slow release of divalent ions into an alginate solution, comprising the steps of: preparing a first solution containing alginate; preparing a second solution containing an anionic surfactant in oil; preparing a third solution containing divalent ions; injecting said first and second solutions into a microfluidic device generating alginate in oil emulsions; collecting and incubating the alginate in oil emulsions in the third solution; inducing solidification of the first solution.
- the present invention provides a method for producing alginate hollow beads, wherein the alginate solidifies via slow release of divalent ions into an alginate solution, comprising the steps of: preparing a first solution containing alginate, consisting of water in oil in alginate double emulsions; preparing a second solution containing an anionic surfactant in oil; preparing a third solution containing divalent ions; - injecting said first and second solutions into a microfluidic device generating alginate in oil emulsions; collecting and incubating the alginate in oil emulsions in the third solution; inducing solidification of the first solution.
- the present invention provides a method for producing two-layer alginate beads, wherein the alginate solidifies via slow release of divalent ions into an alginate solution, comprising the steps of: preparing a first solution containing alginate, consisting of a suspension of alginate beads in the alginate solution; preparing a second solution containing an anionic surfactant in oil; - preparing a third solution containing divalent ions; injecting said first and second solutions into a microfluidic device generating alginate in oil emulsions; collecting and incubating the alginate in oil emulsions in the third solution; inducing solidification of the first solution.
- the present invention also provides a use of the method for producing alginate hollow beads, wherein the first solution containing alginate consists of water in oil in alginate double emulsions. [0014] It provides a use of the method for producing 2-layer alginate beads, wherein the first solution containing alginate consists of a suspension of alginate beads in alginate solution Further, it provides a use of the method for producing alginate beads for co-culturing fluorescent reporter cell-lines and microorganisms, wherein the fluorescent reporter cell-lines and the cells are co-encapsulated into the beads.
- Figure 1 Images of the design for the flow-focusing microfluidic device (FFD).
- Figure 3 Images of the alginate in HFE7500 emulsions deposited in 200 mM CaCh droplets, incubated at room temperature for lh, observed under the bright field microscope of the fluorescence microscope, before (A) and after (B) addition of PFO (20X objective).
- Figure 4 Images of the alginate in HFE7500 emulsions deposited in 200 mM CaCh droplets, incubated at room temperature for 2h, observed under the bright field microscope of the fluorescence microscope, before (A) and after (B) addition of PFO (20X objective).
- Figure 5 Images of the alginate in HFE7500 emulsions deposited in 200 mM CaCh droplets, incubated at room temperature for 4h, observed under the bright field microscope of the fluorescence microscope, before (A) and after (B) addition of PFO (20X objective).
- Figure 6 Images of the alginate in HFE7500 emulsions deposited in 200 mM CaCh droplets, incubated at room temperature for 24h, observed under the bright field microscope of the fluorescence microscope, before (A) and after (B) addition of PFO (20X objective).
- Figure 7 Images of the alginate in HFE7500 emulsions deposited in 200 mM CaCh droplets observed under the bright field microscope of the fluorescence microscope, 4X objective.
- Figure 8 Images of the alginate in HFE7500 emulsions deposited in 200 mM CaCh droplets, after addition of PFO, observed under the bright field microscope of the fluorescence microscope (4X objective).
- Figure 9 Images of the alginate in HFE7500 emulsions deposited in 200 mM CaCh droplets, incubated at room temperature overnight, observed under the bright field microscope of the fluorescence microscope (4X objective).
- Figure 10 Images of the alginate in HFE7500 emulsions deposited in 200 mM CaCh droplets, incubated at room temperature overnight, after addition of PFO observed under the bright field microscope of the fluorescence microscope (4X objective).
- Figure 11 Images of the alginate in HFE7500 emulsions from the collection tube, incubated at room temperature overnight, observed under the bright field microscope of the fluorescence microscope (10X objective).
- Figure 12 Images of the alginate in HFE7500 emulsions from the collection tube, incubated at room temperature overnight, after addition of PFO observed under the bright field microscope of the fluorescence microscope (10X objective).
- Fig. 13 Diagram of the workflow for the production of alginate droplets and solidification via micellar transport of Ca 2+ .
- Fig. 14 Diagram of the workflow for the production of triple emulsions and solidification via micellar transport of Ca 2+ .
- Fig. 15 Diagram of the workflow for the production of 2-layer alginate droplets and solidification via micellar transport of Ca 2+ .
- Fig. 16 Diagram of the workflowforthe viability assay of the encapsulation of epithelial cells in alginate beads via micellar transport of divalent ions.
