WO2021101361A1 - Procédé de génération de topographie définie de microfluidique pour simuler la croissance et le développement d'organismes - Google Patents

Procédé de génération de topographie définie de microfluidique pour simuler la croissance et le développement d'organismes Download PDF

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Publication number
WO2021101361A1
WO2021101361A1 PCT/MX2019/000132 MX2019000132W WO2021101361A1 WO 2021101361 A1 WO2021101361 A1 WO 2021101361A1 MX 2019000132 W MX2019000132 W MX 2019000132W WO 2021101361 A1 WO2021101361 A1 WO 2021101361A1
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WIPO (PCT)
Prior art keywords
growth
development
simulate
organisms
topography
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PCT/MX2019/000132
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English (en)
Spanish (es)
Inventor
María José RIVAS ARREOLA
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Rivas Arreola Maria Jose
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Application filed by Rivas Arreola Maria Jose filed Critical Rivas Arreola Maria Jose
Publication of WO2021101361A1 publication Critical patent/WO2021101361A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J8/00—Chemical or physical processes in general, conducted in the presence of fluids and solid particles; Apparatus for such processes
    • B01J8/18—Chemical or physical processes in general, conducted in the presence of fluids and solid particles; Apparatus for such processes with fluidised particles
    • B01J8/20—Chemical or physical processes in general, conducted in the presence of fluids and solid particles; Apparatus for such processes with fluidised particles with liquid as a fluidising medium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus

Definitions

  • the present invention is related to the field of biology, specifically with a process for generating a defined microfluidic topography that facilitates the recreation of a biomimetic environment to favor the growth and development of biological systems that provides the evaluation of new culture media or supplements. for the development of biological systems, evaluation of cellular antioxidant activity, the cltotoxicity of molecules, biomaterials or new substances in development.
  • Microfluidic systems are based on technology developed since the 1950s for integrated circuits, and their integration into more complex systems during the 1980s, capable of performing a specific function. These systems, between 10 and 1000 microns in size, were called “Micro-Electro-Mechanical Systems (MEMS, Micro Electromechanical Systems). Soon towards the end of the 90's the systems were developed. Laboratory on a Chip (LOC, Lab on a Chip), whose basis was to use scalable principles of chemical, biological, medical, biochemical and biotechnological analysis, and where the use of reagents is minimal.
  • LOC Laboratory on a Chip
  • micro-fluids The study of micro-fluids is a multidisciplinary field that includes parts of Physics, Chemistry, Engineering and Biotechnology. He studies the behavior of fluids at the microscale and also understands the design of systems in which minute amounts of fluid will be used.
  • microfluidic biochips are revolutionizing molecular biology in enzymatic analysis procedures (eg, glucose and lactate assays), DNA analysis (eg, polymerase chain reaction, and high-throughput sequencing ), and proteomics.
  • the basic idea of microfluidic biochips is to integrate assay operations, such as detection, as well as pretreatment sample and sample preparation on one chip.
  • assay operations such as detection, as well as pretreatment sample and sample preparation on one chip.
  • a new area of application for biochips is clinical pathology, especially the immediate point of care for diagnosing diseases.
  • device-based microfluidics are capable of continuous, real-time sampling of air / water sample tests for biochemical analyzes of toxins and other dangerous pathogens.
  • microfluidics In general, microfluidics consists of the manipulation of liquids in micrometric scale spaces. Such manipulation can be achieved, for example, by making small channels through which the liquid is pumped. Another option is to use capillary force to flow the liquid through porous materials, such as papers used in pregnancy tests. In the last two decades, microfluidics has gained important relevance in the research area as a platform for the development of new portable analytical systems for biological tests and applications in the chemical and biotechnology industries. pharmaceutical, energy, and aerospace among many others. The main advantages of this scheme are a low consumption of reagents, samples and energy in a minimum space.
  • LOC technology deals with laboratory experiments performed on a very small scale. It can integrate various laboratory functions on a chip ranging in size from a few millimeters to a few square centimeters; This helps achieve high-performance detection and automation.
  • LOC technology enables the use of small volumes of fluid, helping to reduce costs and reagent analysis and response time. It also allows greater control over sample concentrations, as well as interactions to reduce the amount of chemical waste. This technology can aid the development of highly compact systems through mass production.
  • LOC is an emerging technology and it has some drawbacks. Physical and chemical effects such as surface roughness, capillary forces, and chemical interactions between materials are most significant at the microscale level. This can often result in complications during LOC experiments that would not be expected with traditional laboratory equipment. Detection principles may not be always in accordance with microscale dynamics and this can result in a low signal-to-noise ratio.
  • microdevices for the generation and use of energy, commonly called powerMEMS (micro electromechanical energy systems).
  • powerMEMS micro electromechanical energy systems
  • These include devices that use a few drops of high abundance combustible liquids, such as ethanol, methanol. glucose, or ethylene glycol, to provide enough energy for energy efficient electronic devices.
  • bioMEMS biocompatible microfluidic fuel cells
  • biomaterials and new molecules require knowledge in many areas.
  • the evaluation of cytotoxicity is one of the most commonly used in vitro studies to determine the biocompatibility of a molecule or matter of interest. It is a study that provides valuable information on the materials that should be discarded or those that should be subjected to further studies.
  • One of the typical ways in which the cytotoxicity evaluation is carried out is to place the molecule or material to be evaluated in contact with the cell culture in monolayer, after a period of time the dye 3- (4,5-dimethylthiazol-2-yl) 2 is used.
  • S phenyltetrazolium bromide (MTT) which is reduced by the mitochondrial dehydrogenases of viable cells to produce a purple color easily measurable by absorbance.
  • Microfluidic technology has found many applications, mainly:
  • microfluidic chips allow easy single cell manipulations and rapid drug changes.
  • the invention developed aims to develop a process for the generation of defined microfluidic topography that facilitates the recreation of a biomimetic environment to favor the growth and development of biological systems and that facilitates the evaluation of new culture media or supplements for development. of biological systems, evaluation of cellular antioxidant activity, the cytotoxicity of molecules, biomaterials or new substances in development.
  • the inner chip comprises at least one cell culture unit, wherein each cell culture unit comprises at least two cell culture chambers, a power pump, a plurality of liquid pathway channels, at least two isolation channels and a gas path channel; the channels of the liquid path are connected with the cell culture chambers and the liquid cavities of the cell culture chambers and the energy pump to form a circulating channel; the at least two isolation channels are arranged in the at least two liquid passage channels; and the gas path channel is disposed between the gas cavity and a gas path inlet of the chip.
  • the system comprises the chip and a microfluidic control system, wherein the microfluidic control system comprises a micro gas injection pump and a gas storage unit; and the micro gas injector pump is connected with the gas path input of the chip.
  • the method comprises the following steps: injecting different cells into different cell culture chambers of a cell culture unit; controlling the circulating flow of liquid in the cell culture unit by means of the pump; and regulate the gas pressure output by the pump to achieve the optimal cell growth effect.
  • the method can simulate the environment in vivo to perform an in vitro co-culture on cells.
  • the microfluidic device described in the previous document does not present topography on any of its contact surfaces.
  • the invention developed, object of this document includes a device that has a topography that facilitates adhesion, proliferation and 3D growth. It does not recreate co-crops.
  • several chips containing different types of cell units can be connected. All this allows the evaluation of cell growth, extracellular matrix compounds, as well as compounds or materials on cell growth parameters.
