WO2013021035A1

WO2013021035A1 - Microfluids for cell-based assays

Info

Publication number: WO2013021035A1
Application number: PCT/EP2012/065625
Authority: WO
Inventors: Christoph Merten; Dominik EICHER; Nirupama RAMANATHAN; Vishal Gupta
Original assignee: European Molecular Biology Laboratory (Embl)
Priority date: 2011-08-09
Filing date: 2012-08-09
Publication date: 2013-02-14
Also published as: GB201209924D0

Abstract

A microfluidic apparatus, including at least one cell compartment for cultivating biological cells, and at least one microfluidic channel for transporting at least one substance. The cell compartment is separated from the microfluidic channel by at least one wall section, and the at least one wall section is permeable to the at least one substance. Such apparatus and associated method of operation circumvent limitations of droplet-based microfluidics, facilitating high content image analysis and easy accessibility for washing cells for antibody assays. The apparatus and method can be used to analyze biological cells from an environment, such as a sewage plant or soil.

Description

MICROFLUIDS FOR CELL-BASED ASSAYS

Field of the Invention

[0001] The present disclosure relates to a micro fluidic apparatus and a method for cell- based assays, as well as a method for using the microfiuidic apparatus to grow cells. The present disclosure relates in particular to a microfiuidic apparatus for cultivating biological cells and for providing substances to the cells. In one aspect of the invention, the biological cells are placed in at least one cell compartment, and the substances are provided to the biological cells by diffusion through walls of the cell compartment. In a further aspect of the invention, the biological cells are placed in a first species of droplets, and the substances are provided to the biological cells either by diffusion into the first species of droplets, or by fusing the first species of droplets with a second species of droplets.

Background to the Invention

[0002] Many biological environments (e.g. soil, human gut, natural water sources, etc.) are inhabited by complex, inhomogeneous populations of cells composed of different species. These inhomogeneous cell populations are termed "communities". In the human body, the number of non-human cells (e.g. different E. coli strains in the gut) exceeds by far the number of human cells. However, the characterization of the different cells or members of the community on the phenotypic level and genotypic level often requires homogenous populations. For example, whole genome sequencing of the members that are present at very low abundance within a given community has turned out to be difficult. Furthermore, particular ones of the phenotypes (e.g. an industrially-relevant enzymatic activity that can be detected in a soil sample) can rarely be assigned to a specific genotype during the assaying of inhomogeneous populations of the cells. As a result, many enzymatic activities have been observed (e.g. the conversion/metabolism of a chemical substrate), but neither the enzyme itself causing the enzymatic activity, nor the gene encoding the enzyme is known. Hence the industrial exploitation of the enzyme remains impossible. This phenomenon is discussed by Nichols et al "Use of Ichip for High-Throughput In Situ Cultivation of "Uncultivable" Microbial Species, Applied & Environmental Microbiology, April 2010, p 2445-2450.

[0003] In principle, the homogeneous cell populations of different species in the community can be obtained by generating single cell samples from the community. This can be done, for example by limiting dilution in which the community is significantly diluted such that any sample contains a single cell, followed by clonal expansion or recultivation. This second amplification step (clonal expansion/recultivation) is required as many analytical methods (e.g. whole genome sequencing, enzymatic assays) require more than a single cell for efficient readout. However, many of the cells present in communities in complex environments cannot be grown in an isolated form as the members need factors (e.g. small molecules, proteins) produced by other members of the community. In consequence, such cells have not yet been fully characterized.

Related art

[0004] Droplet microfluidics is an alternative to the use of conventional wells on microtiter plates. Droplet microfluidic systems meet the need for lower costs, higher throughput, and higher sensitivities of biological and medical analyses, as explained in Guo et al., "Droplet microfluidics for high-throughput biological assays", Lab on a Chip (2012), DOI: 10.1039/c21c21147e, by reducing reaction volumes. The droplet microfluidic system lends itself for cell growth assays, bacterial persistence screens, virus-host interactions, antibody screens, directed evolution for developing new enzymes and proteins, and single -template PCR (polymerase chain reaction).

[0005] Lecault et al. discuss the use of single cell analysis based on microfluidics in "Microfluidic single cell anaylsis: from promise to practice", Curr Opin in Chem Bio (2012), 16:1-10, for single cell gene expression measurements, single cell genome analyses, intracellular protein expression measurements and signalling kinetics, single cell growth, and response phenotypes. Single cell analysis is not suited for analysis of a heterogeneous symbiotic community of cells.

[0006] The droplet-based microfiuidics systems allow the generation of highly monodisperse (<3% polydispersity in terms of the volume) water-in-oil (w/o) droplets at rates of up to 10,000/s by flow- focusing a continuous aqueous phase with a second immiscible oil phase 1 -3. Over the last decade, the idea of using these droplets as micro reactors for parallelized reactions within a volume range of picoliters to nanoliters has been exploited in many different applications. These different applications include nanoparticle synthesis, protein crystallization, single molecule PCR, proteome analysis, clinical diagnosis on human physiological fluids, titration of anticoagulants and the encapsulation and screening of cells 4-13. Furthermore, several companies have been established for commercializing droplet-based microfluidics for various applications such as targeted sequencing (e.g., Raindance Technologies, GNUbio) and diagnostics (Droplet Diagnostics).

[0007] International Patent Application No. WO 2010/018499 (Philips) describes a method for providing a gradient of a substance to cells arranged in a micro fluidic channel as well as a microfiuidic device. The cells are localized by trapping them on an inner surface of the microfiuidic channel and incubated in a fluid solution. The fluid solution flows through the microfiuidic channel and provides a spatially and temporally stable gradient of the substance. The fluid solution in the microfiuidic channel is a one -phase solution.

