WO2014198994A1 - Procédé d'analyse rapide de l'activité cellulaire - Google Patents

Procédé d'analyse rapide de l'activité cellulaire Download PDF

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Publication number
WO2014198994A1
WO2014198994A1 PCT/ES2014/070492 ES2014070492W WO2014198994A1 WO 2014198994 A1 WO2014198994 A1 WO 2014198994A1 ES 2014070492 W ES2014070492 W ES 2014070492W WO 2014198994 A1 WO2014198994 A1 WO 2014198994A1
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cells
microspheres
cell
activity
growth
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PCT/ES2014/070492
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English (en)
Spanish (es)
Inventor
Ángel CEBOLLA RAMIREZ
Alba MUÑOZ SUANO
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Biomedal, S.L.
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Publication of WO2014198994A1 publication Critical patent/WO2014198994A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses

Definitions

  • the sector of the technique to which this invention can be applied preferably comprises the research and development sector in biomedicine and biotechnology, in which the analysis of the specific characteristics of different cell types is common. Its use can be extended to the clinical diagnosis sector, the innovative therapies sector and in another field that is different from the environmental and ecological impact sector.
  • this invention can be applied to processes of analysis of a multitude of cellular variables, whether viability, specific expression, production of molecules of interest or descriptive characteristics, whether for basic research purposes, biotechnological advances, clinical applications in a wide range or industrial purposes.
  • the object of this patent is based on cellular characterization processes.
  • the cellular characterization allows the description and identification of different cell types within a heterogeneous population or deviations in an initially homogeneous population, in addition to the identification of mutants, highly secretory or producing clones, hydrolytic capacities, important for biotechnological developments as well as for the improvement of industrial processes.
  • the main problem associated with the identification of the phenotypic characteristics of microorganisms or cell populations is the time required for their determination. In most cases, this determination is subject to a specific growth of said cells under different test conditions.
  • Another clear example of this delay in phenotypic cell analysis is the identification of producing cells, such as hybridomas, eukaryotic cells producing monoclonal antibodies, in which the standard time from fusion to the identification of positive clones is between 2 and 4 weeks, depending on the rate of duplication of the cells (Milstein, C. Bioessays. 1999; 21: 966-73).
  • microencapsulation in agarose or polymeric hydrogels of cells within microspheres preferably between 30 and 1000 microns it is possible to compartmentalize individualized cells and perform growth studies or other cellular activities using nutrients or reagents in low quantity.
  • a flow cytometer is needed.
  • this instrument is usually expensive, far exceeding 50,000 euros and needing very advanced training for its use.
  • the cytometers that can be used for the analysis of microspheres of 30 to 200 microns, such as the COPAS of Biometric Union cost more than 200,000 euros.
  • This technology has the advantage of individually analyzing cell growth, reducing the number of divisions necessary to make visible phenotypic differences between one cell and another. Although it is also true that it has many disadvantages.
  • encapsulations are performed with agarose, which needs a high temperature in order to be used, subjecting the cells to temperatures above 40 ° C.
  • the procedure for the synthesis of the microspheres is by emulsion with an oil, which may necessitate different subsequent steps for the removal of the oil with the consequent cellular suffering and Inability to use the system for cells more sensitive to temperature or stress.
  • a problem added to the system is the variability in the size of the capsules produced, since using the emulsions it is very difficult to control the size of the capsule and, therefore, the amount of cells that can accommodate it.
  • the results are obtained through flow cytometry, a technique that, although very precise and consistent, has the problem of the maximum size that can be analyzed. Taking into account the aforementioned about the little control over capsule size when produced by emulsion, a previous step is required to the filtering analysis, which can lead to an error in the analysis by discarding a population of capsules by their size.
  • Flow cytometry is also a complex technique that requires high investment and specific training for laboratory operators.
  • the generation of monodisperse microspheres can be done with different technologies. For example with ultrasonic nozzles, or in a preferred case, with fluid focusing technology (Flow Focusing).
  • Flow Focusing is a microfluidic manipulation technology for the generation of spheres or monodisperse drops, based on the production of capillary microbeads that are "focused" towards a hole (US6,464,886).
  • This technology allows to design and produce microparticles with selected size, structure and composition.
  • This technology has developed its own instrument (Cellena ®) that allows encapsulation by FlowFocusing of cells maintaining its viability through the use of an ionic gel polymer such as alginate, widely reported as biocompatible in other fields of research and development such as cell therapy.