  • the biomimetic flow apparatus includes a microfluidic flow channel and an inlet in fluid connection with the flow channel to allow fluid to flow into the flow channel.
  • the flow channel has at least one surface with a topography formed therein.
  • the topography of at least one surface of the flow channel is selected to cause cells in a layer of cells on the surface to achieve an arrangement, behavior or morphology. Cell arrangement, behavior or morphology is determined, at least in part, by the topography of at least one surface.
  • the invention described in the aforementioned document does not allow the growth of co-cultures or the connection of several systems between them.
  • the invention developed, object of this document includes a device that has a topography that facilitates adhesion, proliferation and 3D growth. It does not recreate co-cultures, several chips containing different types of cell units can be connected.
  • the method comprises retaining in a micro reactor a cell population comprising one or more effector cells, wherein the content of the micro reactor further comprises a population of reader particles comprising one or more reader particles, incubating the cell population and the population of reading particles within the micro reactor, which analyzes the cell population for the presence of the extracellular effect, in which the population of reading particles or its subpopulation provides a direct or indirect reading of the extracellular effect and determines, in a function of the results of the assay step, whether one or more effectors, whether cells within the cell population exert the extracellular effect on the read particle. If an extracellular effect is measured, the cell population is retrieved for further analysis to determine the cell or cells responsible for the effect.
  • the microfluidic device described. in the previous document does not present topography in any of its contact surfaces.
  • the invention developed, object of this document includes a device that has a topography that facilitates adhesion, proliferation and 3D growth; it does not recreate co-cultures. Additionally, several chips containing different types of cell units can be connected and allows the evaluation of cell growth, extracellular matrix compounds, as well as compounds or materials on cell growth parameters.
  • the objective of the developed invention is to make available a process for the generation of defined microfluidic topography to simulate the growth of biological systems and the development of organisms through connecting various microfluidic devices with topographic patterns defined on a micrometric scale that facilitate cell adhesion. and they favor the recreation of a biomimetic environment.
  • the invention relates to a process that is capable of the following:
  • Figure 1 shows a plate resulting from the process for the generation of microfluidic devices.
  • Figure 2 shows the process for the generation of microfluidic devices and for the simulation of the growth and development of organisms.
  • FIG 1 shows a plate of polystyrene resulting (1) from the process, which contains an inlet (2) and an outlet (5), another inlet (3) and another outlet (4).
  • the resulting polystyrene plate (1) favors growth in 3 dimensions, and allows the connection of several resulting polystyrene plates (1), to generate a microfluidic system between them that contain the same type of cells or that are different; therefore, cell populations ranging from 100-1000 cells are required.
  • the process has application in 3-dimensional cell growth, which helps studies of cell morphology and physiology to understand the development of cell structures and tissue engineering; additionally, it is used to evaluate the effect of substances (cosmetics, chemicals, pharmaceuticals, biomolecules, etc.) on cell growth.
  • Process for the generation of a mlcrofluidic device is used to evaluate the effect of substances (cosmetics, chemicals, pharmaceuticals, biomolecules, etc.) on cell growth.
  • Figure 2 shows the process for the generation of the resulting polystyrene plate or plates (1) which is used as microfluidic devices and which takes the following steps:
  • the design is heat transferred from the acetate film for photocopying or projection to the stainless steel by means of heat between 150 ° C to 180 ° C for 2 to 5 minutes (30),
  • the acid used can be: a. HCi (1: 1 v / v) make a solution with these concentrations, b. HNOa (1: 3 v / v) make a solution with these concentrations c.
  • the resulting polystyrene plate (1) is washed with water (50), the above to remove polystyrene remains and possible residues and achieve better cell adhesion,
  • the resulting polystyrene plate (1) is cut and assembled to complete the microfluidic system (60),
  • the resulting polystyrene plates (1) which make up the device, are subjected to 5 washes in sterile water and dried in the biosafety hood for 8 hours with UV light (70), this includes: a. Immerse the resulting polystyrene plate (1) in a sterile 250 mL beaker with 150 mL of sterile water, with manual shaking for 2 min and this is repeated 5 times; or it can also be carried out using a stirring system at 150 rpm with a sterile magnetic stirrer (71),
  • the cellular structures or microorganisms (80) are included: a.
  • the cells can be placed in the growth area before closing the assemblies of the resulting polystyrene plates (1) or through the inlet channels (2 or 3) (81), b.
  • the culture medium is introduced, where for mammalian cells, basically the culture media contain: proteins, amino acids, growth factors and antibiotics, and a pH indicator (82) can be placed, c.
  • the resulting polystyrene plates assembly (1) is closed and placed in the incubator to later continue with the required tests, where the characteristics are temperature, humidity and CO2 flow; for mammalian cells the characteristics are 34 ° C to 37 ° C, 95% humidity, 5% CO2
  • the culture medium for microorganisms contains water, agar, carbohydrates, amino acids, vitamins, P, Fe, N, Mg, S and can contain buffer solutions and pH indicators (84), e.
  • the culture surface must be covered with the required agar, then place the microorganisms and incubate under the required conditions according to the selected microorganisms (85).
  • the process contained in this invention generates assemblies of the resulting polystyrene plates (1), which have the following characteristics: ⁇
  • the assemblies of the resulting polystyrene plates (1) generates microfluidic devices that are portable, consisting of four channels: two inlet channels (2) and (3) and two outlet channels (4) and (5) and an area with defined topography where cell cultures can easily adhere.
  • ⁇ The inputs (2) and (3) and outputs (4) and (5) of the assemblies of the resulting polystyrene plates (1), allow it to be connected to a multi-syringe infusion pump and also allow connectivity between them.
  • Each of the assemblies of the resulting polystyrene plates (1) can work individually and can be connected in parallel or in series.
  • the deposit has a defined topography that allows to recreate a biomimetic environment, in this way the cells have the possibility of growing in 3D, not only in monolayer.
  • the system can evaluate the effect of a molecules, biomaterials or new substances in development on a particular cell line, on several cell lines or simulate the path of a solution in several cell lines, since the assemblies of the resulting polystyrene plates (1) They can be connected in series to be able to simulate the path of the compounds of the material or biomolecule of interest on cell lines of the main organs of the human body, or independently.