[0008] U.S. Patent No. 7,452,726 (Chou et al, assigned to Fluidigm Corporation) discloses a system and method for the microfiuidic manipulation and/or detection of particles such as cells. The microfiuidic device is provided with a cell growth chamber having a cell inlet with an inlet valve to control fluid flow through the cell inlet into the cell growth chamber. A reagent inlet enables the inputting of a reagent into the cell chamber. [0009] International Patent Application No. WO 2009/149257 (University of Chicago) describes a method for sampling an aqueous environment by introducing a fluid through a first micro channel into an exchange region of a device. An array of plugs is formed in the exchange region and this array of plugs is directed into a second micro channel downstream of the exchange region. The content of at least one of the plugs is subsequently analyzed.

[0010] Nichols et al also teach in their paper a so-called Ichip that is an assembly of fiat plates containing multiple registered through-holes.

[0011] Park et al. present a droplet-based micro fiui die system that encapsulates and co- cultivates subsets of a heterogeneous community, using aqueous droplets dispersed in a continuous oil phase (Park et al.. Microdrop let-Enabled Highly Parallel Co-Cultivation of Microbial Communities. PLoS ONE (2011), 6(2):el7019). The droplets do not exchange nutrients or other molecules by diffusion. No growth occurred in droplets hosting one of two different E. coli auxotroph cells capable of surviving and proliferating only by exchanging molecules. The oil phase surrounding the droplets prevents the diffusion from one droplet to another. Hence, the cultivation environment in individual droplets was completely localized.

[0012] It is an object of the present invention to overcome the disadvantages of the prior art.

Summary of the invention

[0013] The present disclosure provides a method and an apparatus for providing at least one substance to one or more biological cell. The apparatus comprises at least one cell compartment for cultivating one or more biological cells. One or more cells may be present in one cell compartment and a plurality of cell compartments may be provided. The apparatus further comprises at least one micro fiuidic channel for transporting at least one substance. The cell compartment is separated from the at least one microfiuidic channel by at least one wall section, wherein the at least one wall section is permeable to the at least one substance. The permeable wall section allows for diffusion of the substance through the at least one wall section. No opening or fluid contact between the at least one channel and the at least one cell compartment is provided.

[0014] The one or more biological cells can be kept and cultivated in the at least one cell compartment. No perfusion of the cells is required and substances can be brought to cells using the permeable wall section.

[0015] The at least one channel and the at least one permeable wall section may be used for cell medium and substances of the cell media to diffuse to the cells.

[0016] The at least one channel and the at least one permeable wall section may also be used for transporting drugs or other substances to the cells. The at least one substance can be encapsulated in an aqueous plug, i.e. a liquid section comprising the substance is arranged between two oil sections along the at least one channel and the liquid section comprising the substance is brought to the permeable wall section. The at least one substance can then diffuse from the microfluidic channel to the cell compartment and to the at least one cell.

[0017] A plurality of aqueous plugs containing different substances can be applied along the microfluidic channel. A plurality of cell compartments may be arranged in a row or line parallel to the at least one microfluidic channel.

[0018] The cell compartments may be elongated in a direction substantially perpendicular to the at least one microfluidic channel or the at least one wall section.

[0019] The apparatus may comprise two microfluidic channels and a permeable wall section is provided between each one of the two microfluidic channels and the at least one cell compartment. A first microfluidic channel may be used for providing cell medium including for example nutrition to the cells. A second microfluidic channel may be used for providing test substances or drugs to the cells. [0020] The at least one cell compartment may additionally comprise a cell compartment inlet channel and a cell compartment outlet channel for exchanging a liquid surrounding the cell(s).

[0021] The micro flui die apparatus can be used for preparing biological cells for metagenomic analysis.

[0022] The disclosure further relates to a method for analysing at least one biological cell. The method comprises providing the at least one biological cell within a first one of a plurality of droplets of an aqueous solution surrounded by immiscible oil. The method further comprises diffusing at least one substance from at least one second one of the plurality of droplets to the first one of the plurality of droplets through the immiscible oil. 26.

[0023] The method may further comprise providing cell media in the at least one second one of the plurality of droplets and diffusing components of the cell media from the at least one second one of the plurality of droplets to the first one of the plurality of droplets through the immiscible oil (30).

[0024] The method may further comprise fusing the at least one second one of the plurality of droplets and the first one of the plurality of droplets.

Brief description of the drawings

[0025] The invention may be better understood when reading the following detailed description of examples of the present invention which is given with respect to the accompanying figures, in which:

[0026] Figure 1 shows an example of a microfluidic apparatus based on semi- compartmentalization;

[0027] Figure 2 shows a second example of a microfluidic apparatus according to the present disclosure; [0028] Figure 3 shows biological cells in the microfiuidic apparatus of Figure 2 after 0 to 156 hours;

[0029] Figure 4 shows biological cells in the microfiuidic apparatus of Figure 2 after 280hours;

[0030] Figure 5 shows a biological pathway in the cells tested:

[0031] Figure 6 shows the release of reactive oxygen from cells treated with different drugs: and

[0032] Figure 7 shows the correlation between the speed of the spin coater and the resulting thickness of different SU-8 photoresists.

[0033] Figure 8 shows an example of the microfiuidic apparatus used for metagenomics application.

[0034] Figure 9 shows an example of the microfiuidic apparatus using first ones of droplets for hosting biological cells and second ones of droplets for providing substances to the biological cells.

[0035] Figure 10 shows an example of the microfiuidic apparatus with a permeable wall allowing passage of components of media by diffusion.