- Fig. 17 Diagram of the workflow for the viability assay of the encapsulation of epithelial cells in alginate beads via acid dechelation of divalent ions.
- Figure 18 Diagram of the principle of slow release of divalent ions from the third solution to the first solution with the help of the anionic surfactant in oil.
- Figure 19 Diagram of workflow for assessing bead making using divalent cations (Ca 2+ ) versus non-divalent cations (Na + ), prepared with non-ionic surfactant (negative control), anionic surfactant (Tested condition) or chelated calcium suspended in alginate and acid suspended in fluorinated oil (positive control).
- Figure 20 Image of alginate beads solidified via micellar transport of Ca 2+ from the collection solution to alginate.
- Figure 21 Images of the alginate in oil emulsions collected in NaCI or CaCh prepared with a non-ionic surfactant (negative control), anionic surfactant (Test sample) or non-ionic surfactant with the alginate supplemented with calcium chelated to EDTA and the oil supplemented with acetic acid (positive control).
- Figure 22 Images of the alginate in FIFE7500 emulsions collected in culture medium 200 mM CaCh, after collection, after medium exchange and after addition of PFO and removal of oil, analyzed under the bright filed filter (BF), phase contrast (PC) or UV fluorescence (UV) settings of a fluorescent microscope.
- Figure 23 Images of the alginate in HFE7500 emulsions 10% w/w Krytox collected in 9 mL culture medium 200 mM CaCh after collection, medium exchange, and addition of PFO and removal of oil, analyzed using phase contrast or UV fluorescence (UV) settings of a fluorescence microscope.
- emulsion refers to immiscible liquids that coexist in a mixture.
- the term “molecules” refers to enzymes, proteins, antibodies, polymers, nucleic acids that need to be encapsulated and/or analyzed.
- the terms “more concentrated” and “less concentrated” refer to divalent ions which are selectively transported between solutions of 1 mM to 10 M CaCh to a solution with no CaCh-
- the term "micellar transport” refers to the transport of a chemical, between two solutions it is miscible in, through a solution it is not miscible in, by being enclosed in the center of a micelle composed of surfactant molecules.
- double layer emulsion refers to an emulsion within an emulsion.
- bearing refers to a spherical solid object that remains stable in an aqueous solution.
- the term “hollow” refers to an object surrounding an empty space in its center.
- droplet breaker refers to demulsifiers to break the droplets, such as chloroform or lH,lH,2H,2H-Perfluoro-l-octanol. These chemicals displace surfactants from the oil-water interfaces of the droplets and make the droplets unstable.
- alginate refers to a whole family of linear copolymers containing blocks of (l,4)-linked b-D-mannuronate (M) and a-L-guluronate (G) residues.
- the blocks are composed of consecutive G residues (GGGGGG), consecutive M residues (MMMMMM), and alternating M and G residues (GMGMGM).
- the term "cell medium” refers to a medium for culturing cells, a medium for increasing proliferation or differentiation.
- microorganism refers to bacteria, archaea, fungi, yeast, phages, viruses.
- droplet refers to a measure of volume and further relates to an isolated portion of a first fluid (first solution) that is surrounded by a second fluid (second solution).
- two-layer bead refers to a spherical object composed of a solid polymer bead in its center surrounded by a solid polymer layer.
- the term “droplet” also refers to "microfluidic droplet”. Therefore, in the context of a microfluidic system, the term “droplet” also refers to an isolated portion of a "first" solution that is surrounded by a “second” solution, where the first and second solutions are immiscible.
- a "change in fluorescence" refers to a reduction or increase of the fluorescent signal produced by the reporter cell line.
- a reduction of the fluorescent signal refers to a situation where the fluorescence of the reporter cell line is reduced by a stimulus compared to the reporter cell in the exact same conditions minus the stimulus.
- An increase of the fluorescent signal refers to a situation where the fluorescence of the reporter cell line is increased by a stimulus compared to the reporter cell in the exact same conditions minus the stimulus.
- activity on the fluorescent reporter cell line refers to a situation where the microorganism modifies the fluorescent signal produced by the reporter cell by altering a biological pathway.
- post-processing refers to addition of droplets breaker and removal of remaining oil in the collection vial.
- a “dynamic droplet incubator” refers to a device allowing the increased diffusion of calcium to the droplets via cycling of the calcium loaded oil. The production of alginate beads
- Alginate beads are produced using a microfluidic system in which, 2% w/v sodium alginate is cut by fluorinated oil (e.g. HFE7500) containing an anionic fluorinated surfactant producing alginate in oil emulsions and collected in a solution containing 200 mM CaC . Calcium then diffuses from the collection solution to the emulsions through the oil. The calcium then cross-links the alginate, solidifying it and resulting in a hydrogel bead. These beads are collected and released from the oil envelope using perfluoro-l-octanol and resuspended in medium.
