Microparticle for cultivating and testing cells

Abstract

Cultivation and/or test-system comprising at least two cultivation spaces combined in a microparticle, wherein the sperically shaped microparticle comprises, (i) a first cultivation space in the center core of said spherically shaped microparticle, (ii) a second cultivation space in the wall surrounding the core of said microparticle, (iii) wherein the wall surrounding the core allows for the exchange of molecules as, salts, nutrients, peptides, chemicals and other compounds in order for the cells in the first cultivation space to interact with the cells in second cultivation space and vice versa. In the test system, the cells are co-cultivated and the microparticles are then selected based on the phenotype of the cells in the first or second cultivation space.

Claims

1. A method of co-cultivating and/or testing bacterial cells and mammalian cells together in a microparticle system, the method comprising the steps of: i) co-cultivating said bacterial and mammalian cells in said microparticle system, ii) giving the co-cultivated cells time to interact by means of compound diffusion between two cultivation spaces, iii) sorting up to 10.sup.9 of said microparticles and at least 100 of said microparticles in parallel based on a phenotype of one or more groups of cells present in one or more of the microparticles.

2. The method according to claim 1, wherein the phenotype of the one or more groups of cells is determined by detection of probiotic effects of the bacterial cells on the co-cultivated mammalian cells wherein the analysis of probiotic effects of the co-cultivated cells in one microparticle is determined by means from the group consisting of cell proliferation assays, immunomodulatory assays and the composition of different bacterial strains.

3. The method according to claim 2, wherein the probiotic effects are selected from the group consisting of antimicrobial effects, antifungal effects, promotion of growth, immunomodulatory effects and neurological modulatory effects.

4. The method according to claim 1, wherein the microparticles contain prebiotic substances.

5. The method according to claim 4, wherein the prebiotic substance is selected from the group consisting of oligosaccharides, polysaccharides, fibers trans-galactooligosaccharide, inulin and any other substance having prebiotic effects on bacterial and/or mammalian cells.

6. The method according to claim 1, wherein the probiotic effects of at least one or multiple bacterial strains isolated from natural samples from the group consisting of feces microbiome samples, gut microbiome samples, vaginal microbiome samples, soil microbiome samples and skin microbiome samples on mammalian cells are analyzed.

7. The method according to claim 4, wherein the analysis of probiotic effects of the co-cultivated cells in one microparticle is determined by detection of specific substances selected from the group comprising cytokines, interleukins, leukotrienes, hormones, histamine, nitric oxide, neurotransmitters, muropeptides, oligosaccharides, teichoic acids, lipoteichoic acids, volatile fatty acids and lipoproteins.

8. The method according to claim 1, wherein the sorting is done by ultrahigh-throughput flow systems from the group consisting of microfluidics, millifluidics and fluorescence activated cell sorting (FACS).

9. The method according to claim 1, wherein a first cultivation space contains at least 1-1.000.000 cells.

10. The method according to claim 1, wherein a second cultivation space contains between 1 and 25.000 cells.

11. The method according to claim 1, wherein the bacterial cells and the mammalian cells in the microparticles are co-cultivated for at least 12 hours and/or up to two weeks before being sorted.

12. The method according to claim 1, wherein the microparticles are cultivated in mixtures selected from the group consisting of diluted soil samples, diluted feces, and microbial isolates from the vagina, nasopharynx, sinuses, skin and other natural samples.

13. The method according to claim 1, wherein the microparticles include magnetic beads.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows one preferred embodiment of the method according to the invention.

(2) FIG. 2 shows analysis workflow of the method according to the invention.

EXAMPLES

(3) A strain of Escherichia coli (e.g. MG1655) is transformed with a plasmid (named here pTrp) containing the trpABCDE operon under the control of a strong constitutive promoter. The E. coli strain harboring pTrp is able to overproduce L-tryptophan and secrete the amino acid to its surrounding, hereafter referred to as the “producer” strain.