Detailed description of the Invention

[0036] The invention will now be described on the basis of the drawings. It will be understood that the embodiments and aspects of the invention described herein are only examples and do not limit the protective scope of the claims in any way. The invention is defined by the claims and their equivalents. It will be understood that features of one aspect or embodiment of the invention can be combined with a feature of a different aspect or aspects and/or embodiments of the invention.

[0037] The present disclosure uses a method termed "semi-compartmentalization" and is based on an apparatus that can overcome at least some of the limitations of droplet- based microfluidics that were discussed in the introduction. The method and apparatus are based on biological cells grown in a static continuous aqueous phase inside cell channels or cell compartments, in which the biological cells can also attach to the surface of the cell compartments. This facilitates high content image analysis and also easy accessibility to wash the biological cells for antibody assays. Drugs or other substances are encapsulated in aqueous plugs and loaded into an adjacent micro channel from which the drugs or other substances, including so-called "community factors", diffuse towards the neighboring ones of the biological cells. The drugs or other substances are provided in a potentially aqueous environment and encapsulated between oil sections along the microfiuidic channel. This type of encapsulation is termed "aqueous plug" in this disclosure.

[0038] The oil used for the oil sections may be immiscible, in particular with aqueous solutions and may be preferentially fiuorinated.

[0039] Sections of the aqueous plugs, i.e., aqueous solutions comprising identical or different substances or substances at different concentrations, are separated by the oil sections. In this way, potentially thousands of drug or substance gradients can be generated, maintained and screened, as long as no fluid flow is required to supply nutrients in the cell channel (which would disturb the gradients and any form of drug compartmentalization). To demonstrate the feasibility of this approach, cultivated ones of the biological cells were held for up to 12 days in the microfiuidic apparatus without perfusion or exchange of media, but rather by exploiting the diffusion of media contents (drugs, community factors, etc.) through a permeable wall from a neighboring channel through which the media contents was continuously pumped (see Figure 1).

[0040] It is understood that the term "microfiuidic" as used herein refers to systems and devices with sub-millimeter sized structures through which reagents are infused and processed on a picoliter to a nanoliter scale. These microfiuidic systems have their manufacturing techniques originated from the semiconductor industry and are as such known in the art. The microfiuidic structures allow the precise spatial (μηι scale) and temporal (ms scale) control of microenvironments and this concept has been one of the major motivations for research in this field. Micro fluidics is also being exploited for high throughput assays on cells and multi-cellular organisms, as disclosed herein. [0041] Figure 1 shows an example of a micro fiuidic apparatus 10 based on the principle of semi-compartmentalization. The micro fiuidic apparatus 10 may be used without limitation for drug screening or for the analysis of biological cells or enzymes in different environments.

[0042] In one aspect of the disclosure, different substances (e.g. drugs factors A and B) are encapsulated into plugs 40, spaced out by immiscible oil 30 and stored in a first micro fiuidic channel 20 of the micro fiuidic apparatus 10. The substances or drug compounds A and B can diffuse from the plugs 40, through a wall 50 made of polydimethyl siloxane (PDMS), to a neighboring cell microfiuidic channel 60 hosting biological cells 70. The substances A and B diffuse through the PDMS wall 50 separating the first microfiuidic channel 20 from the cell microfiuidic channel 60, thus generating a concentration gradient of the substances in the PDMS wall 50 and the neighboring cell microfiuidic channel 60. A plurality of different substances, each with a concentration gradient, can be applied to the biological cells 70 in cell compartments 65 of the cell microfiuidic channel 60.

[0043] Cell media 85 is infused continuously through a separate media microfiuidic channel 80 and its contents diffuse towards the biological cells 70 in the cell compartments 65 in order to prevent perturbations of the gradients produced. Nutrients from the cell media 85 also diffuse through the PDMS wall 50 and reach the biological cells 70 in the cell channel 60 or the cell compartments 65. Direct perfusion to the biological cells 70 can be omitted.

[0044] The hydrophilicity and hydrophobicity of a particular compound also has an effect on diffusion through the PDMS wall 50. A more hydrophobic compound will diffuse through the PDMS wall 50 faster than hydrophilic compounds due to its solubility in PDMS material. The partition coefficient (log P) is the ratio between the solubility of molecules in organic solvent to water and can determine if the molecule is hydrophilic or hydrophobic. Shim J.U. et al. (201 1) have shown that small molecules with a higher log P value (hydrophobic compounds) are capable of diffusing through the PDMS wall 50 into droplets much faster than compounds with lower log P values (hydrophilic compounds).

[0045] The hydrophilicity of PDMS and the diffusion rates in PDMS may be modified by an oxygen plasma treatment of the PDMS wall 50 prior to use. The PDMS wall 50 may have a thickness typically of 5 to several hundred μηι but is not limited to these dimensions. Furthermore, the transparency of PDMS allows for optical analyses of the cells, e.g. by microscopy.

[0046] While the above example has been described with respect to PDMS as permeable material for the wall 50, which is permeable for substance diffusion, any other material with appropriate diffusion properties may be used for the wall 50, such as but not limited to polytetrafiuoroethylene (PTFE). Diffusion properties of larger molecules or proteins may be limited. Polymeric material may be preferred. If imaging of the biological cells 70 is required, a transparent material may be used.

[0047] Figure 2 shows an example of the micro fluidic apparatus 10 for the cultivation of the biological cells 70 without direct perfusion of the cell media 85. The biological cells 70 are cultivated in the cell microfiuidic channel 60 with a plurality of circular chambers 62 and the cell media 85 is infused into the adjacent media microfiuidic channel 80 from which the nutrients in the cell media 85 diffuse towards the biological cells 70. The microfiuidic apparatus 10 of Figure 2 was designed to assess the possibility of cultivating the biological cells 70 without perfusing or exchanging the cell media 85 (which would abolish any concentration gradient).