  • the present invention describes a method of rapid cell characterization by microencapsulation of cells characterized in that the determination of cell populations, as well as the different products secreted or synthesized by said populations are detected and identified in the capsules themselves or spheres by means of microscopic detection systems, which do not need additional training of personnel for their management and also require less time for analysis, than cytometric techniques.
  • an advantage of the methods described in this invention is the reduction of the total time necessary for a cellular determination, whether it is the determination of the proliferative, secretory or catalytic capacity, among others, of the cells object of the analysis, without resorting to Instrumentation of high economic cost, such as a flow cytometer, or to personnel with specific training for it.
  • Another advantage is that microscopic detection can encapsulate more than one cell per capsule, since the detection technician himself identifies each cell individually, while cytometry has to encapsulate a single cell or microorganism per capsule since said apparatus is not able to differentiate between a large colony or two or three small colonies, identifying each event exclusively as a single cell or a single microorganism, even if the capsule contains more than one.
  • the present invention relates to equipment and methods for performing rapid and microscopic scale cell analysis generating hydrogel microspheres containing the cells and which are subsequently examined by microscopy and image analysis. These analyzes are carried out as an alternative to applications usually performed in liquid cultures or in solid media that need a much larger critical mass of cells to be studied at a macroscopic level, with considerable time and reagent savings.
  • the method tries to determine certain cellular characteristics such as the ability to grow under certain conditions or for the production of specific metabolites such as enzymes, marker proteins, exopolysaccharides, bioplastics, amino acids, antibodies, etc.
  • the characteristic to be studied will correspond to the proliferative capacity of the cells under certain conditions.
  • it refers to the determination of the ability to secrete specific proteins such as antibody fragments, enzymes, etc.
  • the described method refers to the determination of the intracellular catalytic capacity of cells.
  • the invention therefore comprises a method for cellular characterization by means of a microencapsulation system of cells with a hydrogel, a process of treating the microspheres to incubate them once aliquoted with different nutritive and / or reactive substances, and an analysis procedure. programmed through an image analyzer taken from a microscope, optical and / or fluorescent.
  • the microencapsulation process or step consists in the generation of microspheres from a cell suspension to which a compound that gels into a biocompatible polymer is added when the microspheres reach a change in the conditions of the medium. .
  • This gelation of the sphere produces the entrapment of cells in spheres of a micrometric size with the cells or microorganisms, embedded inside.
  • the procedure by which this encapsulation is carried out can be by ionic gelation, emulsion or any other procedure compatible with cell life.
  • the polymer used must also be biocompatible and with the diffusion of biomolecules or metabolites (nucleic acids, nucleotides, oligonucleotides, proteins, amino acids, peptides, carbohydrates, antibiotics, peptides, fatty acids, secondary metabolites) out and into the microsphere.
  • the preferred polymer for this invention is alginate but its composition can be modified to adapt it to the specific conditions required by the different cells to be encapsulated, either by using a combination of polymers or by adding nutrients or growth factors to matrix. This polymer is mixed with the cells under study.
  • the method of generating droplets from the suspension with the cells prior to gelation is performed by applying the jet focusing technology or Flow Focusing®.
  • the next step after microencapsulation is an incubation period, in which the cells are allowed to stabilize and activate biochemically enough to perform the function to be studied.
  • the ability to form microcolonies inside the microspheres would be studied as a test to determine cell growth.
  • certain compounds that affect cell growth and / or the synthesis of certain cellular products such as peptides, enzymes, antibodies, etc., etc. may be added before incubation.
  • the next step of this invention corresponds to the analysis of the microspheres and the microcolonies by means of an image analyzer taken from a microscope. This analysis is done specifically by obtaining photos through a camera associated with a conventional microscope. These images will be analyzed by a specific instrument that will perform the analysis of the microspheres, cells or microcolonies present in the sample by determining the number, color, fluorescence or diameter of the cells or microcolonies.
  • the cells to be encapsulated would be microorganisms in order to identify microorganisms that have a certain phenotype. These microspheres would be incubated in a battery of different conditions to determine the characteristics of the microorganism under study. Once the incubation period was over, the microspheres would be analyzed by capturing a series of images through the microscope to determine the size of the microcolonies. The difference in the diameter of the microcolony formed within the microsphere gives us an idea of the effect that the different conditions tested on the microencapsulated microorganism have on growth. In a preferred aspect of this invention, the analysis of the size and number of Microcolonies in each scanning condition would be done automatically, using the automatic image analyzer.