- fluorinated oil e.g. HFE7500
- the present invention refers to a method for producing alginate beads, wherein the alginate solidifies via slow release of divalent ions into an alginate solution, comprising the steps of: - preparing a first solution containing alginate; preparing a second solution containing an anionic surfactant in oil, wherein said second solution is immiscible with the first solution; preparing a third solution containing divalent ions; injecting said first and second solutions into a microfluidic device generating alginate in oil droplets; collecting and incubating the alginate in oil droplets in the third solution, inducing solidification of the first solution.
- the droplets may be contained in a microfluidic system (on-chip) or in a collector device (off-chip) separate from the microfluidic system.
- the droplets may have a spherical or non-spherical form.
- the alginate beads are solid beads. In a preferred embodiment, the alginate beads are hollow alginate beads or 2-layer beads.
- the first solution comprises sodium alginate at 0.1% w/v to 20% w/v, more preferably 0.5% w/v to 10% w/v, even more preferably 2% w/v to 4% w/v and most preferred 2% w/v.
- the second solution comprises a surfactant and a fluorinated oil.
- the fluorinated oil is HFE7500 and the surfactant Krytox FSH.
- the w/v concentration of Krytox FSH in HFE7500 is defined. The concentration is between 0.01% w/w and 50% w/w, preferably between 1% w/w and 15% w/v, more preferably between 2.5% w/w and 12.5% w/w, even more preferably between 5% w/w and 10% w/w and most preferred 10% w/w.
- the divalent ions are Mg 2+ , Zn 2+ , Hg 2+ , Cu 2+ , Sn 2+ , Fe 2+ , Co 2+ , Mn 2+ , Pb 2+ Ca 2+ and/or Ba 2+ .
- the divalent ions are Ca 2+ and/or Ba 2+ and most preferred Ca 2+ .
- the incubation of the alginate in oil emulsions in the third solution containing CaC is performed for a period of 1 min to 30 days. In a preferred embodiment, for a period of 30 min to 24h. In a more preferred embodiment between lh and 12h. In an even more preferred embodiment, between 2h and 4h and most preferably of 2h.
- the concentration of the CaCh solution is between 1 mM and 10 M, preferably between 10 mM and 1000 mM, more preferably between 100m M and 300m M, even more preferably between 150mM and 250mM and most preferably 200mM.
- a droplet breaker In one embodiment to the alginate in oil emulsion in the third solution is added a droplet breaker, and wherein the droplet breaker is lH,lH,2H,2H-Perfluoro-l-octanol.
- the solidification of the first solution is done via micellar transport of divalent ions from the third solution into the first solution via the second solution.
- the micellar transport of the calcium ions from the third solution to the first solution inducing the solidification is performed at a temperature between 1 °C to 95 °C, preferably between 15 °C to 50 °C, more preferably 17.5 °C to 35°C, even more preferably 20 °C to 30 °C, even more preferably 22 °C to 25 °C and most preferably 22 °C.
- the first solution comprises sodium alginate in cell culture medium and cells are resuspended in said first solution.
- the cells are from any adherent cell line.
- the cells are gut cells.
- the present invention refers to the use of the method for producing alginate beads for co culturing fluorescent reporter cell-lines and microorganisms, wherein the cell-lines and microorganisms are co-encapsulated.
- the fluorescent reporter cell-line is encapsulated in the solid alginate bead.
- the microorganism is tested for activity on the fluorescent reporter cell line.
- the fluorescent reporter cell-line is analyzed for a change in fluorescence.
- the first solution comprises sodium alginate in cell culture medium and cells are resuspended in said first solution.
- the cells are from any adherent cell line.
- the cells are gut cells.
- Alginate hollow beads are produced using a succession of microfluidic systems to first produce triple emulsions.
- An aqueous solution is cut by a fluorinated oil in a microfluidic system producing simple emulsions.
- the single emulsions are reinjected in a second microfluidic system and cut by 2% w/v sodium alginate producing double emulsions.
- the double emulsions are reinjected into a third microfluidic system and cut by fluorinated oil (e.g. HFE7500) containing an anionic fluorinated surfactant producing water in oil in alginate in oil triple emulsions.
- fluorinated oil e.g. HFE7500
- the alginate in the triple emulsions then needs to be solidified.
- the triple emulsions are collected in a solution containing 200 mM CaC . Calcium then diffuses from the collection solution to the alginate through the outer oil layer. The calcium cross-links the alginate, solidifying it and resulting in a hollow bead. These capsules are then released from the oil shell using perfluoro-l-octanol and resuspended in medium.
