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WO2016166282A1 - Substrat pour la culture de cellules - Google Patents

Substrat pour la culture de cellules Download PDF

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Publication number
WO2016166282A1
WO2016166282A1 PCT/EP2016/058342 EP2016058342W WO2016166282A1 WO 2016166282 A1 WO2016166282 A1 WO 2016166282A1 EP 2016058342 W EP2016058342 W EP 2016058342W WO 2016166282 A1 WO2016166282 A1 WO 2016166282A1
Authority
WO
WIPO (PCT)
Prior art keywords
substrate
cells
patch clamp
cell
patch
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/EP2016/058342
Other languages
German (de)
English (en)
Inventor
Thomas Knott
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
CYTOCENTRICS BIOSCIENCE GmbH
Original Assignee
CYTOCENTRICS BIOSCIENCE GmbH
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 CYTOCENTRICS BIOSCIENCE GmbH filed Critical CYTOCENTRICS BIOSCIENCE GmbH
Publication of WO2016166282A1 publication Critical patent/WO2016166282A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
    • G01N33/48728—Investigating individual cells, e.g. by patch clamp, voltage clamp
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2535/00—Supports or coatings for cell culture characterised by topography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants

Definitions

  • the invention relates, inter alia. a substrate for cultivating cells according to the preamble of claim 1.
  • This substrate according to the invention is intended in particular for culturing a collection or an aggregate of cells for the determination of preferably biologically relevant measured variables on these cells or their cell membranes.
  • the accumulation or aggregate of cells may also be referred to as tissue.
  • the substrate serves to culture so-called adherent cells, such as fibroblasts, endothelial cells, neurons, muscle cells, and the like, which grow on surfaces, as opposed to cells that grow freely in the nutrient medium.
  • the substrate is characterized in that it has at least one surface which, at certain positions, preferably at certain addressable positions, has an affinity, in particular an improved affinity, for adhering the cells to the substrate surface.
  • addressable is to be understood according to the invention that the corresponding position on the surface of the substrate (again) can be found, and so can be controlled, for example, in a subsequent measurement method to a cell adhering to this position / position specifically to the appropriate measurement or investigation.
  • the substrate according to the invention is suspendable in at least one solvent or at least one solvent mixture, i. the corresponding substrates can be converted into a suspension.
  • these are a buffered solvent or a buffered solvent mixture, i. a solvent or solvent system that does not (strongly) change its pH within certain limits on addition of an acid or a base.
  • the substrate according to the invention is preferably planar or planar. In the context of the present invention, this should mean that the substrate has a smaller, in particular significantly smaller, dimension in one spatial direction than in the two other spatial directions.
  • the substrate according to the invention is, in particular, a disk-shaped or annular substrate, wherein the substrate may preferably be composed of at least two disks or rings connected to one another.
  • the density of the MMS in this case controls the lowering, floating or rising in a suspension of the substrates in a solvent or solvent mixture, preferably in buffer solution
  • the relative density of the MMS compared to culture medium, washing and measuring buffers and compared to the respective cells an adjustable parameter for optimizing the cell culture processes (sinking or hovering or ascending) and the measuring processes, in particular the patch-clamp processes (MMS acts as a kind of sinker or parachute or hot-air balloon for the cells) from the perspective of the present invention.
  • the patch-clamp technique is an electrophysiological method of measuring the current flowing through individual ion channels in the cell membrane of a cell. can make.
  • the term "patch" refers to the small membrane section under the so-called patch pipette, which also serves as a measuring electrode, so that it is possible with this method to measure the function of cells in their natural environment in the living organism -Clamp technique according to the so-called cytocentering method (EP 1 31 1 655), cell suspension is pipetted onto a chip having microstructured openings By negative pressure, a cell of the cell suspension is selectively positioned on the opening of the chip and electrically
  • the method offers a number of advantages: In addition to the significantly higher throughput rate, the integration of microfluidic channels, for example, enables automated drug addition, as is the case in the Pharmafor The perfusion is also the intracellular egg This makes it much easier to investigate drugs that act intracellularly.
  • EP 131 1655 is hereby expressly incorporated by reference into the content of this description.
  • the "floating substrate" according to the invention for cultivation is introduced into a cell-repellent and hydrophobic culture dish or a device of comparable functionality, in particular growth of the cells on the cell Bottom of the culture dish excluded.
  • a concave culture dish is preferred to collect the substrates and the cells to be cultured at the lowest point of the dish.
  • a dish with many concave (micro) wells is more preferred.
  • the substrate according to the invention may vary in size, shape as well as in the coating.
  • these parameters are crucial for the growth or the amount of growing cells on the substrate.
  • the microstructuring can take place by means of geometric shape (provision of depressions, elevations and the like in the surface, possibly also in regular structures) and / or structured coating (possibly also as regularly repeating structures).
  • the coating can be carried out by physical, chemical, biochemical, molecular biological or biological methods.
  • the composition of the coating can be defined chemically or biochemically. This allows further control of cell growth.
  • Microstructured surfaces have already been used with cells in recent years. Especially for the implantation of implants, this technology opens up new possibilities in the targeted control of cell growth. This can be used, for example, for the treatment of peripheral nerve lesions in that the artificial nerve implant has microstructured surfaces that allow sequential growth of the severed nerve endings.
  • Another application is the targeted differentiation of endothelial and smooth muscle cells for use in vascular implants.
  • an extracellular environment is created by the microstructured surface, which should favor the formation of artificial vessels.
  • the present invention applies such microstructured surfaces in a novel way to the growth of susceptible micro-tissues for analytical purposes.
  • the growth of the cells is possible without having to give the substrate in suspension.
  • the adverse influence of shear forces, which occurs in a cultivation of cells in a moving suspension avoided. Only for the measurement, the substrate is transferred into suspension. For cultivation, however, the substrate is gently rinsed only with the appropriate nutrient medium.