[0048] To test the influence of plasma treated surfaces on growth of the biological cells 70, HEK 293 cells were seeded directly onto the glass and PDMS surfaces immersed in the cell media 85. Since the biological cells 70 have to be injected into the microfiuidic cell channel 60 using a syringe, the effect of shear force was tested in the same experiment by forcing the biological cells 70 through a needle onto the glass surface and the PDMS surface. [0049] Figures 3 and 4 show cells cultivated in the micro fluidic device washed with the cell media 85. Time lapse images showing the biological cells 70 in the micro fluidic devices 10 from 0 h (when the biological cells 70 were loaded) up to 156 h (see Fig. 3) and after 280 h (see Fig. 4). Scale bars represent 50 μηι.

[0050] The micro fluidic device 10 of the present disclosure can be used to prepare biological cells 70 from a heterogeneous community for metagenomic analysis. The individual members of the heterogeneous community from an environment are encapsulated into aqueous micro compartments, e.g. aqueous ones of droplets 110 surrounded by immiscible oil 30, in the cell microfiuidic channel 60 of the micro fluidic device 10, as shown in Fig. 8. Nutrients, or community factors, for the biological cells 70 forming the individual members are provided in the media microfiuidic channel 80 and can be diffused to the cell microfiuidic channel 60 through the permeable wall 50. The complex inhomogeneous community can continue to be grown in a continuous aqueous phase. Over time, the community factors produced by the complex inhomogeneous community diffuse through the permeable wall 50 into the droplets 1 10 and provide the encapsulated biological cells 70 with, e.g., nutrients, required for efficient growth.

Examples

[0051] Example 1 : A drug library comprising 12000 compounds is converted into an array of compound plugs spaced out by sections of immiscible oil 30 (s. for example reference 15, Clausell-Tormos et al., 2010). The drug library is screened for cytostatic properties using the microfiuidic apparatus 10 and method. For this purpose, the biological cells are cultivated for several days and monitored for cell proliferation. Furthermore, at the end of the screen, the cells chamber is flushed with fluorescently- labelled antibodies and subsequently with a washing solution to stain for specific cell markers. Then, an optical readout, e.g. a fluorescence readout, is performed. Assuming a total assay volume of approximately 6 microliters for the entire chip, there is an almost 20000-fold reduction in consumables and costs compared to microtiter plate -based formats requiring an assay volume of ΙΟμΙ for each sample. Furthermore, the small assay volumes also allow the screening of samples that are not available at large scale such as primary cells (e.g., patient material, tumour biopsies) or stem cells.

[0052] Similar screens can be performed to screen drugs for their influence on cellular senescence, stem cell proliferation and cell reprogramming (e.g., the generation of induced pluripotent cells).

[0053] Example 2: A drug screen is performed as described in Example 1 , with the following modifications: The array of compound plugs 40 is loaded into the first microfluidic channel 20 prior to seeding/cultivating any of the biological cells 50 in the same microfluidic device 10. The microfluidic device 10 is incubated at elevated temperature to trigger diffusion of the compounds into the surrounding material. Subsequently the array of plugs 40 is flushed out of the first microfluidic channel 20 and the biological cells 50 are seeded into the microfluidic channel 20, hence growing on specific compound spots, previously generated by diffusion.

[0054] Example 3: A drug screen is performed as described in Example 1 , with the following modifications: The array of compound plugs 40 is loaded into the first microfluidic channel 20 prior to seeding/cultivating any of the biological cells 50 in the same microfluidic device 10. The microfluidic device 10 is exposed to an electric field to achieve migration of the compounds into the surrounding channel by electrophoretic forces. Subsequently the array of plugs 40 is flushed out of the first microfluidic channel 20 and the biological cells 50 are seeded into the same channel 20, hence growing on specific compound spots, previously generated by application of the electric field.

[0055] Example 4 (as shown in Fig. 8): The microfluidic device 10 of this disclosure can be used for screening water samples from a sewage plant for specific enzymatic activities (e.g. the degradation of toxic chemicals). These water samples are heterogeneous, i.e. contain many different species (members) in the community of the sewage plant. The water samples are diluted in media and the species from the water samples are subsequently encapsulated into the droplets 1 10 at the single cell level, together with components of an assay for the enzymatic activity of choice (e.g. using a fluoro genie substrate generating a fluorescence signal upon enzymatic conversion). The media is an isotonic buffer that has a composition for avoiding bursting or contraction of the biological cells due to osmosis. The encapsulation of the biological cells from the sewage plant is done, for example, in a fiuorinated oil 30 or hydrocarbon oils 30 with surfactants added for stabilizing the droplets 110 (s. reference 12, Clausell-Tormos et al., 2008). The density of the species in the initial water sample is not exactly known. The samples of the species can be "over-diluted" so that only a small fraction of the droplets 1 10 is occupied by a species. Subsequently the droplets 110 are stored in the cell microfiuidic channel 60 next to the media microfluidic channel 80.

[0056] It is conceivable that the media microfiuidic channel 80 is filled with an undiluted heterogeneous water sample as the cell media 85. During incubation for several hours or even days, any so-called community factors (i.e. nutrients) produced in the heterogeneous water sample diffuse through the permeable wall 50 separating the cell microfiuidic channel 60 from the media microfiuidic channel 80 into the droplets 1 10. This allows proliferation of the species encapsulated in the droplets 1 10 that cannot be grown in isolated form.

[0057] It is furthermore conceivable that the undiluted heterogeneous water samples are provided in second ones of the droplets 1 10 (the droplets 110 hosting the biological cells 70 from the diluted water sample are called 'first ones of droplets 110' in the remaining description of Example 4). The second ones of the droplets 110 host multiple ones of the biological cells 70. These biological cells 70 belong to either one single or to different ones of species.

