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WO2011012119A1 - Procédé et dispositif pour détecter le mouvement et l'agglutination de cellules et de particules sur des couches cellulaires, tissulaires et d'implant lors de la simulation de conditions de circulation physiologique - Google Patents

Procédé et dispositif pour détecter le mouvement et l'agglutination de cellules et de particules sur des couches cellulaires, tissulaires et d'implant lors de la simulation de conditions de circulation physiologique Download PDF

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Publication number
WO2011012119A1
WO2011012119A1 PCT/DE2010/000900 DE2010000900W WO2011012119A1 WO 2011012119 A1 WO2011012119 A1 WO 2011012119A1 DE 2010000900 W DE2010000900 W DE 2010000900W WO 2011012119 A1 WO2011012119 A1 WO 2011012119A1
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WO
WIPO (PCT)
Prior art keywords
cells
particles
cell
tissue
interaction zone
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/DE2010/000900
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German (de)
English (en)
Inventor
Sandy Mosig
Knut Rennert
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.)
Universitaetsklinikum Jena
Original Assignee
Universitaetsklinikum Jena
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Publication of WO2011012119A1 publication Critical patent/WO2011012119A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • 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
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/02—Membranes; Filters
    • 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
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/10—Perfusion

Definitions

  • the invention relates to a method and a device for detecting movement and attachment processes of cells and particles at cell, tissue and implant layers in the simulation of flow conditions.
  • Such movement and attachment processes of the cells and particles are important for the study of cell and tissue physiology and effects caused by drugs, pathogens or other agents and the behavior of these cells and particles as they flow through the Influence organism.
  • the simulation devices should be able to simulate the flow conditions occurring in the body as realistically as possible in order to
  • TM Bocan Animal Mode of Atherosclerosis and Interpretation of Drug Intervention Studies, Curr Pharm Des 4, 1998, 37-52, AC McMahon, L. Critharides and HC Lowe Cardiovasc Haematol Disord 5, 2005, 433-440, JC Russell and S. Proctor: Small Animal Mode of Cardiovascular Disease: Tools for the Study of the Role of Metabolic Syndrome, dyslipidemia, and atherosclerosis, Cardiovasc Pathol 15, 2006, 318-330).
  • the microscope with condenser is directed to a focal plane above the interaction surface and can only look at this plane.
  • the observation is, however, restricted exclusively to this defined level, so that the cells are persecuted in their movement does not continuously in real time "can. In the considered level, it is only possible to detect the cells essentially as a static size of individual measurements.
  • the deliberately chosen flow rate in the flow chamber in DE 100 19 833 C2 allows the adjustment of flow and pressure conditions only to a very limited extent due to the device.
  • laminar flow conditions can not be built up and evaluated at the interaction surface.
  • Single observations of cells are not spatially resolvable due to the high dynamics of the cell movement.
  • this device only reproduces the flow conditions of the bloodstream and can thus observe cells that move in this simulated bloodstream and possibly hang on the wall and attach. Cells, however, which migrate through this wall and thus can be found below the interaction surface, can not be detected and evaluated and are thus not accessible to the investigation. However, such a statement is of great interest for the analysis of, for example, drug delivery for pharmaceuticals.
  • Endothelial cells Single cell layers of endothelial cells cultured in commercially available cell culture inserts. Before the start of the experiment, the endothelial cells are incubated with the substances to be tested by placing them directly on the cells. Subsequently, the substances are washed away and the actual attempt is started. The flow of the medium is generated via a syringe pump. Adhesion and transmigration are observed by a phase-contrast microscope placed at a perpendicular angle above the interaction surface. Endothelial cells need time to adapt to flow conditions.
  • Endothelial cells grown under static conditions have a different physiology than cells produced under the influence of flow conditions (eg BJT Butcher, AM Penrod, AJ Garcia and RM Nerem: Unique morphology and focal adhesion development of valvular endothelial cells in static and fluid flow environments, Arterioscler Thromb Vase Biol 24, 2004, 1429-1434).
  • flow conditions eg BJT Butcher, AM Penrod, AJ Garcia and RM Nerem: Unique morphology and focal adhesion development of valvular endothelial cells in static and fluid flow environments, Arterioscler Thromb Vase Biol 24, 2004, 1429-1434).
  • the devices provide no requirement to attract the cells under flow conditions. There is thus no possibility for longer cultivation of endothelial cells under flow conditions and the results are only very limited comparable with the in vivo situation.
  • transmigrated cells are collected in a container. When penetrating the interaction surface, however, transmigrated cells remain attached to their underside, which are ignored in the evaluation. Gravity is not enough to cause all transmigrated cells to fall down in order to be completely quantitatively evaluated. Thus, adhesion and migration processes are only inaccurately detectable.
  • transmigrant cells can not be detected and analyzed after adhesion.
  • the cell examination is given only on the said monocell layer.
  • the pressure and flow conditions in the capillary system can only be influenced to a limited extent. Even with this method, no long-term studies are possible.
  • transmigrating cells also escape elimination of the observation and the subsequent evaluation in this device as well. Again, only monolayers are used for investigation. Real-time analysis of cell observation is also not possible. The adjustment and adjustment of temperature and pH are not given. The cell layer can be incubated with the substances to be tested only from the apical side before starting the experiment. No dynamic perfusion is possible during the experiment.
  • the disadvantages of the animal experiments are, in particular, that the animals are killed during or at the end of the experiments and that a reproducible local effect of pathogens, drugs and other agents on disease patterns at defined points of the body even when using inbred strains, due to the individual Differences of the individual animals used, are very difficult to reproduce. This leads to the necessity of carrying out a large number of individual experiments in order to compensate for this disadvantage by using statistical aids.
  • the individual observation of flowing and accumulating cells in standardisable examination devices causes great difficulties due to the one-level visual observability and the high dynamics of the cell movement, so that the evaluation essentially amounts to the acquisition of statistical quantities ,
  • the invention is therefore based on the task of more accurately and completely detecting the movement and attachment processes of cells and particles in the simulation of flow conditions.
  • the movement and attachment processes should be able to be simulated and evaluated under conditions that are much closer to the pressure and flow behavior in the organism to be replanted or the body's own vessel, even for long-term investigations.
  • the adhesion and migration behavior of the cells and particles should also be able to be detected as completely as possible and with high evaluation accuracy.