  • Another application of the invention is the study of non-cultivable microorganisms.
  • the fastest growing microorganisms usually dominate the culture plates with rich media when they are isolated from the environment, not letting the slow-growing ones grow.
  • the fast-growing microorganisms that exceed the limits of the sphere can be washed, and leave the longest time for the slow-growing microorganism to continue growing.
  • compounds typical of their usual environment or the creation of a 3D environment that provides them with specific requirements can be added to the microsphere by modifying the crosslinkable polymer composition and microscopic analysis of the resulting microcolonies allows the study of said microorganisms. Also by controlled modification of the encapsulation environment, such as temperature, extracts from normal habitat, it is possible to determine the specific requirements without which microbial growth cannot be carried out.
  • the identification of microbial activities is carried out, one of these specific activities, but not exclusive, is the identification of enzymatic activity as the activity hydrolytic
  • studies can be carried out on viable cell consortia without whose components no growth occurs.
  • This can be applied to non-cultivable microorganisms by classical methods as well as to eukaryotic tumor cells that need accessory cells for growth.
  • combinatorial and controlled encapsulation of different cell types allows analyzing the conditions in which growth occurs and who are the mandatory participants of the consortium for proliferation to occur.
  • the ease of analysis by identifying microcolonies through images from microscopy facilitates the achievement of results in a short period of time.
  • the use of the method to detect genetic signaling or exchanges between cells of different origin, which give rise to an activity detectable by means of fluorescent or colorimetric signals, can also be part of the invention.
  • the identification of secretory cells is performed.
  • the cells are hybridoma producing antibodies, and the selection thereof is made by modifying the encapsulation matrix so that it is able to specifically retain the monoclonal antibodies under study.
  • the identification of the positive clones will be done after the microspheres have been labeled with an antibody marker, this marker can be a secondary antibody conjugated with a fluorophore, or any other equivalent marker. After incubation with this fluorescent marker, only microspheres containing hybridomas producing antigen-specific antibodies fixed on the microcapsule matrix will retain antibodies in its matrix and will be specifically labeled by the fluorescent marker used for staining.
  • These positive microspheres will be easily identifiable by fluorescence microscopy and the selected clones will be easily isolated by means of a microinjection needle.
  • cell in the present invention refers to the smallest structural unit of an organism that is capable of functioning independently or to a single-celled organism, which is composed of one or more nuclei, cytoplasm and various organelles, surrounded by a cell membrane. semipermeable or a cell wall.
  • the cell can be eukaryotic, prokaryotic, animal, plant or archeobacteria.
  • the cells can be primary or from cell lines.
  • microencapsulation is the process of coating molecules, solid particles, or liquid globules with materials of different nature to give rise to particles of micrometer size, that is between one and a thousand microns.
  • microencapsulation refers to the process of coating cells for microsphere formation.
  • FF jet focused or Flow Focusing
  • microencapsulation technology selected for microencapsulation in the context of this invention.
  • the preference for this technology is fundamentally based on the fact that they achieve particles below 200 microns and up to a maximum of 1000 microns, being a gentle technique and with less known effects on cell viability, exerting less influence on the vital conditions of the encapsulated cell and providing objectivity to encapsulation.
  • microspheres in the present invention refers to spherical particles of homogeneous and semipermeable size that contain within one or more of a cell.
  • microcolony in the context of this invention refers to the result of the proliferation of a cell inside a microsphere, thus maintaining a size smaller than 200 microns and therefore being invisible to the naked human eye. It generally adopts a spherical and opaque conformation in the translucent context of the microsphere, which makes it easily distinguishable using a microscope with phase contrast.
  • compound refers to that substance to be analyzed to determine its effect on the microencapsulated cell. Fundamentally, its effect on the proliferation of the microencapsulated cell will be analyzed, although its effect on its secretion or enzymatic activity can also be analyzed.
  • the microencapsulation procedure can be by ultrasound, drip, emulsions, or jet focusing.
  • the preferred method for cell microencapsulation in the context of this invention is jet focusing technology or Flow Focusing (FF, US 6,464,886).
  • the preferred material for this encapsulation process is ionically crosslinkable polymers.