  • the MMS or "floating substrate” according to the invention has a density which is smaller than the density of the cells to be cultivated, then the cells can settle on the substrate according to the invention without problems, for example the typical density of cells at 1 .1 g / mL, so that in these cases a density of the "floating substrate” of 1.05 g / mL is preferred. It has also proven to be particularly advantageous if the density of the "floating substrate” has a density which is greater than the density of the buffer, then the substrate according to the invention can settle out in this solvent / solvent mixture.
  • the substrate according to the invention is preferably "soft", but stable against cellular forces and / or not sharp-edged Ideally, the Shore hardness of the substrate is adjustable since the substrate hardness is a mechanical parameter that influences the differentiation of stem cells.
  • Both resorbable, biocompatible, biosynthetic material and non-resorbable material can be used as material, as is known from the prior art for so-called microcarriers for adherent cells.
  • Known materials are DEAE (diethylaminoethyl) -dextran, glass, polystyrene plastics, acrylamide plastics, collagens, alginates and the like. Also combinations of materials are included.
  • gases or vacuum may also be sealed into the material, in particular in order to vary the density of the substrate. Weight of the substrate divided by total volume (including inclusion volume) is referred to as the resulting density of the substrate.
  • the structuring or coating of the substrates according to the invention can be carried out such that both a 2-dimensional and a 3-dimensional cell growth is possible.
  • the size of the micro-substrates lies within the size ranges specified in the claims, advantageously between 1 ⁇ m to a few 100 ⁇ m.
  • FIG. 1 is a schematic representation of a culture dish with various substrates according to the invention, on which cells are already partly cultivated, and
  • FIG. 2 shows the schematic representation of an arrangement for a patch clamp measurement, in which cells to be examined are cultivated on a substrate according to the invention, and assigned to a patch clamp chip.
  • FIG. 1 shows a schematic plan view of a culture dish 6 into which various substrates 1, 2, 3 and 4 according to the invention have been introduced. Some of these substrates already have cells 5 cultured, i. these cells 5 are adhesively anchored to the respective substrates 2, 3 and 4.
  • the substrate 1 is a disk-shaped substrate which is not (yet) covered with a cell.
  • the substrate 2 is also disk-shaped and has a slightly larger diameter than the substrate. 1 On the surface of this substrate 2, a cell 5 is fixed.
  • the substrate 3 according to FIG. 1 is an annular substrate on which a cell 5 is likewise cultivated.
  • the substrate 4 according to the invention according to FIG. 1 is constructed from annular elements, specifically from five annular elements in the manner of the olympic rings. At this substrate 4, three cells 5 are already cultured, which are joined together at the corresponding location of the substrate 4 to a cell collection or a cell composite.
  • the respective surfaces of the substrates 1, 2, 3 and 4 are modified by coatings and / or microstructures so that the substrate has sufficient affinity to allow good adhesion of the cells 5.
  • corresponding coatings and / or microstructures are already sufficient on annular substrates in order to allow cultivation of the cells on the substrates according to the invention.
  • the inner surface of the culture dish 6 is cell-repellent and hydrophobic, so that the growth of the cells takes place on the substrates and not on the inner surface of the culture dish 6.
  • the cultivation of the cells takes place in the appropriate culture medium, and only after the cultivation of the cells 5 on the corresponding substrates 1, 2, 3 and 4 are the substrates cultured with the cells transferred into suspension in order to provide them in a suitable manner for the subsequent analysis.
  • the corresponding methods are generally carried out in such a way that the substrates according to the invention are introduced into the (cell) culture dish 6, in particular are sprinkled in, before the sowing of the cells takes place.
  • the substrates may be magnetically equipped to optimize the fixation at the bottom of the culture dish 6 accordingly.
  • the substrates according to the invention are transferred into suspension, for example by whirling, and, for example, into the corresponding patch clamp chip, for example the CytoPatch TM chip of US Pat Applicant, transferred.
  • FIG. 2 shows, in a schematic sectional view, how cells 5, which are cultivated on an annular substrate 2 according to the invention, can be supplied to a patch clamp measurement.
  • the annular substrate 2 is shown with two cells 5 cultured on this substrate 2.
  • the two cells 5 in the substrate 2 are cultured together to form a cell collection consisting of two cells.
  • the method described below can be carried out in the same way according to the invention with individual cells, as shown for example in Figure 1.
  • an anchoring element 7 is shown, by means of which the substrate 2 can be handled, and transferred in the desired manner in the patch-clamp device and can be positioned on or in the patch clamp chip. This transfer and positioning is indicated by the two arrows in Figure 2 graphically.
  • Such a patch clamp chip 8 is shown schematically in sectional view in the lower part of FIG.
  • this patch clamp chip in addition to the actual patch clamp openings 9, 9 'suction openings 10, 10' with their schematically indicated suction channels 1 1, 1 1 'for sucking the cells to be examined and cell membranes to the patch clamp openings 9, 9 '.
  • the channels 12, 12 'connected to the patch clamp openings 9, 9' are also indicated in the drawing.
  • These channels 12, 12 'takes place necessary for the measurement contact of the cell membranes with the measuring devices.
  • optical windows on the underside of the patch clamp chip 8 with the aid of which the docking of the cells 5 to the openings 9, 9 'can be monitored and, if necessary, documented and possibly optically analyzed.
  • the patch clamp chip 8 has devices which cooperate with anchor elements 7 of the substrate 2 in order to ensure an exact positioning of the substrate 2 in or on the patch clamp chip 8.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Physics & Mathematics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Organic Chemistry (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Hematology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un substrat utilisé pour la culture d'au moins une cellule, en particulier pour la culture d'une accumulation ou d'un agrégat de cellules, pour déterminer des grandeurs de mesure biologiquement pertinentes au niveau desdites cellules ou membranes de cellule. Le substrat présente au moins une surface qui, en des emplacements déterminés, de préférence en des emplacements déterminés adressables, présente une affinité pour adhérer aux cellules. L'invention concerne également un procédé permettant la culture d'au moins une cellule, en particulier la culture d'une accumulation ou d'un agrégat de cellules, selon lequel le substrat décrit selon l'invention est introduit dans un dispositif de culture hydrophobe repoussant les cellules, et les cellules sont cultivées sur ce substrat. Le substrat et le procédé décrits selon l'invention sont en particulier appropriés pour des procédés analytiques, qui utilisent la technique patch-clamp.
PCT/EP2016/058342 2015-04-15 2016-04-15 Substrat pour la culture de cellules Ceased WO2016166282A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102015206765 2015-04-15
DE102015206765.3 2015-04-15