  • Implant layer optically recorded and evaluated in the entire at least one interaction zone (and not only by view from above).
  • Detection is preferably carried out by several
  • Interaction zone in an area not only above, but also within and possibly below the same visually spatially resolved and fully monitored.
  • This monitoring may also be accompanied by spectroscopic detection of the movement and attachment of the cells and particles, including the zone of interaction with their cell, tissue and implant layers themselves, in that a spectroscopy unit is coupled to at least one of said observation beam paths.
  • Cells and particles transmigrating through the cell, tissue and implant layer of this at least one interaction zone which can be optically traced and possibly analyzed spectrometrically, are collected in at least one perfusion chamber below the interaction zone and can thus be fed to a further evaluation.
  • the flow conditions of the flow medium for the flow through the interaction zone for example by adjusting the pH, temperature, oxygen saturation and nutrient supply for the cells and particles, specially adjusted, for example by a metered control.
  • the invention allows an individual adaptation of physiological or even pathological flow conditions for the cells to be examined over desired periods of time.
  • this system allows the freely controllable addition of various substances not only from the apical but also from the basolateral side of the cell / tissue or implant layer. This is important because z. B. subcellular bioactive substances, such as modified LDL, have a strong influence on the interaction of leukocytes with the entire tissue association.
  • the study of the effects of various drugs, biologically active molecules, chemical noxae and microbiological organisms, such as bacteria, parasites and fungi is not limited only to the interaction of cells in the flow medium with the apical cells of the vascular wall, but also allowed an investigation of the vascular wall transmigrating and / or tissue differentiating cells.
  • the perfusion chambers integrated in the invention and the cell traps contained therein it is possible to absorb completely migrated cells vigorously and to make subsequent analytical and / or functional examinations accessible.
  • the invention has a freely controllable control of the environmental parameters pH value, temperature, amount of oxygen and nutrient addition, which physiological conditions can be adjusted or by the adjustable environmental parameters related pathological events, such as alkalosis, acidosis, nutrient restriction, hypoxia standardized and close to the in vivo situation can be analyzed.
  • Diseases such as cancer and sepsis, whose spread through blood flow, can be studied much more accurately, thoroughly and comprehensively with this invention than with previous in vitro methods.
  • the invention makes it possible to replicate the metastasis of tumor cells in a cost-efficient, standardized manner and without the use of animal models.
  • physiological conditions can be simulated to obtain a comprehensive picture of the spread of tumor cells in vivo.
  • Fig. 1 chamber body with three below the interaction zone
  • Fig. 2 frontal view of the chamber body with indicated supervisory Lens and Tubussystem
  • Fig. 3 lateral view of the chamber body in the longitudinal axis with indicated supervisory and lateral objective
  • Tube systems including fluorescence unit
  • FIG. 4 shows a lateral view of the chamber body in the longitudinal axis with indicated objective lens and tube systems and lateral Raman microscope and laser.
  • FIG. 5 schematic representation of the liquid circulations
  • Fig. 1 shows a chamber body 1 with three interaction zones 2, 3, 4, each consisting of a human body replicated cell, tissue and implant layer.
  • the chamber body 1 has an inflow 5, via which a flow medium 6 with the cells to be examined and particles (indicated by arrow) is introduced into the chamber body 1, so that the cells and particles of the inflowing flow medium 6 successively the interaction zones 2, 3, 4, under which three perfusion chambers 7, 8, 9 arranged are, flow through and come into contact with the respective cell, tissue and implant layer.
  • the flow medium exits via a drain 10 again.
  • the chamber body is heated by an integrated heating / cooling water channel system 11.
  • the interaction of the cells and particles of the flow medium with the interaction zones 2, 3, 4 can be observed by a microscope system 12 arranged laterally or on the side (see Fig. 2).
  • a microscope system 12 both stereomicroscope systems and confocal laser scanning microscopes or other microscope and spectroscopy systems can be used in a conventional manner.
  • the observation of the interaction zones 2, 3, 4 by the microscope system 12 is effected by interchangeable glass or quartz inserts 13, 14, 15 provided in the chamber body 1 and assigned to the interaction zones 2, 3, 4.
  • the microscope system 12 (means not shown in the drawing for reasons of clarity, for example rail-like guide elements known per se for transversal position change and also pivoting or rotation elements known per se for changing the angular position, via the side of the chamber body 1 with the glass body). or quartz inserts 13, 14, 15.
  • the microscope system 12 can monitor and evaluate the adhesion, migration and differentiation of the cells and particles of the flow medium and the cell, tissue and implant layers of the interaction zones 2, 3, 4 in real time.
  • FIG. 3 shows two microscope systems 12 which observe the interaction zones 2, 3, 4 of the chamber body 1, both in the direction of supervision and as well as laterally (observation beam path 36).
  • the two microscope systems 12 are each coupled via a camera port 16 or 17 to a computer system 18 (see FIG.
  • tissue and implant layers of the interaction zones 2, 3, 4 and / or the perfusion chambers 7, 8, 9, these areas are stimulated by a suitable and fluorescent dyes Light source 19 irradiated.
  • tissue and implant layers of the interaction zones 2, 3, 4 and / or the perfusion chambers 7, 8, 9, spectroscopy measurements can also be carried out.
  • the optical excitation by a laser 20 see Fig .. 4).
  • the measurement of the spectra is carried out by a detector 21, which can be integrated into the system, for example in the form of a known Raman microscope.
  • the detector 21 is coupled to a transducer system 22 for signal digitization, which is also in communication with the computer system 18 for data evaluation.
  • a cell reservoir 23 is used, which is coupled to a heatable / coolable medium reservoir 24 (see schematic illustration FIG.
  • a control of pH value and pO 2 value can be effected by gassing systems which may be integrated in the medium reservoir 24 or also in the cell reservoir 23 (not shown explicitly in the drawing for reasons of clarity).
  • An addition of substances into the flow medium can take place through a substance application valve 25.
  • FIG. 5 shows this medium circuit 26, in which also a sensor chamber 27 with sensor elements for detecting the cell culture parameters, such as pH values, temperature and Nutrient supply, is arranged for the existing in the flow medium 5 cells and particles.
  • the said cell culture parameters are controlled in the medium circuit 26 and, for example, as mentioned above, can be controlled in an application-specific manner via the abovementioned fumigation systems of the medium reservoir 24 or of the cell reservoir 23.
  • perfusion chambers 30, 31, 32 are arranged (see also perfusion chambers 7, 8, 9 in Fig. 1) are attached to the same (corresponding to the three said interaction zones), to each of which a reservoir 33, 34, 35 for additionally introduced into the medium circuit 26 test substances and other applications is connected.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Sustainable Development (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Dispersion Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