- a further aspect of the present invention refers to a method for producing hollow alginate beads, wherein the alginate solidifies via slow release of divalent ions into an alginate solution, comprising the steps of: preparing a first solution containing alginate, consisting of water in oil in alginate double emulsions; preparing a second solution containing an anionic surfactant in oil; preparing a third solution containing divalent ions; - injecting said first and second solutions into a microfluidic device generating alginate in oil emulsions; collecting and incubating the alginate in oil emulsions in the third solution, inducing solidification of the first solution.
- the first solution comprises sodium alginate at 0.1% w/v to 20% w/v, more preferably 0.5 % w/v to 10% w/v, even more preferably 2% w/v to 4% w/v and most preferred 2% w/v.
- the second solution comprises a surfactant and a fluorinated oil.
- the fluorinated oil is HFE7500 and the surfactant Krytox FSH.
- the w/v concentration of Krytox FSH in HFE7500 is defined. The concentration is between 0.01% w/w and 50% w/w, preferably between 1% w/w and 15% w/v, more preferably between 2.5% w/w and 12.5% w/w, even more preferably between 5% w/w and 10% w/w and most preferred 10% w/w.
- the divalent ions are Mg 2+ , Zn 2+ , Hg 2+ , Cu 2+ , Sn 2+ , Fe 2+ , Co 2+ , Mn 2+ , Pb 2+ Ca 2+ and/or Ba 2+ .
- the divalent ions are Ca 2+ and/or Ba 2+ and most preferred Ca 2+ .
- the incubation of the alginate in oil emulsions in the third solution containing CaC is performed for a period of 1 min to 30 days. In a preferred embodiment, for a period of 30 min to 24 h. In a more preferred embodiment between 1 h and 12 h. In an even more preferred embodiment, between 2 h and 4 h and most preferably of 2 h.
- the concentration of the CaCh solution is between 1 mM and 100 M, preferably between 10 mM and 1000 mM, more preferably between 100 mM and 300 mM, even more preferably between 150 mM and 250 mM and most preferably 200 mM.
- a droplet breaker In one embodiment to the alginate in oil emulsion in the third solution is added a droplet breaker, and wherein the droplet breaker is lH,lH,2H,2H-Perfluoro-l-octanol.
- the solidification of the first solution is done via micellar transport of divalent ions from the third solution into the first solution via the second solution.
- the micellar transport of the calcium ions from the third solution to the first solution inducing the solidification is performed at a temperature between 1 °C to 95 °C, preferably between 15 °C to 50 °C, more preferably 17.5 °C to 35 °C, even more preferably 20 °C to 30 °C, even more preferably 22 °C to 25 °C and most preferred 22 °C.
- the fluorescent reporter cell-line is encapsulated in the hollow alginate bead.
- the fluorescent reporter cell-line is encapsulated in the core of the hollow alginate bead.
- the microorganism is encapsulated in the shell of the hollow alginate bead.
- the microorganism is encapsulated in the core of the hollow alginate bead.
- the microorganism is tested for activity on the fluorescent reporter cell line.
- the fluorescent reporter cell-line is analyzed for a change in fluorescence.
- the first solution comprises sodium alginate in cell culture medium and cells are resuspended in said first solution.
- the cells are from any adherent cell line.
- the cells are gut cells.
- Alginate 2-layer beads are produced using a microfluidic system in which, 2% w/v sodium alginate is cut by fluorinated oil (e.g. HFE7500) containing an anionic fluorinated surfactant producing alginate in oil emulsions and incubated in a dynamic droplet incubator that cycles calcium loaded oil to the droplets. Calcium then diffuses from the oil to the emulsions through the oil. The calcium then cross-links the alginate, solidifying it and resulting in a hydrogel bead. These beads are collected and released from the oil shell using perfluoro-l-octanol and resuspended in 2% w/v sodium alginate solution.
- fluorinated oil e.g. HFE7500
- the alginate beads in alginate solution suspension is then is cut by fluorinated oil (e.g. HFE7500) containing an anionicfluorinated surfactant in a microfluidic device producing alginate beads in alginate solution in oil emulsions and incubated in a dynamic droplet incubator that cycles calcium loaded oil to the droplets. Calcium then diffuses from the oil to the emulsions through the oil. The calcium then cross-links the alginate, solidifying it and resulting in a 2-layer hydrogel bead. These beads are collected and released from the oil shell using perfluoro-l- octanol and resuspended.
