WO2009147482A2 - Système et procédé pour une stimulation dynamique et/ou un contrôle de cellules et de tissus dans une culture - Google Patents

Système et procédé pour une stimulation dynamique et/ou un contrôle de cellules et de tissus dans une culture Download PDF

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Publication number
WO2009147482A2
WO2009147482A2 PCT/IB2009/005702 IB2009005702W WO2009147482A2 WO 2009147482 A2 WO2009147482 A2 WO 2009147482A2 IB 2009005702 W IB2009005702 W IB 2009005702W WO 2009147482 A2 WO2009147482 A2 WO 2009147482A2
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WO
WIPO (PCT)
Prior art keywords
bottom wall
culture
culture chamber
deformable
cells
Prior art date
Application number
PCT/IB2009/005702
Other languages
English (en)
Other versions
WO2009147482A3 (fr
Inventor
Marco Pizzi
Diego Bruno
Marco Cerrato
Original Assignee
Techfab S.R.L.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Techfab S.R.L. filed Critical Techfab S.R.L.
Publication of WO2009147482A2 publication Critical patent/WO2009147482A2/fr
Publication of WO2009147482A3 publication Critical patent/WO2009147482A3/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • 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
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/26—Constructional details, e.g. recesses, hinges flexible
    • 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
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli

Definitions

  • the present invention relates to a system for the dynamic stimulation of cells and tissues in a culture.
  • the invention relates to a device that enables application of mechanical stimuli to the cells with a fine control of the degree of deformation imposed thereon or else measurement of the mechanical load to which the cells themselves subject the substrate to which they adhere, said device being compatible with the techniques and apparatuses of optical microscopy in use in the sector.
  • this stimulus of a physical/mechanical nature can lie at the root of phenomena such as differentiation and cell growth or growth of a tissue, and hence its presence is essential for the culture conditions to reproduce in the most faithful way possible the corresponding conditions to which the tissue itself would be exposed in vivo.
  • the cells subjected to dynamic loads must generally be taken away from the culture environment and transferred to a distinct apparatus for conducting measurements and tests (for example, for enabling observation under the microscope) .
  • a system capable of overcoming the aforesaid drawbacks.
  • a system comprising a device that enables subjection of cells and tissues to dynamic stimulation in a finely controlled and precise way using as supports for cells and tissues containers for cell culture typically in use in the art.
  • the system according to the invention enables precise detection of the degree of the load applied by the cells or tissues in a culture to the substrate to which they adhere.
  • a system for dynamic stimulation of cells and tissues in a culture that is compatible with the techniques of optical microscopy in use in the sector.
  • a device for dynamic stimulation of cells and tissues according to
  • Claim 1 In addition, according to the invention, a system is provided according to Claim 12.
  • Figure 1 is a perspective and cutaway view of the device for dynamic stimulation of cells and tissues according to the invention
  • Figure 2 is a schematic representation of the system for analysis of cell response to the dynamic stimulation according to the invention, comprising the device of Figure 1;
  • Figure 3 shows schematically the flexible elements 5 of a device according to the invention to which cells present within the culture chamber adhere;
  • Figure 4 is a perspective view that illustrates a detail of the flexible elements made on the bottom wall of the device according to the invention to which a cell adheres;
  • Figure 5 shows a further embodiment of the flexible elements of the bottom wall of the device according to the invention.
  • Illustrated in Figure 1 and designated as a whole by 1 is a device for dynamic stimulation and/or control of cells or tissues in a culture. It comprises a culture chamber 2, in which cells or portions of tissues can be introduced, the bottom wall 3 of which is made at least in part of a deformable material and integrally provided of a single piece on which is at least one diffraction grating 4.
  • said diffraction grating 4 is deformable together with the at least one deformable part of the bottom wall 3, when a mechanical stimulus is applied thereto, outside or inside the culture chamber 2.
  • the deformable diffraction grating 4 comprises a plurality of elements 5, each of which is made so as to be flexible independently of the others, made on the bottom wall 3 on the side of a face 6 thereof set in use inside the culture chamber 3.
  • the elements 5 which are individually flexible in a mutually independent way are made so as to be deformable in response to a stimulus of a mechanical type applied thereto by at least one cell/particle 101 or portion of tissue present in the culture chamber 2 and adherent to the elements themselves ( Figure 5) .
  • particles or “particle” are meant to indicate a micrometric or nanometric entity, whether natural or artificial, such as cells, subcellular components, viruses, liposomes, niosomes, microspheres and nanospheres, or even smaller entities, such as macro- molecules, proteins, DNA, RNA, etc.
  • the term "cell” will be used, but, where not otherwise specified, it shall be understood as a non- limiting example of particles in the wider sense referred to above.
  • the term “cell” or “cells” is understood both unicellular organisms and single cells of pluricellular organisms.
  • the culture chamber 2 of the device 1 can house generic particles, where by the term “particle” is understood, here and in what follows, a biological entity, such as cells, micro-organisms, viruses or parts thereof, for instance, portions of nucleic acids or proteins, plasmids, etc.
  • the position of at least one of the flexible elements 5 is altered with respect to the corresponding original position.
  • the degree of said alteration, i.e., of said deformation can be set in relation, once the mechanical properties of the cell/portion of tissue 101 and of the flexible elements 5 themselves are known, to the degree of the load applied by the cell/portion of tissue thanks to the presence of the diffraction grating 4, as will be seen.
  • the deformable diffraction grating 4 comprises a plurality of elements 5, each of which is made so as to be flexible and which are made on the bottom wall 3 on the side of a face 7 thereof set in use on the outside of the culture chamber 2.
  • the device 1 comprises actuator means 8 for applying a mechanical stimulus "external" to the at least one deformable part of the bottom wall 3.
  • the device 1 enables application from outside of a mechanical stimulus that induces a deformation of the at least one deformable part of the bottom wall and, consequently, of the cells/portions of tissue that adhere to the bottom wall on the side of the face 6 set in use within the chamber.
  • the deformation at least partially involves also the diffraction grating 4, which may be a simple grating or else result from the composition of two gratings with linearly independent spatial orientations (for example, orthogonal) .
  • the optical system will hence enable evaluation also of anisotropic deformations obtained by operating the actuators designed for deformation of the membrane in a different way.
  • the deformation of the elements 5 of the grating 4 is equivalent to an alteration of the corresponding pitch p typical of the grating 4, which can be detected as will be seen in what follows and set in relation with the deformation imposed on the cells/portions of tissues by operating the actuator means 8 ( Figure 1) or else imposed on the flexible elements 5 by the cells themselves ( Figure 3) .
  • the at least one deformable part of the culture chamber 2 is made of a transparent deformable material .
  • it is made of PDMS or a biocompatible silicone.
  • the culture chamber 2 is provided with a lid 14, made of a transparent material.
  • the culture chamber 2 has, for example, a diameter a little smaller than that of the 2 -inch Petri capsules typically used in the sector so that it can be coupled with the lid of a typical Petri capsule. In this way, a possibility of play is guaranteed between the lid 14 and the chamber 2.
  • the culture chamber 2 is constituted by a first cuplike body 9 identified by the bottom wall 3 and by a first side wall 10. Said cuplike body 9 is fixed with respect to a second cuplike body 11 identified by the bottom wall 3 itself and by a second side wall 12 that is substantially concentric and radially external to the first.
  • the actuator means 8, when they are present, are housed substantially within corresponding compartments 13 made within the side wall 12 of the second cuplike body 11.
  • said actuator means 8 are constituted by bimorph piezoelectric actuators .
  • Said actuators are a particular class of piezoelectric devices characterized by the capacity of producing flexural deformations much greater than those on the length and thickness for each individual piezoelectric layer. They are made up of two layers of piezoceramic material facing one another in such a way that, by applying a voltage to the electrodes, there will be determined deformation of the layers in opposite directions, which results in a flexural deformation of the actuator.
  • the device 1 is moreover provided with an annular load-bearing structure 15 having tangential appendages 16 projecting radially for housing motor means 17 and means 18 for supplying the actuator means 8.
  • the device 1 is provided with a system 19 for control of the actuator means 8, which are designed for fine adjustment of the degree of the load applied and, consequently, of the deformation induced in the at least one deformable part of the culture chamber 2 and in the cells or portions of tissues that adhere thereto in a way substantially fixed with respect thereto and which hence exchange therewith loads of tension, compression, etc.
  • Illustrated schematically in Figure 2 and designated as a whole by 100 is a system for determining the mechanical loads imposed on the cells/tissues in a culture by deformation of the substrate to which they adhere or else for determining the loads applied by the cells/tissues in the culture to the substrate to which they adhere.
  • the system 100 comprises a device 1 according to the invention, within which cells 101 or portions of tissues 101' can be housed.
  • the system 100 moreover comprises a laser source 102 and an optical detection system 103 designed to measure the light intensity and to detect the displacement of the orders of diffraction of the diffracted laser beam.
  • the deformation of the respective flexible elements 5 corresponds to a variation of the pitch p and, consequently, results in a displacement of the various orders of diffraction, with the obvious exclusion of the order 0.
  • the pitch p of the diffraction grating 4 is chosen, for example, as a function of the wavelength ⁇ of the laser light beam to be used for the measurement and of the angle ⁇ of the incident energy beam (laser beam) .
  • the optical detection system 103 is an optical sensor with four quadrants that enables evaluation of the direction and the degree of deformation.
  • Figure 2 is a schematic illustration of the structure thereof and in which the quadrants are identified by the letters A to D
  • a grating 4 When it is desired to measure only the deflection in a single direction it is sufficient for the grating 4 to be a simple binary grating. If it is desired to evaluate the deformation also in an orthogonal direction, a grating 4 is used constituted by a composite grating, for example, by two- dimensional periodic structures.
  • the culture chamber 2 contains cells/portions of tissue that adhere to the flexible elements 5 of a diffraction grating 4 made integrally on the at least one deformable part of the bottom wall 3 on a surface 6 set in use inside the chamber 2, said cells apply to the flexible elements 5 mechanical stimuli that determine a deformation thereof .
  • Said deformation in turn brings about a local variation of the pitch p of the corresponding diffraction grating 4, which can be detected in a way altogether similar to what has been described previously for the case of the deformation imposed from outside.
  • the system according to the present invention enables the focal plane to be kept unvaried during the deformation and makes it possible to measure precisely the degree of said deformation.
  • the system according to the invention can moreover be implemented and/or used both under a microscope and within an incubator, and consequently makes it possible to implement experimental protocols that envisage successive steps of dynamic deformation/stimulation between which one and the same cell culture can be observed and controlled under the microscope before being again inserted into the incubator.
  • the device 1 of the system 100 according to the invention is compatible also with the use of atomic- force- microscopy apparatuses .
  • the elements 5 are constituted by slats made by etching at least in part the bottom wall 3, in the case of the embodiment of Figure 5 the elements 5 are made as projections of an elongated shape of the bottom wall 3 , which extend in cantilever fashion therefrom towards the inside of the chamber 2. In this way, each projection element 5 is free to bend with respect to its root portion independently of the others, at least to the extent in which said deflection does not exceed the pitch p between them.
  • the wall 3 can be provided also as a substrate made of semiconductor material, by making the elements 5 using known photoetching techniques. It is consequently possible, if need be, to integrate in the wall 3 also possible optical or capacitive sensors.
  • the microstructures will be obtained by moulding on biocompatible silicone using moulds provided with silicon-micromachining techniques typical of microelectronics .