[0058] The second ones of the droplets 110 do not contain any assay reagents and hence do not interfere with the later optical readout for enzymatic activity. To allow for a clear distinction between the second ones of the droplets 110 and the first ones of the droplets 1 10, the second ones of the droplets 1 10 can also be generated in a way that their size differs, e.g. by using a bigger droplet maker for the second the droplets 1 10. [0059] Both the first ones of the droplets 1 10 and the second ones of the droplets 1 10 are incubated together in the cell compartment 65 or in the cell microfiuidic channel 60. During incubation for several hours or even days, community factors produced in the second ones of the droplets 1 10 diffuse through a thin layer 31 of the immiscible oil 30 (see Fig 9A), separating the first ones of the droplets 110 and the second ones of the droplets 1 10, from the second ones of the droplets 1 10 to the first ones of the droplets 1 10. This enables growth of the biological cells 70 in the first ones of the droplets 1 10 that cannot grow in isolated form.

[0060] It is furthermore conceivable to provide the community factors or nutrients to the first ones of the droplets 1 10 by droplet fusion. For example, supernatants can be taken from the complex heterogeneous community (e.g. after centrifugation) and encapsulated into supernatant ones of the droplets 1 10 that are fused with the first ones of the droplets 1 10 hosting individual ones of the biological cells 70. The community factors are not supplied continuously in this aspect of the invention, but rather stepwise by the fusion of the supernatant ones of the droplets with the first ones of the droplets 1 10.

[0061] Subsequent to the incubation period, a optical readout of the individual first ones of the droplets 110 is performed and those first ones of the droplets 1 10 showing a high fluorescence signal (indicating the desired enzymatic activity), as well as the first ones of the droplets 1 10 showing a low fluorescence signal are specifically (and separately) collected using a fluorescence activated droplet sorting method, as described in reference 17, Baret et al, 2009.

[0062] The sorted first ones of the droplets 110 of each type (high and low fluorescence) are pooled, broken and the content of the first ones of the droplets 1 10 applied to whole genome sequencing. This allows the identification of genes present in all of the positive (high fluorescence) samples, but absent in the negative samples. Such genes are good candidate genes for the enzymatic activity of interest. Further downstream, biochemical characterization of the corresponding gene products ultimately enables assignation of the enzymatic activity to one or more of the candidate genes and thus the biotechnological production of the enzyme itself.

[0063] Example 5: The micro fluidic device 10 of the disclosure can be used for whole genome sequencing of species present in soil samples. The soil samples are diluted in a soil media and the species from the diluted soil samples are encapsulated into the droplets 1 10 at the single biological cell level. The term "soil media" in this context means a buffer or a buffer with soil extracts. The buffer with soil extracts can be made by rinsing a buffer over the soil to dissolve ingredients from the soil into the buffer. Optionally additional nutrients can be added to the soil media. Subsequently the droplets 1 10 are stored in the cell micro fluidic channel 60 next to the media microfiuidic channel 80 filled with (undiluted) heterogeneous soil samples. Optionally the undiluted soil media can be enriched with water content.

[0064] During incubation for several hours or even days, nutrients or community factors produced in the heterogeneous soil sample diffuse through the permeable wall 50 separating the cell micro fluidic channel 60 from the media microfiuidic channel 80 into the droplets 1 10. This allows proliferation of the encapsulated species that cannot be grown in isolated form. Subsequent to the incubation period, qPCR reagents are delivered to the droplets 110 (e.g., by fusion with a second droplet species containing these qPCR reagents) and the droplets 1 10 are thermocycled (either on-chip or off-chip). In the example, the PCR mix contains specific primers for ribosomal 16s RNA genes of species that have not yet been fully sequenced (for many species the 16s RNA sequence is known, while the entire genome has not yet been sequenced due to the inability to generate homogeneous populations). Furthermore, the mix includes SYBR green. As a direct result, only those droplets 1 10 hosting the species with the particular 16s RNA sequences show an increased fluorescence signal after PCR. These droplets 1 10 with increased fluorescence species are specifically sorted and collected by the fluorescence- activated droplet sorting method of reference 17, Baret J-C et al., 2009. Subsequent to this step, the sorted droplets are pooled, broken and the content applied to whole genome sequencing. As a result, the genomic sequence of the species for which so far only sequence fragments have been available (e.g. the 16s RNA sequences) can be obtained.

[0065] Example 6 (as shown in Fig. 9): It is conceivable to provide E. coli cells 70 expressing β-Galactosidase within the first ones of the droplets 1 10 comprising minimal media 85 and a fiuorogenic substrate for β-Galactosidase, e.g. fluorescein di-β-ϋ- galactopyranoside, wherein one of the first ones of the droplets 110 hosts one single or no E. coli cell 70. The E. coli cells 70 require additional glucose for growth. In a first set-up (see Fig. 9A), the first ones of the droplets 110 hosting the E. coli cells 70 are placed in the cell compartment 65 in the immiscible oil 30. Second droplets 1 11 containing minimal media 85 with 1% glucose are added to the cell compartment 65. The glucose diffuses from the second droplets 1 11 through the immiscible oil 30 to the first ones of the droplets 110. The E. coli cells 70 can metabolize the glucose and subsequently proliferate. The β-Galactosidase produces fluorescein that can be detected, e.g. by fluorescence microscopy. The more fluorescein is produced the stronger the fluorescence.