  • Materials used for this invention include, but are not limited to, natural alginate and polysaccharides such as chitopectin, gellan gum, xanthan gum, hyaluronic acid, heparin, pectin and carrageenan.
  • ionically crosslinkable polyanions for use in the practice of the present invention include but are not limited to, polyacrylic acid and polymethacrylic acid.
  • the ionically crosslinkable polycations such as polyethyleneimine and polylysine are also suitable for the present invention.
  • alginate (the alginate referred to here corresponds to alginic acid salts) will be used as an ionically crosslinkable polymer, at a concentration range between 0.5% and 5% w / v.
  • the alginate is provided in a concentration range between 1.5% and 2% w / v.
  • the ionically crosslinkable polymer crosslinking process is carried out by adding the encapsulation mixture of multivalent cations, such as calcium, zinc, barium, strontium, aluminum, iron, manganese, nickel, cobalt, copper, cadmium, lead, or mixtures of any 2 or more thereof.
  • the encapsulation mixture of multivalent cations such as calcium, zinc, barium, strontium, aluminum, iron, manganese, nickel, cobalt, copper, cadmium, lead, or mixtures of any 2 or more thereof.
  • calcium is used as a crosslinker of the ionically crosslinkable polymer of the encapsulation mixture.
  • this alginate can be prepared in water, or in a specific medium for the growth of the cells to be encapsulated.
  • the encapsulation medium can be derivatized with any complement that is necessary for the survival of a certain cell type.
  • the alginate can be derivatized to contain substrates or compounds that react specifically with molecules or enzymes from the encapsulated cell.
  • the encapsulation mixture consists of an ionically crosslinkable polymer (alginate being preferred for this invention) and the specific cells we want to encapsulate.
  • the proportion of cells used for the encapsulation mixture will determine the amount of cells present in a microcapsule. In one aspect of the present invention, dilutions of the cells with alginate will be made with the aim of finding 1 cell per microcapsule; In another aspect of the present invention, the objective will be to have between 2 and 3 cells per capsule and in another aspect other than the present invention the objective will be to encapsulate the maximum number of cells per microcapsule.
  • the microspheres can be collected by various methodologies.
  • the microspheres can be centrifuged and washed thoroughly with water before transferring them to the specific culture medium for the encapsulated cell type or in a second aspect of this invention, the cells can be filtered through a 70um filter.
  • the preferred embodiment of this invention is the filtering of the microspheres, since in addition, by means of this filtering, any satellite or irregularity that may have occurred during the encapsulation process is eliminated.
  • the fundamental objective is the incubation of the microspheres in a specific medium to ensure the functionality of the microencapsulated cells to be studied.
  • this medium may contain a certain compound under study, this compound is a mutagen such as hydroxylamine or acridine orange, or an inhibitor of cell proliferation such as antibiotics, anticancer, or an activator such as substrate of an activity, a growth factor, a hormone or a specific marker among others.
  • the different incubation conditions will be performed with microspheres from a single encapsulation.
  • the calculation of the number of microspheres required for the different conditions necessary for performing a complete analysis is essential for an optimal development of the present invention. This calculation is performed statistically but can be optimized by empirical results depending on the composition of the encapsulation matrix and the size of the cells to be encapsulated, as well as the necessary concentration thereof.
  • the medium will be distributed in conical tubes with a capacity 10 times greater than the volume of medium to be added (50 ml tubes for 5 ml_ of culture).
  • the microspheres will be distributed homogeneously among all conditions.
  • the microspheres will be resuspended in a given volume of culture medium specific for the cell type to be studied. Equal volumes of capsules will be distributed in each condition.
  • the incubation time will be determined empirically for each type of cell phone.
  • the minimum incubation time will be that necessary to identify microcolonies of approximately one third of the total volume of the microsphere in the control condition of the analysis, under optimal growth conditions without any conditions in the environment. This method supposes a considerable reduction of the incubation time, in the case of microbiological cultures they would pass from 16-24h for the observation of colonies in Petri dishes at 4-8h. In the case of eukaryotic cells, time would pass from several weeks to several days.
  • the object of study is the formation of viable consortia that facilitate the proliferation of a specific cell type.
  • This specific cell type can be traditionally noncultivable microorganisms or tumor cells, existing in both cases. Examples of cells that are not able to grow in vitro due to the lack of environmental factors essential for their growth, some growth factor or inefficient coupling of cell biochemistry to the nutrients provided in the environment.