Abstract

L'invention porte sur un dispositif pour une stimulation dynamique et/ou un contrôle de cellules dans une culture comprenant une chambre de culture comprenant une paroi inférieure faite au moins en partie d'un matériau déformable et sur laquelle, disposée d'un seul tenant avec celle-ci, se trouve au moins une grille de diffraction, qui peut subir en conséquence une déformation conjointement avec ladite au moins une partie de la paroi inférieure, en réponse à un stimulus mécanique appliqué à l'extérieur ou à l'intérieur de la chambre de culture. La grille de diffraction déformable est formée par une pluralité d'éléments réalisés de façon à être flexibles indépendamment les uns des autres, réalisés sur la paroi inférieure sur le côté d'une face de celle-ci réglés en utilisation à l'intérieur ou sinon à l'extérieur de la chambre de culture. Dans le premier cas, le dispositif est approprié pour la mesure de charges mécaniques appliquées par les cellules dans la culture sur la structure à laquelle elles adhèrent. Dans le deuxième cas, le dispositif est utile pour évaluer avec précision le degré d'une charge appliquée aux cellules dans la culture par la mesure des déformations auxquelles elles sont soumises. L'invention porte en outre sur un système et un dispositif pour une stimulation dynamique et/ou un contrôle de cellules et de tissus dans une culture.
PCT/IB2009/005702 2008-05-26 2009-05-25 Système et procédé pour une stimulation dynamique et/ou un contrôle de cellules et de tissus dans une culture WO2009147482A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ITTO20080395 ITTO20080395A1 (it) 2008-05-26 2008-05-26 Sistema e dispositivo per la stimolazione e/o il controllo dinamici di cellule e tessuti in coltura
ITTO2008A000395 2008-05-26