(4) A strain of Saccharomyces cervisiae that is auxotrophic for L-tryptophan and Histidine (e.g. YFL040W) is transformed with a plasmid (named here as pFluor) containing the coding sequence of a fluorescent protein (e.g. GFP, eGFP, mCherry, RFP) under the control of a strong constitutive promoter (P.sub.TEF1) as well as the gene or gene operon that allows for intracellular production of histidine. Such complementation of the histidine auxotroph allows for positive selection of S. cervisiae harbouring the pFluor plasmid. When cultured in the presence of L-tryptophan but in the absence of Histidine, the auxotrophic Saccharomyces cervisiae strain harbouring pFluor proliferates and expresses the fluorescent protein intracellularly. The proliferation of this strain can be monitored via fluorescence measurements, namely illuminating the cells with light of a wavelength or range of wavelengths and measuring the amount of light emitted by the cells at a wavelength or range of wavelengths greater than the wavelength(s) used for illumination. This auxotrophic Saccharomyces cerevisiae strain will be referred to hereafter as the “detector strain.”

(5) The producer strain is inoculated into a minimal medium (e.g., M9 minimal medium with 4 g/L glucose). This culture is grown for 4-8 hours at 37° C. with shaking at 200 rpm, then diluted to an OD600 of 0.02 using the same minimal medium. The detector strain is inoculated into a synthetically defined medium containing L-tryptophan (to allow for cell growth) but missing histidine (to ensure maintenance of the pFluor plasmid). This detector strain culture is grown for 4-8 hours at 30° C. with shaking at 200 rpm. The detector strain culture is then washed with an isotonic buffer and resuspended using a synthetically defined medium missing both L-tryptophan and histidine.

(6) Alginate core-shell capsules, 30 μm in diameter, are produced using a microfluidic system in which two aqueous solutions, an inner phase containing the producer strain in medium missing histidine and L-tryptophan and an outer phase containing the detector strain in medium missing histidine and L-tryptophan, 2% w/v sodium alginate and 100 mM Ethylenediaminetetraacetic acid calcium disodium salt (hereafter Ca-EDTA), are cut by fluorinated oil (e.g. HFE7500) containing a fluorinated surfactant and 0.15% v/v acetic acid. The acetic acid in fluorinated oil releases the calcium from the EDTA complex. The calcium then binds to the alginate, solidifying it and resulting in a core-shell hydrogel within its core the producer strain and in the hydrogel the detector strain. These capsules are collected and released from the oil shell using perfluoro-1-octanol and resuspended in the same medium missing L-tryptophan as used after washing of the detector strain. Oil is then removed from the solution and the capsules incubated at 30° C. to allow for growth of the producer strain, production of L-tryptophan, subsequent growth of the detector strain, and concomitant production of the fluorescent protein. The capsules are then analyzed and potentially sorted using a FACS (fluorescence activated cell sorting) device, that has the capability of dispensing individual capsules into wells of a microtiter plate. The fluorescence of each capsule is analyzed by illuminating the capsule with a laser having a wavelength corresponding to the excitation maximum of the fluorescent protein of interest and measuring the amount of light emitted by the capsule at a range of wavelengths longer than the wavelength used for illumination/excitation. Capsules exhibiting higher fluorescence must contain higher concentrations of fluorescent protein and must therefore contain a higher number of cells of the detector strain. One may also infer that droplets containing higher numbers of detector strain cells must also contain producer strain cells which generated higher amounts of L-tryptophan. Using the FACS device, capsules exhibiting high levels of fluorescence are separated and/or detected from the remainder of the capsule pool and can be dispensed individually into wells of a microtiter plate as shown in FIG. 2.

(7) Material

(8) Reagents: Saccharomyces cerevisiae Meyen ex E. C. Hansen (ATCC® 201168™, atcc), auxotrophic for leucine, histidine and tryptophan; pD1217; pRFSD-mCherry (Biomillenia produced plasmid) (ATUM) Yeast Transformation Kit YEAST1 (Sigma-Aldrich); Yeast Nitrogen Base Without Amino Acids yeast classification medium (Sigma). Yeast synthetic drop-out medium without amino acids (Sigma-Aldrich); Yeast Synthetic Drop-out Medium Supplements without tryptophan (Sigma-Aldrich); Yeast Synthetic Drop-out Medium Supplements without histidine (Sigma-Aldrich); E. coli with pSC101-trp.I15 (ATCC); Fluoresceinamine, isomer I (Sigma-Aldrich); N-Hydroxysulfosuccinimide sodium salt (Sigma-Aldrich); MES hemisodium salt (Sigma-Aldrich); N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (Sigma-aldrich); SnakeSkin™ Dialysis Tubing, 3.5K MWCO, 22 mm (Thermofisher).