[0066] In a second set-up (see Fig. 9B), the second droplets 1 11 are not provided with glucose. Therefore, no glucose can diffuse from the second droplets 1 1 1 to the first ones of the droplets 1 10 hosting the E. coli cells 70. As a result, the E. coli cells 70 cannot grow and less fluorescein is produced by β-Galactosidase. This results in a weaker fluorescence signal. The comparison of the fluorescence signals in the first and the second set-up indicates whether diffusion between the first ones of the droplets 110 hosting the E. coli cells 70 and the second droplets 1 11 occurs.

[0067] Example 7 (as shown in Fig. 10): the micro fluidic apparatus 10 comprises a plurality ofgenetically modified HeLa cells 70 in the cell microfluidic channel 60. The growth medium 85 is placed in the medium microfluidic channel 18, which is separated from the cell microfluidic channel 60 by the permeable wall 50, made of PDMS.

[0068] A drop channel 200 is also separated from the cell microfluidic channel 60 by a second wall 50'. The drop channel 200 comprises a tetracycline drop 220 in a first part of the drop channel 200 and a sulforhodamine drop 230 in a second part of the channel 200. The tetracycline drop 220 is separated from the sulforhodamine drop 230 by an oil section 210. Results show that the tetracycline drop 220 allows tetracycline to diffuse into the cell microfluidic channel 60 in the area adjacent to the tetracycline drop 220. The tetracycline induces expression of GFP in the genetically modified HeLa cells 70 in the area adjacent to the tetracycline drop 220. No expression of the GFP is induced in the area of the cell microfluidic channel 60 adjacent to the sulforhodamine drop 230. The sulforhodamine drop 230 acts as a control. This demonstrates that the tetracycline is able to pass through the wall 50' and that the genetically modified HeLa cells 70 grow because components of the growth medium 85 defusing through the wall 50.

MICROFLUIDIC DEVICE FABRICATIONS AND EXPERIMENTAL SET UP

[0069] Device fabrication by lithography: The microfluidic device 10 fabrication was done using photolithography. A dried silicon wafer (Siltronix) was used as a substrate on which a layer of SU-8 negative photoresist (Microchem) was deposited using a spin coater (Laurell). The spin speed determines the thickness of the photoresist layer and was adjusted accordingly (see Fig. 7).

[0070] Following spin coating and prior to exposure, soft baking hardened the photoresist layer. The time of soft baking is proportional to the thickness of the photoresist layer. Table 1 shows the correlation between the thickness of the photoresist layer and the required soft baking time.

Table 1: SOFT BAKE TIMES

115-150 5 20-30

160-225 7 30-45

230-270 7 45-60

280-550 7-10 60-120

[0071] The chip layout was designed using AutoCAD software and subsequently printed as a mask. The pattern was projected onto the photoresist layer, resulting in specific polymerization in the exposed regions. The exposure dose correlates with the thickness of the photoresist layer and can be determined according to Table 2. UV exposure was done using an MA-45 (Suess) mask aligner.

Table 2: EXPOSURE DOSE

[0072] To complete the photoreaction, another post exposure baking step was performed. Table 3 shows the time needed for post exposure baking according to the thickness range.

Table 3: POST EXPOSURE BAKE TIMES Post exposure bake times

Thickness

(65^°C) (95 ^°C)

Microns

Minutes Minutes

25-40 1 5-6

45-80 1-2 6-7

85-110 2-5 8-10

115-150 5 10-12

160-225 5 12-15

230-270 5 15-20

280-550 5 20-30

[0073] SU-8 developer (Microchem) was used to develop the mold that solubilizes the non-polymerized photoresist layer leaving the pattern on the substrate. The time for developing varies according to the thickness (Table 4).

Table 4: DEVELOPMENT TIMES USING SU-8 DEVELOPER

[0074] For multi-layer designs, the thinner layer was prepared first. Following exposure of the first layer, around 10 ml of SU-8 photoresist (specifications according to the desired depth; see Fig. 7 for details) was poured directly on the wafer with the first layer and was spun to the required thickness of the second (thicker) layer. All other procedures were the same as for single layer molds except that an additional alignment was done before exposing. The alignment was done with the help of the microscope optics on the mask aligner and the alignment marks (fiducials uniformly designed on both layers) on the mask. Subsequently, multi-layer molds were developed.

[0075] The quality of the mold was checked under the microscope. The entire photolithography procedure was carried out in a clean room that was UV light protected and hence the developing process can be done as long as the mold was not exposed to UV light. After the entire non-polymerized photoresist layer was washed away, hard baking was done remove any cracks. This was done by gradual heating the mold to high temperature of about 150°C and then cooling the mold by simply switching off the hot plate. The mold thus prepared was subsequently used for producing the microfiuidic devices 10.

[0076] Micro-molding of PDMS (polydimethyl siloxane) was done by mixing the polymer base Sylmar 184 and the curing agent in the ratio 9:1. The mixture was then poured on the mold, degassed using a vacuum desiccator and allowed to solidify at 65 °C overnight. Once the PDMS solidified, it was carefully cut using a scalpel and peeled off the mold. Access holes to channels were punched using biopsy punches (Harris Unicore). The PDMS was then covalently bonded to a glass substrate by bringing both in contact with each other soon after oxygen plasma treatment using a Diener Femto plasma oven.

[0077] Microfiuidic set-up: Polytetrafluoroethylene (PTFE) tubing was connected to BD plastic syringes via needles. The syringes were filled, free of air, with the reagents that have to be infused into the device. The filled syringes were then mounted onto syringe pumps (Havard Apparatus PHD 2000). Following this, the syringes were primed (to avoid air infused) by infusing until liquid was pumped out through the other end of the tubing that was then connected to the microfiuidic device. The flow of liquid inside the micro fluidic device 10 was monitored using a light microscope. Images and videos were recorded using a Mikrotron camera and Motion Blitz control software. Pumps were either controlled manually or using a Labview program.