- fluorinated oil e.g. HFE7500
- a further aspect of the present invention refers to a method for producing 2-layer alginate beads, wherein the alginate solidifies via slow release of divalent ions into an alginate solution, comprising the steps of: preparing a first solution containing alginate, consisting of a suspension of alginate beads in an alginate solution; preparing a second solution containing an anionic surfactant in oil; preparing a third solution containing divalent ions; injecting said first and second solutions into a microfluidic device generating alginate in oil emulsions; collecting and incubating the alginate in oil emulsions in the third solution; inducing solidification of the first solution.
- the first solution comprises sodium alginate at 0.1% w/v to 20% w/v, more preferably 0.5% w/v to 10% w/v, even more preferably 2% w/v to 4% w/v and most preferred 2% w/v.
- the second solution comprises a surfactant and a fluorinated oil.
- the fluorinated oil is HFE7500 and the surfactant Krytox FSH.
- the w/v concentration of Krytox FSH in HFE7500 is defined. The concentration is between 0.01% w/w and 50% w/w, preferably between 1% w/w and 15% w/v, more preferably between 2.5% w/w and 12.5% w/w, even more preferably between 5% w/w and 10% w/w and most preferably 10% w/w.
- the divalent ions are Mg 2+ , Zn 2+ , Hg 2+ , Cu 2+ , Sn 2+ , Fe 2+ , Co 2+ , Mn 2+ , Pb 2+ Ca 2+ and/or Ba 2+ .
- the divalent ions are Ca 2+ and/or Ba 2+ and most preferably Ca 2+ .
- the incubation of the alginate in oil emulsions in the third solution containing CaC is performed for a period of 1 min to 30 days. In a preferred embodiment, for a period of 30 min to 24h. In a more preferred embodiment between lh and 12h. In an even more preferred embodiment, between 2h and 4h and most preferably of 2h.
- a droplet breaker In one embodiment to the alginate in oil emulsion in the third solution is added a droplet breaker, and wherein the droplet breaker is lH,lH,2H,2H-Perfluoro-l-octanol.
- the solidification of the first solution is done via micellar transport of divalent ions from the third solution into the first solution via the second solution.
- the micellar transport of the calcium ions from the third solution to the first solution inducing the solidification is performed at a temperature between 1 °C to 95 °C, preferably between 15 °C to 50 °C, more preferably 17.5 °C to 35 °C, even more preferably 20 °C to 30 °C, even more preferably 22 °C to 25 °C and most preferred 22 °C.
- the fluorescent reporter cell-line is encapsulated in the 2-layer alginate bead.
- the fluorescent reporter cell-line is encapsulated in the center of the 2- layer alginate bead.
- the fluorescent reporter cell-line is encapsulated in the outer layer of the 2-layer alginate bead.
- the microorganism is encapsulated in the 2-layer alginate bead.
- the microorganism is tested for activity on the fluorescent reporter cell line.
- the fluorescent reporter cell-line is analyzed for a change in fluorescence.
- the first solution comprises sodium alginate in cell culture medium and cells are resuspended in said first solution.
- the cells are from any adherent cell line. In a preferred embodiment, the cells are gut cells.
- a further embodiment of the present invention refers to methods for producing alginate beads , hollow alginate beads or 2-layer alginate beads, wherein the second solution contains a mixture of anionic surfactants in oil.
- a further embodiment of the present invention refers to methods for producing alginate beads , hollow alginate beads or 2-layer alginate beads, wherein the second solution contains a mixture of anionic surfactant(s) with non-anionic surfactant(s) in oil.
- a further embodiment of the present invention refers to methods for producing alginate beads , hollow alginate beads or 2-layer alginate beads, wherein the second solution is immiscible with the first or the third solution, as is obvious to the skilled person in the art, since the second solution is an oil while both the first and the third solution are aqueous solutions.
- the experimentation is based on the key feature of microfluidic droplet systems that is the use of water-in-oil emulsion droplets to compartmentalize reagents into nanoliter to picolitre volumes.
- Droplet-based microfluidics manipulate discrete volumes of fluids in immiscible phases with low Reynolds number and laminar flow regimes.
- Two immiscible phases used for the droplet generation are referred to as the continuous phase (medium in which droplets are generated) and dispersed phase (the droplet phase).
- the size of the generated droplets is mainly controlled by the flow rates of the continuous phase and dispersed phase, interfacial tension between two phases and the geometry used for the droplet generation.
- the oil that separates the aqueous phase droplets can prevent cross-contamination between reagents in neighboring droplets and reduce the non-specific binding between channel surface and the reagents.
- the same technical principles for fluid control and monitoring of droplets using fluorescence microscope were employed.