  • the cells are encapsulated under controlled conditions in the presence of other cells that are in their original environment in which if they grow, for example, in the case of soil microorganisms, controlled consortia of species that normally cohabit , some of them belonging to the category of non-cultivable.
  • tumors in which it is very difficult to expand the cells in vitro for subsequent characterization.
  • the encapsulation of cells adjacent to the tumor in the same capsule could give the environment necessary to mimic the tumor microenvironment on a small scale and allow the expansion of the tumor cell under study.
  • the ultimate goal may be the identification of secretory clones within a heterogeneous cell population; in that specific case, the encapsulation matrix will be made in a way that is able to specifically retain the secreted product and facilitate its subsequent identification.
  • the capsules will be incubated for a certain time that allows the production of the secreted product, either a compound or molecule or a specific enzymatic activity of the cell type studied. This time will vary from 24 to 96 h depending on the secreted molecule or enzymatic activity object of the study.
  • a preferred form of the invention is to make an alginate copolymer that allows the binding of a ligand in the microsphere networks to which the secreted protein must bind.
  • Secretion identification would be performed using a specific ligand against the secreted protein. After washing to remove excess secreted protein not bound to the ligand, the labeled ligand would be retained against the secreted protein and the microsphere would be detected. suitable for the accumulation of marker in the sphere with more specific protein retained.
  • the last step of the selected analysis method is an analysis of images obtained from the microspheres under a microscope with phase or fluorescence contrast.
  • the microspheres that have been incubated for a certain time are preferably transferred, but not exclusively, to a slide, where they will be examined by microscopy to determine the size of the microcolonies in the condition Analysis control
  • the preferred microscope for this invention is an optical microscope with a 4x objective, although a 2.5x or a 10x can also be used.
  • the microscope is inverted, although non-inverted and magnifiers can be used.
  • the microscope can be fluorescent and thus allow the detection of cellular activities associated with a fluorescent product whose concentration generated will be proportional to the cellular activity.
  • fluorescent products can be fluorescent proteins such as GFP, YFP, DsRED, etc. or compounds that are substrates of enzymatic reactions coupled to methyl umbelliferone, such as 4-Methylumbelliferyl-p-D-galactopyranoside or 4- Methylumbelliferyl a-D-glucopyranoside.
  • photos of the microspheres will be taken after incubation, taking photos to preferably have between 100 and 1000 microcolonies.
  • the photos will be taken using a camera connected directly to the microscope.
  • the analysis of the microcolonies will be carried out preferably by determining a series of parameters.
  • Number of microspheres first, an identification and counting of the microspheres present in the preparation will be carried out. Through the microsphere count an internal control of microsphere distribution between the different conditions will be carried out. The number of microspheres present in the different conditions must be equivalent to give the results valid. A heterogeneous distribution of capsules between different conditions can lead to a bias in the number of microcolonies that does not correspond to reality. A percentage of variation of less than 20% between microsphere counts in different conditions is desirable.
  • the count of the microcolonies will be similar among all the conditions of the analysis.
  • the colon count will be lower, since the number of clones that will lead to a microcolony tends to be 0. In extreme cases the number of microcolonies will be 0. Size of the microcolonies: the second parameter to analyze is the size of the microcolonies. As in the count we can find two possibilities:
  • the count of the microcolonies will be similar between all the conditions of the analysis. However, the size of the microcolonies will be significantly reduced. At least a reduction in the diameter of 2 times the size of the microcolonies in the optimal culture condition will be identified, b. If the culture conditions do not have the effect described in point one of this list, but completely block cell proliferation, the colon count will be lower, since the number of clones that will lead to a microcolony tends to be 0. In extreme cases the size of microcolonies will be 0.
  • the identification of some isolated microcolony within the population will occur.
  • the general diameter of the microcolonies will be undetectable; however, the above-mentioned clones, which have mutations, may have a size similar to that observed in the control condition. In this case, we must relate the size of the microcolonies with the number of them and with the total number of capsules in the sample.
  • the preferred representation of the data in the methodology of this invention would be the normal distribution of the sizes obtained in each of the conditions including the control.
  • Another way of representing the data would be the representation of the average diameters of the microcolonies present in each condition.
  • the analysis of the standard error of the mean, as well as the average values of the maximum and minimum sizes, and the coefficients of variation of the microcolonies in each condition, provide fundamental information for obtaining objective results.