[0078] Loading of cells: HEK (human embryonic kidney) 293 cells were loaded in the micro fluidic device 10 using syringe pumps (Havard Apparatus PHD 2000) at a flow rate of 500 μΐ hr. Before loading cells, the chip was soaked overnight in media at 65°C. This was done to avoid evaporation of the infused reagents or their aspiration into the PDMS mesh. Any leached un-polymerized PDMS in the microfiuidic channels was washed by purging in 1 ml of media at a flow rate of 150 μΐ hr. The cell density used for loading was 3 x 106 cells/ml. The biological cells 50 were maintained in suspension within the syringes by constant stirring using a custom-made magnetic stirrer. The entire procedure of loading the biological cells 50 was carried out in sterile conditions inside a laminar flow hood. The entire chip loaded with the biological cells 50 was kept immersed in media throughout experimentation and incubated at 37°C under a 5% C0₂ atmosphere. While HEK 293 cells were used in the example shown, a person skilled in the art may apply any other suitable biological cell lines.

[0079] While the above examples have been shown with specific biological cells and a cell preparation, a person skilled in the art will understand that other biological cells or specimens can be equally used in the practice of the present disclosure. Fabrication and cell culturing and treatment methodology can be modified as known in the art.

References

1. Umbanhowar PB, Prasad V, Weitz DA. Monodisperse emulsion generation via drop break off in a cofiowing stream. Langmuir. Jan 25 2000; 16(2):347-351.

2. Thorsen T, Roberts RW, Arnold FH, Quake SR. Dynamic pattern formation in a vesicle-generating microfiuidic device. Phys Rev Lett. Apr 30 2001; 86(18):4163-4166.

3. Anna SL, Bontoux N, Stone HA. Formation of dispersions using "flow focusing" in microchannels. Applied Physics Letters. Jan 20 2003; 82(3):364-366.

4. Wheeler A, Moon H, Bird C, et al. Digital microfiuidics with in-line sample purification for proteomics analyses with MALDI-MS . Anal. Chem. 2005; 77(2):534- 540.

5. Beer N, Hindson B, Wheeler E, et al. On-chip, real-time, single-copy polymerase chain reaction in picoliter droplets. Nat. Methods. 2006;3:551-559.

6. Srinivasan V, Pamula VK, Fair RB. Droplet-based microfiuidic lab-on-a-chip for glucose detection. Analytica Chimica Acta. Apr 1 2004; 507(1): 145-150.

7. Chen DLL, Ismagilov RF. Microfiuidic cartridges preloaded with nanoliter plugs of reagents: an alternative to 96-well plates for screening. Current Opinion in Chemical Biology. Jun 2006; 10(3):226-231.

8. Song H, Li H, Munson M, Van Ha T, Ismagilov R. On-chip titration of an anticoagulant argatroban and determination of the clotting time within whole blood or plasma using a plug-based microfiuidic system. Anal. Chem. 2006; 78(14):4839-4849.

9. Martin K, Henkel T, Baier V, et al. Generation of larger numbers of separated microbial populations by cultivation in segmented-fiow microdevices. Lab on a Chip. 2003; 3(3):202-207.

10. Grodrian A, Metze J, Henkel T, Martin K, Roth M, Kohler J. Segmented flow generation by chip reactors for highly parallelized cell cultivation. Biosensors and bioelectronics. 2004; 19(1 1): 1421-1428.

1 1. Huebner A, Srisa-Art M, Holt D, et al. Quantitative detection of protein expression in single cells using droplet microfiuidics. Chemical communications. 2007; 2007(12):1218-1220. 12. Clausell-Tormos J, Lieber D, Baret J, et al. Droplet-based microfluidic platforms for the encapsulation and screening of mammalian cells and multicellular organisms. Chemistry & biology. 2008; 15(5):427-437.

13. Frenz L, El Harrak A, Pauly M, Begin-Colin S, Griffiths AD, Baret JC. Droplet- based microreactors for the synthesis of magnetic iron oxide nanoparticles. Angew Chem Int Edit. 2008; 47(36):6817-6820.

14. Vyawahare S, Griffiths AD, Merten CA. Miniaturization and parallelization of biological and chemical assays in microfluidic devices. Chem Biol. Oct 29 2010; 17(10): 1052-1065.

15. Clausell-Tormos J, Griffiths AD, Merten CA. An automated two-phase microfluidic system for kinetic analyses and the screening of compound libraries. Lab Chip. May 21 2010; 10(10): 1302-1307.

16. Shim J.U, Patil S.N, Hodgkinson J.T, Bowden S.D, Spring D.R, Welch M, Huck W.T.S, Hollfelder F, Abell C. 2011. Controlling the contents of microdroplets by exploiting the permeability of PDMS. Lab Chip 11 : 1132-1 137.

17. Baret JC, Miller OJ, Taly V, Ryckelynck M, El-Harrak A, Frenz L, Rick C, Samuels ML, Hutchison JB, Agresti JJ, Link DR, Weitz DA, Griffiths AD. Fluorescence- activated droplet sorting (FADS): efficient microfluidic cell sorting based on enzymatic activity. Lab Chip. 2009; 9(13): 1850- 1858.

Claims

A micro fiui die apparatus (10), the apparatus comprising:

at least one cell compartment (65) for cultivating one or more biological cells; at least one microfiuidic channel (20) for transporting at least one substance, wherein the at least one cell compartment (65) is separated from the at least one microfiuidic channel (20) by at least one wall section (50), wherein the at least one wall section (50) is permeable to the at least one substance.

The microfiuidic apparatus (10) of claim 1 , wherein the at least one microfiuidic channel (20) is filled with at least one aqueous solution (40) encapsulated between sections of oil (30) along the channel.