- a master mold of 21 pm in height is fabricated for preparing a microfluidic flow-focusing device (FFD) (Fig. 1).
- FFD microfluidic flow-focusing device
- a glass wafer was heated at 200 °C for 5 min. Approximatively 5 mL of SU8- 2025 were spin coated at 500 rpm for 5 sec and at 3500 rpm for another 30 sec; it was then heated at 65 °C for 3 min and at 95 °C for 5 min. The coated wafer was then exposed to 365 nm wavelength UV light at 21.1 mW/cm2 through a quartz slide flush with the mask design for the flow-focusing device. The wafer was then heated at 65 °C for 1 min and at 95 °C for 5 min. The glass wafer was incubated in propylene glycol methyl ether acetate (PGMEA) in a beaker for 4m in while rotating at 200 rpm. The glass wafer was then cleaned with isopropanol and dried with an airgun; the glass wafer was heated again at 200 °C for 5 min.
- PMEA propylene glycol methyl ether acetate
- ROMS was peeled off the master mold. ROMS was cut around the design of the microfluidic chip. 0.75 or 1 mm holes in inlets 1 and 2 and the outlet were punctured. The prepared ROMS slab and a glass slide were cleaned using isopropanol and dried using an air gun.
- the molecule or organism of interest was added into the first solution and mixed.
- the first solution is loaded into the inlet 1 of the FFD microfluidic device (fig. 1) from a pressurized reservoir through PTFE (Polytetrafluoroethylene) tubing driven by a pressure pump using compressed air.
- the second solution was loaded into inlet 2 of the FFD microfluidic device also from a pressurized reservoir through PTFE tubing driven by a pressure pump using compressed air. Flowrates of the first and second solutions are adjusted in order to let the second solution cut the first solution at the T- junction in order to generate monodisperse droplets of suitable size depending on the application of the method.
- alginate beads were prepared via micellar transport of Ca 2+ form the collection solution to alginate.
- An image of solidified alginate beads via micellar transport of Ca 2+ from the collection solution to alginate is shown in Fig. 20.
- PDMS Poly-(dimethylsiloxane) microfluidic FFDs were fabricated from master molds. The molds were produced by photolithography technique as described in example 1.
- the master mold was placed in the center of an aluminum disc and the edges that were not covered by the master mold folded up like a tart dish.
- 50 g of polydimethylsiloxane (PDMS) were poured into a plastic disc.
- 5 g of cross-linker were poured into the plastic dish with PDMS and mixed thoroughly.
- the mix was poured onto the master mold and placed in a vacuum chamber until bubbles were removed and then placed in a 70 °C incubator for at least 4 hours.
- the master mold was removed from the incubator, the aluminum was peeled off from the master mold.
- Cross-linked PDMS was peeled off the master mold.
- PDMS was cut around the design of the microfluidic chip.
- Fig. 2 shows that the droplets are relatively polydisperse but the majority are approximatively between 18 and 23 pm. Better control of the droplet generation is obtained via using a droplet dye and monitoring the droplet size in combination with flow control, in order to obtain monodisperse droplets.
- the droplets After 1 hour of incubation, the droplets slightly shrunk in size (fig. 3A). Upon addition of PFO the droplets are solid (fig. 3B). However, the produced droplets are polydisperse which makes it difficult to determine if the solidification occurs upon contact with the continuous phase or via micellar transport. In fig. 3B, the resuspended droplets are not homogeneous and non-spherical. Thus, the droplets solidify upon contact with the continuous phase. Inhomogeneous beads are an indication of fast solidification.
- Fig. 4A compared to fig. 3A, shows that the droplets have significantly shrunk in size. This is due to osmosis.
- the continuous phase has a high concentration of CaCh, whereas the droplets do not contain CaCh-
- a buffer solution with various salts concentration not containing divalent ions could be used to counteract this effect.
- Fig. 4B shows that the droplets are homogeneous and spherical.
- the droplets solidify slowly and via micellar transport of CaCh.
- the resuspended droplets are spherical and homogeneous (fig. 5B, fig. 6B). As described before, the droplets solidify via micellar transport of CaCh-
- PDMS Poly-(dimethylsiloxane) microfluidic FFDs were fabricated from master molds. The molds were produced by photolithography technique as described in example 1. [0168] The master mold was placed in the center of an aluminum disc and the edges that were not covered by the master mold folded up like a tart dish. 50 g of polydimethylsiloxane (ROMS) were poured into a plastic disc. 5 g of cross-linker were poured into the plastic dish with ROMS and mixed thoroughly. The mix was poured onto the master mold and placed in a vacuum chamber until bubbles were removed and then placed in a 70 °C incubator for at least 4 hours.