Publications (1)

Publication Number Publication Date
WO2016166282A1 true WO2016166282A1 (fr) 2016-10-20

Family

ID=55913577

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2016/058342 Ceased WO2016166282A1 (fr) 2015-04-15 2016-04-15 Substrat pour la culture de cellules

Country Status (1)

Country Link
WO (1) WO2016166282A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1311655A2 (fr) 2000-07-05 2003-05-21 Nmi Naturwissenschaftliches Und Medizinisches Intitut An Der Universität Tübingen In Reutlingen Dispositif et procede pour mettre en contact electrique des cellules biologiques en suspension dans un liquide

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1311655A2 (fr) 2000-07-05 2003-05-21 Nmi Naturwissenschaftliches Und Medizinisches Intitut An Der Universität Tübingen In Reutlingen Dispositif et procede pour mettre en contact electrique des cellules biologiques en suspension dans un liquide

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HIRO-TAKA MASUDA ET AL: "Coating extracellular matrix proteins on a (3-aminopropyl)triethoxysilane-treated glass substrate for improved cell culture", BIOTECHNIQUES, 1 January 2014 (2014-01-01), England, pages 172 - 179, XP055280664, Retrieved from the Internet <URL:http://www.biotechniques.com/multimedia/archive/00232/BTN_A_000114156_O_232281a.pdf> DOI: 10.2144/000114156 *
NANCY K KLEENE ET AL: "A method for measuring electrical signals in a primary cilium", CILIA, BIOMED CENTRAL LTD, LONDON, UK, vol. 1, no. 1, 3 September 2012 (2012-09-03), pages 17, XP021117410, ISSN: 2046-2530, DOI: 10.1186/2046-2530-1-17 *
WOLFGANG WALZ ET AL: "Patch-Clamp Analysis ADVANCED TECHNIQUES Second Edition Planar Patch Clamping", 1 January 2007 (2007-01-01), pages 411 - 433, XP055281093, Retrieved from the Internet <URL:http://www.nanion.de/images/stories/papers/PlanarPatchClamping.pdf> [retrieved on 20160616] *

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