  • the growth of hardly cultivable cells will also be analyzed by microscopy. By analyzing the photos taken, the optimal conditions for the culture of said cell types will be decided.
  • the culture of these cells by this procedure opens new avenues for their characterization since they can be incubated using extracts from the usual environment of the cells by requiring less amount of medium, increasing the chances of achieving more cell divisions. In the case of tumor cells, encapsulating them with their cellular microenvironment can generate more effective divisions, making it more feasible to make studies of sensitivity to antitumor compounds in cell types where it is currently not possible.
  • a specific method for the present invention of interest in the identification of cells by the production of a specific metabolite in this case the images of these capsules are not going to be analyzed looking for the formation of microcolonies, but rather looking for positivity against the specific marking of the secreted product or the desired enzymatic activity.
  • the marking will preferably be fluorescent due to its greater sensitivity, thus reducing the time needed to accumulate a sufficiently visible signal in each capsule.
  • the hydrogel will have to be based on copolymers that allow ligand binding.
  • Some of the possible ligands to derivatize the monomers that form the copolymer can be biotin and thus bind streptavidin polypeptide, NTA or NDE and thus bind polyhistidine-bound proteins, DEAE to bind proteins bound to choline-binding domains, glutathione that would allow protein binding. linked to GST, etc. It is possible, for example, to obtain copolymer tsar DEAE dextran with alginate and bind specifically GFP linked to LYTAG. Likewise, an antigen of interest can be attached to the copolymer ligands and select encapsulated hybridomas that secrete antibodies that bind to said antigen.
  • Antibody bound to the copolymer antigen could be detected with specific antibodies against the immunoglobulin heavy chain, conjugated to fluorophores such as FITC, phycoerythrin, rhodamine, or any commercially available fluorophore.
  • the positive cells can be isolated by micropipettes using the microscope or by screening the microspheres by isolating them from the rest and expanding them in the optimal conditions for each cell type.
  • FIG. 1 Example of monodisperse capsules produced by jet focusing technology or Flow Focusing, used in this invention.
  • Figure 3 Example of the detection of enzymatic activity of encapsulated microorganisms.
  • Figure 4. Scheme of the procedure described in this invention.
  • Figure 5. Identification of secretory clones of the 1xZ domain of protein A.
  • the microspheres containing bacteria capable of producing the recombinant protein are labeled with the fluorescent antibody in red.
  • Figure.6. Comparison of counting capacity and identification of clones encapsulated in microspheres by flow cytometry and optical microscopy. The figure shows that by means of the flow cytometry technique a single clone is identified within the microsphere, when it is appreciated that there are two, while by microscopy all independent colonies present in each microsphere are clearly identified.
  • the first object of the present invention relates to a method of cellular characterization comprising the following steps:
  • the process of the invention is characterized in that the substance capable of producing hydrogels is a polymeric substance selected from any of the following: alginate, agarose, chitosan, chitopectin, gellan gum, xanthan gum, hyaluronic acid, heparin, pectin, carrageenan, polyacrylic acid, polymethacrylic acid, polyethyleneimine and polylysine.
  • the substance capable of producing hydrogels is alginate.
  • microencapsulator produces the microspheres by any of the following methods: ultrasound, drip, emulsion, or jet focusing.
  • the microencapsulator produces the microspheres by the jet focusing technique.
  • microspheres are 50 to 500 microns in size.
  • cells are selected from any of the following: eukaryotic, prokaryotic, animal, plant or archeobacterium.
  • this is characterized in that the cells present in their genome genetic modifications that give rise to mutant or hybrid organisms. Said genetic modifications are obtained by means of genetic engineering techniques, commonly known to those skilled in the present technical field. Such mutant or hybrid organisms may show an increase or inhibition of the expression of a gene, protein, etc., both homologous and heterologous.
  • this is characterized in that at steps a) or c) at least one compound capable of being detected by microscopy is added when reacting with the cells and / or products secreted by them within the microspheres
  • this is characterized in that the compound capable of being detected by microscopy is selected from any of the following: dye, chromogen and / or fluorophore.
  • dye any of the following: dye, chromogen and / or fluorophore.
  • this is characterized in that the compound capable of modifying the metabolism of the cell is selected from any of the following: mutagen, proliferation inhibitor or activator, growth factors and / or enzymatic substrates .