The microfiuidic apparatus (10) of claim 1 or 2, comprising a plurality of cell compartments (65) substantially arranged in a row.

The microfiuidic apparatus (10) of claim 3, wherein the at least one microfiuidic channel (20) is arranged along the row of the plurality of cell compartments (65), and wherein a plurality of permeable wall sections (50) is provided between the plurality of cell compartments (65) and the at least one microfiuidic channel (20).

The microfiuidic apparatus (10) of any one of claims 3 or 4, comprising a first microfiuidic channel (20) and a second microfiuidic channel (80), wherein the first microfiuidic channel (20) and the second microfiuidic channel (80) are arranged substantially parallel to the row of the plurality of cell compartments (65).

The microfiuidic apparatus (10) of claim 5, wherein at least one first permeable wall section (50) is provided between the first microfiuidic channel (20) and the plurality of cell compartments (65) and wherein at least one second permeable wall section (50) is provided between the second microfluidic channel (80) and the plurality of cell compartments (65).

7. The microfluidic apparatus (10) of any one of the preceding claims, wherein the at least one wall section (50) is made from polydimethyl siloxane (PDMS).

8. The microfluidic apparatus (10) of any one of the preceding claims, wherein the at least one cell compartment (65) is elongated in a direction perpendicular to the at least one wall section (50).

9. The microfluidic apparatus (10) of any one of the preceding claims, wherein the at least one cell compartment (65) comprises a cell compartment inlet channel and a cell compartment outlet channel.

10. The microfluidic apparatus (10) of any one of the preceding claims, wherein the at least one microfluidic channel (20) is adapted to provide community factors to the at least one cell compartment 65).

1 1. The microfluidic apparatus (10) of any one of the preceding claims, further comprising a plurality of at least one cell compartments (65) having different contents.

12. The microfluidic apparatus (10) of any one of the preceding claims, wherein the cell compartment is in use formed by biological cell containing droplets (1 10).

13. A method for providing at least one substance to at least one biological cell (70), the method comprising:

providing the at least one biological cell (70) in at least one cell compartment (65) with a permeable wall section (50);

transporting the at least one substance in a microfluidic channel (20) to the permeable wall section (50); diffusing the at least one substance from the micro fluidic channel (20) through the permeable wall section (50) to the at least one cell compartment (65).

14. The method of claim 13, wherein transporting the at least one substance comprises encapsulating an aqueous solution (40) comprising the at least one substance between two oil sections (30) along the microfluidic channel.

15. The method of claim 14, further comprising encapsulating a plurality of substances in an aqueous solution (40), each one of the plurality of substances encapsulated between two oil sections (30) along the microfluidic channel (20).

16. The method of claim 15, further comprising arranging the plurality of substances in the aqueous solution (40) along a row of a plurality of the at least one cell compartment (65).

17. The method of any one of claims 13 to 16, further comprising providing a cell media (85) in a cell media microfluidic channel (80), wherein a permeable wall section (50) is provided between the cell media microfluidic channel (80) and the at least one cell compartment (65).

18. The method of any one of claims 13 to 17, further comprising a step of limiting dilution of a sample comprising the at least one biological cell (70) prior to providing the at least one biological cell (70) to the at least one cell compartment (65).

19. The method of any one of claims 13 to 18, the providing of the at least one biological cell (70) further comprising encapsulating the at least one biological cell (70) in a droplet (110) of an aqueous solution surrounded by immiscible oil (30).

20. The method of claim 19, further comprising providing the cell media (85) in a second droplet (110) in the at least one cell compartment (65), and diffusing components of the cell media (85) from the second droplet (110) to the droplet (1 10) hosting the at least one biological cell (70) through the immiscible oil (30).

21. The method of claim 20, further comprising providing the cell media (85) in the second droplet (1 10) in the at least one cell compartment (65), and fusing the second droplet (110) hosting the cell media (85) and the first droplet (110) hosting the at least one biological cell (70).

22. The method of any one of claims 13 to 21 , wherein a plurality of the at least one biological cell (70) is extracted from an environment.

23. The method of any one of claims 13 to 22, wherein the at least one substance is a community factor enabling the growth of the at least one biological cell (70).

24. The method of any one of claims 13 to 23, wherein the community factor is obtained from the environment from which the plurality of the at least one biological cells (70) is extracted.

25. A method for analysing at least one biological cell (70), the method comprising: providing the at least one biological cell (70) within a first one of a plurality of droplets (1 10) of an aqueous solution surrounded by immiscible oil (30);

diffusing at least one substance from at least one second one of the plurality of droplets (110) to the first one of the plurality of droplets (1 10) through the immiscible oil (30).

26. The method according to claim 25, further comprising providing cell media (85) in the at least one second one of the plurality of droplets (1 10), and diffusing components of the cell media (85) from the at least one second one of the plurality of droplets (1 10) to the first one of the plurality of droplets (1 10) through the immiscible oil (30).

27. The method of claim 26, further comprising fusing the at least one second one of the plurality of droplets (1 10) and the first one of the plurality of droplets (110).

28. The method of any one of claims 25 to 27, wherein the first ones of the plurality of droplets comprise substantially single ones of the at least one biological cell (70).

29. The method of any one of claims 25 to 27, wherein the second ones of the plurality of droplets comprise a plurality of different ones of the at least one biological cells (70).

30. The method of any one of claims 25 to 29, wherein the first ones of the plurality of droplets substantially comprise single cells and the second ones of the plurality of droplets comprise multiple cells.

31. Use of the micro fluidic device and the method of any of the preceding claims for the analysis of biological cells (70) from an environment.

32. The use of claim 31 wherein the environment is a soil sample or water from a sewage plant.