- ROMS polydimethylsiloxane
- the master mold was removed from the incubator, the aluminum was peeled off from the master mold.
- Cross-linked ROMS was peeled off the master mold.
- ROMS was cut around the design of the microfluidic chip. 0.75 or 1 mm holes in inlets 1 and 2 and the outlet were punctured.
- the prepared ROMS slab and a glass slide were cleaned using isopropanol and dried using an air gun.
- Krytox FSH seems to completely dissolve in HFE7500. Droplets were successfully prepared and were stable until the outlet. Krytox can be used as a surfactant to prepare water in HFE7500 emulsions.
- Fig. 7 A shows droplets of various sizes at the outskirts of the CaCh droplet, while in Fig. 7B, it is shown droplets of relatively monodisperse size in the center of the droplet There seems to be some coalescence due to the mechanical stress of pipetting and deposition in the droplet. Not all the emulsion pockets in the droplets were as polydisperse as in fig. 7 A.
- Fig. 9 shows that the droplets have shrunk overnight compared to after deposition in the CaCh droplet (Fig. 7).
- the difference in CaCh concentration between the droplets and surrounding aqueous phase resulted in osmosis leading to shrinking of the emulsions.
- the alginate solution was prepared in MilliQ. Using a buffer solution such as IX PBS (1.05 mM KH 2 PO 4 , 155 mM NaCI, 3 mM Na2HP04-7(H20)) when preparing the alginate solution might counteract the osmosis effect. Solidification will be used to encapsulate cells, therefore media will be used instead of MilliQ. The media might contain enough salts to counteract the osmosis effect.
- the resulting particles are relatively monodisperse, spherical and homogeneous. These properties indicate that the solidification was a slow process and therefore solidification of alginate via micellar transport was successful.
- Disparity in size can be observed between the different droplet which could be explained by the distance the emulsions are from the interface of HFE7500 and 200 mM CaCh. The closer the droplet are from the surface the easier osmosis can occur. The size difference could also be explained by droplet size disparity during droplet making or coalescence.
- the droplets were solidified and becoming beads after 1 min to 24 h. In a preferred embodiment the droplets were solidified and becoming beads after 5 min to 12 h. In a more preferred embodiment the droplets were solidified and becoming beads after 30 min to 6 h. In an even more preferred embodiment the droplets were solidified and becoming beads after 1 h to 4 h and most preferably after 2 h.
- the aqueous suspension solution was then removed and the beads resuspended in an alginate solution.
- the beads in alginate solution was cut in a microfluidic flow focusing device with oil containing an anionic surfactant to produce beads in alginate in oil emulsions.
- the emulsions were collected in a 1.5 mL microfuge tube and added to a dynamic droplet incubator for solidification as previously described.
- a cell line sampled from the gut was grown in recommended medium until 90% confluency in a flask. The cells were detached from the surface of flask using trypsin-EDTA (Ethylenediaminetetraacetic acid) and resuspended in two separate solutions. A solution containing medium and alginate and a second containing medium, alginate and divalent ions complexed with a chelating agent (Ca-EDTA).
- trypsin-EDTA Ethylenediaminetetraacetic acid
- the solution containing only cells and alginate was cut in a microfluidic flow-focusing device with oil containing an anionic surfactant to produce alginate in oil emulsions.
- the emulsions were collected in medium containing a high concentration of calcium and incubated 2 hours before being resuspended in the aqueous phase using the droplet breaker as previously described.
- the oil was removed and the percentage viability was assessed by incubating the beads in medium containing calceinAM and ethidium homodimer-1 for 20 min followed by observation under the fluorescence microscope (fig. 16).
- Emulsions of 2% w/v sodium alginate, complete medium, IX coumarin in 2% w/w surfactant, HFE7500 were produced by flowing the alginate and HFE7500 solutions into a flow-focusing device and therefore cutting the alginate flow with the solution of HFE7500 at the flow-focusing area.
- Emulsions of 2% w/v sodium alginate, complete medium, IX coumarin in 10% w/w Krytox FSH, HFE7500 were produced by flowing the alginate and HFE7500 solutions into a flow-focusing device and therefore cutting the alginate flow with the solution of HFE7500 at the flow-focusing area.
- Emulsions of 2% w/v sodium alginate, complete medium, 100 mM Ca- EDTA, IX coumarin in 2% w/w surfactant, HFE7500, 1% v/v acetic acid were produced by flowing the alginate and HFE7500 solutions into a flow-focusing device and therefore cutting the alginate flow with the solution of HFE7500 at the flow-focusing area.