  • the cellular characterization is directed to the analysis of the viability and / or cell growth.
  • this is characterized in that cell viability is analyzed by cellular consortia capable of facilitating the growth of a specific cell type.
  • the specific cell types are preferably non-cultivable cells or tumor cells.
  • this is characterized in that the cellular characterization is directed to the identification of cells that have an activity selected from any of the following: secretory, hydrolytic or catalytic.
  • this is characterized in that the secretory activity results in the secretion of molecules that are selected from any of the following: peptides, enzymes, antibodies and / or antibody fragments.
  • this is characterized in that the identification of the cells with secretory activity and / or of the secreted molecules is performed by detecting the binding of said cells and / or molecules to the compound capable of being detected by microscopy as described above throughout this document.
  • this is characterized in that for the identification of the catalytic or hydrolytic activity, a substrate subject to the cellular activity sought is previously added in step b). In another preferred embodiment of the process of the invention, this is characterized in that the substrate reacts with the compound capable of being detected by microscopy as previously described herein.
  • this is characterized in that the microscope is optical or fluorescent.
  • this is characterized in that the image analyzer is automatic.
  • the image analyzer identifies the microspheres and detects the activity and / or condition of the cells and / or secreted products.
  • This example describes how to screen cells that have undergone a genetic change that can be selectable within the microsphere. It starts from a growing cell culture. Previously, the necessary solutions for encapsulation have been prepared. The 1.5% alginate has been prepared, this alginate can be prepared in water or in specific medium for cell growth. The alginate is sterilized by filtration through a pore size of 20um. The 3% calcium chloride in water has also been prepared, which is also sterilized by filtration. An inoculum of between 0.5 and 1x10 cells / ml_ of alginate is made. The mixture is gently stirred by inversion to ensure a homogeneous distribution of the cells in the alginate without producing bubbles in the mixture.
  • the encapsulation is carried out, using a 200um nebulizer that produces particles between 100 and 120 ⁇ in diameter.
  • a 200um nebulizer that produces particles between 100 and 120 ⁇ in diameter.
  • an air flow at a pressure of 60mBar is required.
  • the encapsulation is carried out at a flow of 3mL / h. The particles fall into a 3% calcium chloride bath while stirring.
  • E. coli resistant to rifampin are identified that result from a timely mutation.
  • the microcolonies are analyzed by microscopy and the clones that have had a differential growth with respect to the rest in the presence of 10 mg / L of rifampin are identified, either by an increased or decreased growth.
  • microcapsule with the clone of interest is selected and passed to a well of a 96 plate with optimal culture medium for the encapsulated microorganism. Allowing it to grow the necessary time, the microcolony will be released from the microcapsule and will lead to a conventional axenic liquid culture, which can be expanded for subsequent characterization, DNA extraction or phenotypic analysis.
  • Example 2 Identification of secretory clones. Selection of hybridomas.
  • Encapsulated cells are hybridomas produced after a fusion of splenocytes from a mouse immunized against the green fluorescent protein (GFP) with myeloid cells. Some of these hybridomas produce an antibody specific for the antigen of interest. The identification and selection of these hybridomas is a slow process that takes between 2 and 4 weeks. The application of the present invention would reduce the time necessary until the identification of producing clones to 2-3 days.
  • GFP green fluorescent protein
  • the encapsulation matrix has to be derivatized with a compound that allows antigen retention in the alginate matrix.
  • DEAE dextran is used, which retains the peptide antigen bound to LYTAG.
  • DEAE dextran alginate and a solution of a GFP fusion would be added to -in the C-terminal end to the choline binding domain and also to DEAE, LYTAG (Biomedal SL) .
  • the microspheres will carry alginate as a gelling agent and DEAE-Dextran that would bind the GFP antigen through the LYTAG that has affinity for DEAE.
  • the hybridomas to be encapsulated would be added and encapsulation would proceed.
  • a 200 um nebulizer would be used that gives a microcapsule size between 100 and 120um.
  • the dilution of the cells will be that which allows the encapsulation of 1 cell per capsule.
  • microcapsules After subsequent washing and filtering, the microcapsules would now be maintained in the hybridoma selection medium (Medium HAT) and 24 well plates would be dispensed. The cells are incubated under these conditions for a period of 3-5 days depending on the recovery rate of the cells. After this time, these microcapsules would be incubated with a secondary anti-mouse Ig antibody (or the corresponding producer) fluorescently labeled with rhodamine. Microcapsules containing hybridomas producing specific antibodies will have the same retained in the microcapsule matrix by specific recognition with the GFP antigen fixed therein.