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  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
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  • Chemical Kinetics & Catalysis (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Sustainable Development (AREA)
  • Virology (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

La perte de fonctionnalité d'un organe ou d'un tissu est un des problèmes de santé majeurs. Les centres et les instituts de recherche, ainsi que les entreprises biopharmaceutiques et cosmétiques utilisent des lignées cellulaires pour améliorer la connaissance du développement des tissus, l'évaluation de molécules en développement et/ou de nouveaux produits. La présente invention a pour objectif de rendre disponible un procédé pour la génération de topographie définie de microfluidique pour simuler la croissance de systèmes biologiques et le développement d'organismes à travers la connexion de divers dispositifs de microfluidique avec des motifs topographiques définis à l'échelle micrométrique qui facilitent l'adhésion cellulaire et favorisent la recréaction d'un environnement biomimétique. L'invention concerne un procédé capable de : développer des structures 3D pour simuler l'environnement biomimétique pour favoriser la croissance et le développement de cultures d'organismes cellulaires (bactéries, cellules de mamifère ou cellules végétales), simuler l'interaction des organismes cellulaires telle qu'elle se produit dans les systèmes biologiques, réduire le volume de moyens et de suppléments pour les cultures de systèmes biologiques.
PCT/MX2019/000132 2019-11-22 2019-11-25 Procédé de génération de topographie définie de microfluidique pour simuler la croissance et le développement d'organismes WO2021101361A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
MX2019014003A MX2019014003A (es) 2019-11-22 2019-11-22 Proceso para la generación de topografía definida de microfluídica para simular el crecimiento y desarrollo de organismos.
MXMX/A/2019/014003 2019-11-22

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090298166A1 (en) * 2008-05-30 2009-12-03 Ye Fang Cell culture apparatus having variable topography
US20170252701A1 (en) * 2016-03-03 2017-09-07 Micromedics Inc. Biomimetically Designed Modular Microfluidic-Based Capillaries & Lymphatic Units for Kidney & Liver Dialysis Systems, Organ Bio-Reactors and Bio-Artificial Organ Support Systems

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090298166A1 (en) * 2008-05-30 2009-12-03 Ye Fang Cell culture apparatus having variable topography
US20170252701A1 (en) * 2016-03-03 2017-09-07 Micromedics Inc. Biomimetically Designed Modular Microfluidic-Based Capillaries & Lymphatic Units for Kidney & Liver Dialysis Systems, Organ Bio-Reactors and Bio-Artificial Organ Support Systems

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BRUCE K. GALE: "A review of current methods in microfluidic device fabrication and future commercialization prospects", INVENTIONS, vol. 3, no. 60, 2018, XP055687418, Retrieved from the Internet <URL:www.mdpi.com/journal/inventions> [retrieved on 20200704], DOI: 10.3390/inventions3030060 *
DUSSEILLER M R ET AL.: "An inverted microcontact printing method on topographically structured polystyrene chips for arrayed micro-3-D culturing of single cells", BIOMATERIALS, vol. 26, no. 29, 10 January 2005 (2005-01-10), AMSTERDAM, NL, pages 5917 - 5925, XP025280144, ISSN: 0142-9612, DOI: 10.1016/j.biomaterials.2005.02.032 *
GENCTURK ELIF, MUTLU SENOL, ULGEN KUTLU O.: "Advances in microfluidic devices made from thermoplastics used in cell biology and analyses", BIOMICROFLUIDICS, vol. 11, no. 5, 31 August 2017 (2017-08-31), USA, pages 1 - 41, XP055827833, ISSN: 1932-1058, DOI: 10.1063/1.4998604 *

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