- Emulsions were collected in 150 mI_ medium with 200 mM CaCh or medium with 200 mM NaCI in 1.5 mL microfuge tubes.
- Fig. 19 presents the workflow of this experiment as diagram.
- the tubes were then incubated 24 h, the collection medium removed and 150 mI_ non- supplemented medium added. 150 mI_ of PFO were then added and the tube gently inverted multiple times. The tubes were then centrifuged with a tabletop centrifuge for 5 min and the oil removed.
- Emulsions were observed after collection, change of medium and addition of PFO and removal of oil under the bright phase contrast microscope.
- Emulsions of 2% w/v sodium alginate, complete medium, IX coumarin in 10% w/w Krytox FSH, HFE7500 were produced by flowing the alginate and HFE7500 solutions into a flow-focusing device and therefore cutting the alginate flow with the solution of HFE7500 at the flow-focusing area.
- Emulsions were dripped from above into 300 mI_ medium with 200 mM CaCh in 1.5 mL microfuge tubes. [0209] After collection, the collection medium was removed and 300 mI_ non-supplemented medium added. 150 mI_ of PFO were then added and the tube gently inverted multiple times. The tubes were then centrifuged with a tabletop centrifuge for 5 min and the oil removed.
- spherical fluorescent objects and larger non-fluorescent non-spherical objects could be observed (Fig. 22 after collection).
- the smaller fluorescent objects correspond to stable emulsions produced on the microfluidic chip and the larger objects to solidified alginate in oil emulsions. This is likely due to emulsions coalescing and mixing with the continuous phase. A portion of the emulsions are destabilized upon contact with the collection medium after dripping, leading to larger emulsions and solidified objects upon mixing of alginate and calcium.
- FSH, HFE7500 were produced by flowing the alginate and HFE7500 solutions into a flow-focusing device and therefore cutting the alginate flow with the solution of HFE7500 at the flow-focusing area.
- Emulsions were collected in 9 mL medium with 200 mM CaC in 1.5 mL microfuge tubes. [0217] The collection medium was then removed and 500 mI_ non-supplemented medium added. 150 mI_ of PFO were then added and the tube gently inverted multiple times. The tubes were then centrifuged with a tabletop centrifuge for 5 min and the oil removed.
- Emulsions were observed after collection, change of medium and addition of PFO and removal of oil under the bright field filter or phase contrast and UV filter of the fluorescence microscope.
- Emulsions were stable when preparing alginate in oil emulsions with 10% w/w Krytox as a surfactant, as fluorescent spherical objects could be observed (Fig. 23 after collection). The emulsions remained stable after medium exchange as fluorescent spherical objects persisted (Fig. 23 medium exchange). [0221] After breaking of the emulsions via addition of PFO followed by removal of oil, spherical non- fluorescent objects could be observed of similar size to the emulsions prepared with 10% Krytox collected in 9 mL medium, 200 mM CaCh (Fig. 23 after PFO).
- Beads can successfully be prepared via micellar transport when collected in a large volume of medium with CaCh without incubation when prepared with 10% w/w Krytox.
- the methods according to the present invention can find different applications:
- microorganisms such as probiotics or drugs against pathogens or acidic environments when ingested by individuals. Protection of encapsulated cells or microorganisms in hot but more importantly cold environments such as when freezing for storage.
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EP20185532.7A Withdrawn EP3940072A1 (en) | 2020-07-13 | 2020-07-13 | Alginate beads and production thereof |
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CN117187153A (en) * | 2022-05-30 | 2023-12-08 | 香港城市大学 | Hydrogel-based cell encapsulation method, cells or cell-encapsulated hydrogel particles, systems therefor, uses thereof and kits |
CN115322037B (en) * | 2022-08-29 | 2023-05-05 | 西南交通大学 | Preparation method of Pickering emulsion type calcium alginate capsule slow-release fertilizer |
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ES2912545T3 (en) * | 2011-06-02 | 2022-05-26 | Massachusetts Inst Technology | Modified alginates for cell encapsulation and cell therapy |
WO2015088299A1 (en) * | 2013-12-09 | 2015-06-18 | Vilnius University | Method for production of biopolymer-based droplets and particles in a microfluidic system |
WO2016145242A1 (en) * | 2015-03-11 | 2016-09-15 | Agenus Inc. | Methods and compositions for high throughput screening of biomolecules using gel microdrops |
US20190282510A1 (en) * | 2018-03-19 | 2019-09-19 | Wake Forest University Health Sciences | Method of encapsulating cells using a microfluidic encapsulation device |
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2021
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WO2022013254A1 (en) | 2022-01-20 |
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