(9) Equipment

(10) Microfluidics

(11) 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 picoliter 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.

(12) Preparation of a Microparticle Generating Chip

(13) Soft-lithography in poly(dimenthylsiloxane) (PDMS) was used to prepare the microparticle generating device (1). A SU-8 photoresist mould was used to prepare the PDMS. To prepare the SU-8 mold, a layer of SU-8 was spin coated on a silicon wafer. The wafer was covered by a designed mask and exposed to UV for a certain period of time. After full development and baking the wafer, the SU-8 mould was ready for PDMS. The SU-8 thickness for microparticle making chip in this example was 25 μm. The microparticle volume generated by the chip depends on the SU-8 thickness. To generate microparticles, the thickness can vary from 500 μm to 20 μm. After preparation of the SU-8 mould, PDMS was casted on the mould and bound to a glass slide. The inside part of microfluidic channel was treated by a commercial surface coating agent (Trichloro-(1H,1H,2H,2H-perfluorooctyl)-silane, Sigma-Aldrich) to make the channel surface hydrophobic or layer-by-layer deposition of polyelectrolytes (e.g. Poly(diallydimethylammonium chloride and Poly(sodium 4-strenesulfonate).

(14) Procedure

(15) Staining of Alginate with Fluoresceinamine

(16) For image analysis of the alginate microparticles after production. This will help see if the microparticles are core-shell.

(17) Alginate can be marked fluorescently using the EDC-NHS reaction. Fluorescent molecules can be conjugated with alginate by the binding the amino group of the fluorescent molecule to the carboxylic group of alginates. This reaction is catalyzed by carbodiimides such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). EDC binds to the carboxylic group to form a very reactive intermediate. The addition of N-Hydroxysulfosuccinimide (Sulfo-NHS) results in an ester Sulfo-NHS intermediate, less susceptible to hydrolysis than its EDC ester precursor. When adding a fluorescent molecule such as fluorescenamine the amine group reacts with the Sulfo-NHS intermediate interchanging the Sulfo-NHS with fluorescenamine to give marked alginate molecules.

(18) Protocol

(19) The labelling of alginate was performed as described in a previous method with modifications (Strand et al. J. Microencapsul. 2002(19):615-630).

(20) The addition of the reagents was performed under mixing with a magnetic stirrer. 2.05 g of alginate were dissolved in 60 mL of distilled water. 50 mL of MES buffer (0.2M MES, pH 5.5) were added to the alginate solution and left overnight. The pH of the MES buffer was adjusted by addition of 1M NaOH or 1M HCl. 0.208 g of EDC and 0.235 g of Sulfo-NHS were dissolved in 3 mL solutions of 9 mM MES buffer and were added successively to the alginate solution. The solution was stirred at room temperature for 30 minutes. 0.188 g of fluorescenamine are then dissolved in 4 mL of 9 mM MES and added to the alginate solution to a concentration of 4.5 mM. The solution was left for the reaction to occur for 18 hours.

(21) Unreacted molecules were removed by dialysis. The alginate solution was added to a 3,500 MWCO dialysis membrane and dialyzed against deionized water with changing of water bi-daily until the water is clear.

(22) The pH of the solution is then adjusted to pH 7.2-7.4 by addition of 1M NaOH or 1M HCl and freeze-dried with protection from light. The dried alginate, labelled with fluorescenamine, is then kept at room temperature and protected from light.

(23) Transformation of S. cervisae with red fluorescent protein

(24) Amplification of plasmid with specific mCherry gene sequence and addition of flanking Sapl restriction sites.