L'invention vise à détecter de manière plus précise et complète les processus de mouvement et d'agglutination de cellules et particules lors de la simulation de conditions de circulation physiologique. Selon l'invention, le mouvement et l'agglutination des cellules et particules sont observés, surveillés et évalués de manière optique dans leur totalité dans au moins une zone d'interaction (2, 3, 4), les cellules et particules transmigrantes sont capturées (7, 8, 9) à des fins de contrôle et d'évaluation et, notamment pour des analyses prolongées, les conditions de culture du milieu destiné à circuler dans la zone d'interaction (2, 3, 4) sont régulées par exemple par ajustement de la valeur du pH, de la température et de l'apport en nutriments pour les cellules et particules. L'invention est utilisée par exemple pour tester des produits pharmaceutiques, des substances pathogènes et diverses substances actives.
PCT/DE2010/000900 2009-07-30 2010-07-29 Procédé et dispositif pour détecter le mouvement et l'agglutination de cellules et de particules sur des couches cellulaires, tissulaires et d'implant lors de la simulation de conditions de circulation physiologique Ceased WO2011012119A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102009035502.2 2009-07-30
DE102009035502A DE102009035502A1 (de) 2009-07-30 2009-07-30 Verfahren und Vorrichtung zur Erfassung der Bewegung und Anlagerung von Zellen und Partikeln an Zell-, Gewebe- und Implantatschichten bei der Simulation von Flussbedingungen

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WO2011012119A1 true WO2011012119A1 (fr) 2011-02-03

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WO (1) WO2011012119A1 (fr)

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DE102013200613A1 (de) * 2013-01-16 2014-07-17 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Vorrichtung und Verfahren zum Bestimmen einer Stärke einer Adhäsion eines biologischen Materials
DE102014106423A1 (de) 2014-05-08 2015-11-12 Universitätsklinikum Jena Verfahren und Vorrichtungen zur In-vitro-Herstellung von Anordnungen von Zellschichten

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