  • Hybrida selection medium Medium HAT
  • the labeled clones will be selected by fluorescence microscopy for red emission and with microinjection tools they will be transferred to an independent plate where their hybridoma selection and culture will occur.
  • the higher intensity of the red color emitted could also allow the selection of the best secretors.
  • Example 3 Identification of enzymatic activity.
  • cells with a specific enzymatic activity are identified within a mixed population of cells.
  • the encapsulation matrix has to be derivatized, the mixture being carried out in the presence of the substrate subject to the enzymatic activity sought.
  • the enzymatic activity that is sought is glutenase activity
  • the substrate that is added to the encapsulation matrix is gliadin, one of the component proteins of gluten.
  • the cells are encapsulated in this matrix and incubated in the optimal culture medium for the corresponding cell type.
  • staining is performed with a specific fluorescently labeled antigliadin antibody.
  • Example 4 Identification of secretory clones (recombinant proteins).
  • This example describes how to perform a screening of cells that have undergone a genetic change and that can be selected within the microsphere
  • transformed bacteria that are capable of producing a specific recombinant protein will be selected, in this case, with the antibody binding z-domain of protein A.
  • This protein A is useful in biotechnology for its ability to bind antibodies
  • Staphylococcus aureus protein A (GenBank: AAB05743.1) has been used.
  • GenBank: AAB05743.1 GenBank: AAB05743.1
  • the solutions necessary for encapsulation are prepared, specifically, 1.5% alginate is dissolved in water and sterilized by filtration through a pore size of 20um.
  • a solution of 3% calcium chloride in water is prepared, which is also sterilized by filtration.
  • a concentration of 0.5x10 6 cells / mL of alginate is encapsulated.
  • the encapsulated cells After obtaining the encapsulated cells, they are subjected to a mild osmotic shock to break up the cell membranes and so that the diffusion of the proteins and antibodies in and out of the cells occurs.
  • the encapsulated cells are subsequently incubated with rabbit secondary antibodies labeled with a fluorophore that emits in green. Those capsules with green fluorescence have inside a bacterial colony secreting the protein A of interest ( Figure 5).

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Abstract

La présente invention se rapporte à un procédé de détermination rapide d'activités ou d'états cellulaires tels que la synthèse de biomolécules, la viabilité et la croissance cellulaire au niveau microscopique. La présente invention concerne ainsi un procédé spécifique de détection et de mesure de cellules incorporées dans des microsphères d'hydrogel soumises à des conditions expérimentales d'intérêt et analysées au moyen d'un processeur d'images couplé à un microscope. L'invention a également pour objet un dispositif comprenant un microencapsuleur qui produit des microsphères monodispersées, un microscope optique ou à fluorescence, et un analyseur d'images permettant l'identification des microsphères contenant les cellules, et identifiant ses caractéristiques d'intérêt.
PCT/ES2014/070492 2013-06-13 2014-06-13 Procédé d'analyse rapide de l'activité cellulaire WO2014198994A1 (fr)

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Citations (6)

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WO1989010566A1 (fr) * 1988-04-22 1989-11-02 Massachusetts Institute Of Technology Procede pour former et pour utiliser des microgouttes
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WO1999030832A1 (fr) * 1997-12-17 1999-06-24 Universidad De Sevilla Microjet capillaire stabilise et dispositifs et procedes pour produire ce microjet
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US4916060A (en) * 1985-09-17 1990-04-10 Massachusetts Institute Of Technology Process for chemical measurement in small volume samples by fluorescent indicators
WO1989010566A1 (fr) * 1988-04-22 1989-11-02 Massachusetts Institute Of Technology Procede pour former et pour utiliser des microgouttes
EP0411038A1 (fr) 1988-04-22 1991-02-06 Massachusetts Inst Technology Procede pour former et pour utiliser des microgouttes.
US6464886B2 (en) 1996-05-13 2002-10-15 Universidad De Sevilla Device and method for creating spherical particles of uniform size
WO1999030832A1 (fr) * 1997-12-17 1999-06-24 Universidad De Sevilla Microjet capillaire stabilise et dispositifs et procedes pour produire ce microjet
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