(25) For primer: tacacgtacttagtcgctgaagctcttctatgGTGAGCAAGGGCGAGGAG (SEQ ID NO.1)

(26) Rev primer: taggtacgaactcgattgacggctcttctaccCTAAAGCTTGTACAGCTCGTC (SEQ ID NO.2)

(27) Uppercase: sequences complementary to extremities of mCherry gene.

(28) Lowercase: sequences for addition of Sapl restriction sites flanking the PCR-product.

(29) Insertion into pD1217

(30) Sapl restriction enzymes, the PCR product, pD1217 and the reaction buffer are combined and incubated at 37° C. for 20 minutes. The plasmid is then transformed into competent E. coli and plated on culture plates of LB with kanamycin and incubated overnight at 37° C. A liquid culture complemented with kanamycin and inoculated with the competent cells containing pD1217-mCherry is prepared and the plasmid recovered using a miniprep kit.

(31) Yeast Transformation Protocol

(32) Yeast transformation protocol has been carried out according to the manufacturer's procedure (Sigma-Aldrich) using Yeast Synthetic Drop-out Medium Supplement without histidine for the last step.

(33) Yeast Transformation

(34) For yeast transformation, the following steps have been performed:

(35) 1. Prepare YPD and synthetic complete (SC) drop-out medium plates and autoclave them separately.

(36) 2. Inoculate yeast cells from plates into 20 ml of YPD medium in a 100 ml sterile flask.

(37) 3. Grow overnight with shaking.

(38) 4. Dilute cells from above culture into 100 ml of YPD medium until the OD600 is 0.3.

(39) 5. Pellet cells gently.

(40) 6. Resuspend in 7-8 ml of 1× TE-LiAc solution and rotate at 23° C. for 1-1.5 hours.

(41) 7. Add 10 μL of 10 mg/ml salmon testes DNA (Catalog Number D9156, Sigma-Aldrich) in sterile microfuge tubes designated for transformation and one for a negative control.

(42) 8. Add 0.1 μg of yeast plasmid DNA (to be studied) to each tube and 1000 of competent cells into each tube and then vortex.

(43) 9. Add 600 μL of fresh ly prepared PEG-TE-LiAc solution, vortex, and incubate at 30° C. for 30 minutes with shaking.

(44) 10. Optional-DMSO (Catalog Number D8418, Sigma-Aldrich) can be added to 10% (v/v); followed by heat shock for 15 minutes at 42° C.

(45) 11. Spin for 3 seconds, resuspend cells in sterile water and plate using appropriate SC drop-out medium.

(46) Growth of S. cervisae in Alginate Mixed with Yeast Synthetic Drop-Out Medium Supplemented with Yeast Synthetic Drop-Out Medium Supplements without Histidine

(47) Add marked alginate to 2% w/v to Yeast synthetic drop-out medium supplemeneted with Yeast Synthetic Drop-out Medium Supplements without histidine

(48) Inoculate with yeast and check red fluorescence, fluorescence microscope

(49) Negative control no inoculation, positive control in YPD with/without alginate

(50) Growth of E. coli

(51) Growth in Luria-Bertani (LB)

(52) Capsule Making with Trypto Deficient mCherry Producing Yeast and Trypto Producing E. coli

(53) 1. Culture E. coli transformed with pSC101-trp.I15 overnight in yeast Synthetic Drop-out Medium supplemented with yeast Synthetic Drop-out Medium Supplements without L-tryptophan.

(54) 2. Culture transformed yeast overnight in yeast Synthetic Drop-out Medium supplemented with yeast Synthetic Drop-out Medium Supplements without histidine, 2% w/v alginate, 100 mM Ca-EDTA.

(55) 3. Just before encapsulation the transformed yeast is washed with an isotonic buffer and resuspended in Synthetic Drop-out Medium supplemented with yeast Synthetic Drop-out Medium Supplements without tryptophan.

(56) 4. Produce core-shell capsules with yeast in the alginate matrix and E. coli in the core and resuspend in yeast Synthetic Drop-out Medium supplemented with yeast Synthetic Drop-out Medium Supplements without Tryptophan.

(57) 5. Check mCherry fluorescence during or just after encapsulation and after incubation under fluorescence microscope.