Publications (2)

Publication Number Publication Date
WO2009147482A2 true WO2009147482A2 (fr) 2009-12-10
WO2009147482A3 WO2009147482A3 (fr) 2010-08-26

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IT (1) ITTO20080395A1 (fr)
WO (1) WO2009147482A2 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011044872A1 (fr) * 2009-10-14 2011-04-21 Forschungszentrum Jülich GmbH Dispositif d'analyse de cellules comprenant un élastomère ainsi qu'utilisation du dispositif
WO2014074067A1 (fr) * 2012-11-08 2014-05-15 Agency For Science, Technology And Research Procédés de culture de cellules ou de tissus et dispositifs pour la culture de cellules ou de tissus
WO2020159899A3 (fr) * 2019-01-28 2020-09-17 Cook Medical Technologies Llc Produits, procédés, système et appareil de traitement de matériau

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995003538A1 (fr) * 1993-07-20 1995-02-02 Balzers Aktiengesellschaft Matrice pour biocapteur optique
JPH10155475A (ja) * 1996-12-02 1998-06-16 Toru Takemasa シリコンベルトを使った培養細胞用伸縮刺激負荷装置
EP1428869A1 (fr) * 2001-08-30 2004-06-16 Takagi Industrial Co., Ltd. Incubateur pour cellules et structures
WO2004094586A2 (fr) * 2003-04-18 2004-11-04 Carnegie Mellon University Substrat de croissance cellulaire souple tridimensionnel et procedes associes
EP1679366A1 (fr) * 2005-01-07 2006-07-12 Ecole Polytechnique Federale de Lausanne EPFL-SRI Dispositif de culture de cellules sur des surfaces deformables
US20060281172A1 (en) * 2005-05-30 2006-12-14 Kosuke Kuwabara Cell culturel vessel, production process thereof and cultured cell

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995003538A1 (fr) * 1993-07-20 1995-02-02 Balzers Aktiengesellschaft Matrice pour biocapteur optique
JPH10155475A (ja) * 1996-12-02 1998-06-16 Toru Takemasa シリコンベルトを使った培養細胞用伸縮刺激負荷装置
EP1428869A1 (fr) * 2001-08-30 2004-06-16 Takagi Industrial Co., Ltd. Incubateur pour cellules et structures
WO2004094586A2 (fr) * 2003-04-18 2004-11-04 Carnegie Mellon University Substrat de croissance cellulaire souple tridimensionnel et procedes associes
EP1679366A1 (fr) * 2005-01-07 2006-07-12 Ecole Polytechnique Federale de Lausanne EPFL-SRI Dispositif de culture de cellules sur des surfaces deformables
US20060281172A1 (en) * 2005-05-30 2006-12-14 Kosuke Kuwabara Cell culturel vessel, production process thereof and cultured cell

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011044872A1 (fr) * 2009-10-14 2011-04-21 Forschungszentrum Jülich GmbH Dispositif d'analyse de cellules comprenant un élastomère ainsi qu'utilisation du dispositif
WO2014074067A1 (fr) * 2012-11-08 2014-05-15 Agency For Science, Technology And Research Procédés de culture de cellules ou de tissus et dispositifs pour la culture de cellules ou de tissus
WO2020159899A3 (fr) * 2019-01-28 2020-09-17 Cook Medical Technologies Llc Produits, procédés, système et appareil de traitement de matériau

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Publication number Publication date
ITTO20080395A1 (it) 2009-11-27
WO2009147482A3 (fr) 2010-08-26

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