EP2433123A1 - Procédé d'analyse de la croissance neuritique - Google Patents

Procédé d'analyse de la croissance neuritique

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Publication number
EP2433123A1
EP2433123A1 EP10720130A EP10720130A EP2433123A1 EP 2433123 A1 EP2433123 A1 EP 2433123A1 EP 10720130 A EP10720130 A EP 10720130A EP 10720130 A EP10720130 A EP 10720130A EP 2433123 A1 EP2433123 A1 EP 2433123A1
Authority
EP
European Patent Office
Prior art keywords
regions
cells
neuron
neurons
substrate
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.)
Withdrawn
Application number
EP10720130A
Other languages
German (de)
English (en)
Inventor
Jonathan West
Jean-Philippe Frimat
Julia Sisnaiske
Jan G. Hengstler
Christoph Van Thriel
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.)
Leibniz Institut fuer Analytische Wissenschaften ISAS eV
Forschungsgesellschaft fuer Arbeitsphysiologie und Arbeitsschutz eV
Original Assignee
Leibniz Institut fuer Analytische Wissenschaften ISAS eV
Forschungsgesellschaft fuer Arbeitsphysiologie und Arbeitsschutz eV
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
Priority claimed from DE200910021876 external-priority patent/DE102009021876A1/de
Application filed by Leibniz Institut fuer Analytische Wissenschaften ISAS eV, Forschungsgesellschaft fuer Arbeitsphysiologie und Arbeitsschutz eV filed Critical Leibniz Institut fuer Analytische Wissenschaften ISAS eV
Priority to EP10720130A priority Critical patent/EP2433123A1/fr
Publication of EP2433123A1 publication Critical patent/EP2433123A1/fr
Withdrawn legal-status Critical Current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
    • 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
    • G01N33/5044—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 involving specific cell types
    • G01N33/5058—Neurological cells
    • 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
    • G01N33/502—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 for testing non-proliferative effects
    • G01N33/5032—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 for testing non-proliferative effects on intercellular interactions

Definitions

  • the invention relates to a method for analyzing neurite outgrowth.
  • Neurit refers to the following:
  • neural cell encompasses the diversity of the nervous system cells of humans, mice, other mammals and the cells of the nervous system that are of non-mammalian origin.
  • the functional integrity of the nervous system is guaranteed on the one hand by different types of nerve cells (eg neurons, glial cells) and on the other hand by the interaction between these different cells.
  • the structural and functional basis of this interconnection are axons and dendrites, which carry information from one neuron to another.
  • These networks or circuits are highly adaptive and in both the developing brain and the adult brain, new connections through axons and dendrites are created almost continuously.
  • Exogenous effects on the cells by foreign substances, such as environmental chemicals, drugs, chemical agents or drugs, as well as physical influences, such as electromagnetic radiation can either promote the process of interconnection (neurogeneration) or inhibit (neurotoxicity).
  • neurite outgrowth assays detect the formation of neurites as potential precursors of neuronal connections through axons and dendrites in the nervous system. These measuring methods are used in many ways because they are easy to handle and lead to relatively low material costs.
  • Neurite outgrowth can be measured by the number, length or branching (or other measures of neurite complexity) of the neurites at a particular time or by the neurite outgrowth rate. From these measurements a neurite growth index can then be determined.
  • these processes can be used in parallel through the use of robot technology and microtiter plates and thus the products can be investigated in a high-throughput process.
  • the cells are cultured randomly and unstructured and lack spatial coordinates that support identification of neurites. As a result, highly specific staining is usually needed to visualize the neurites.
  • a selective neurite marker expensive antibody
  • the company Millipore Corporation, Massachusetts, USA distributes a so-called "assay kit" which labels neurites for analysis by automated image processing in a high-throughput process.
  • the distance varies strong between adjacent cells.
  • the cells that touch each other are too close together, on the other hand, cells are separated by large distances so that there are different gradients for soluble factors that regulate neurite outgrowth. These differences in distance make it difficult to detect substance-induced behavioral changes in the cells.
  • US 2002/0072074 A1 discloses a method in which neurons are cultivated on a substrate having a raster pattern and their neurite outgrowths are analyzed.
  • cells do not deposit on first regions coated with peptides but on second regions surrounding the first regions.
  • the cells are then exposed to one, several or no test substance.
  • the substrate has first regions of standardized size and position which may be equidistant.
  • the arrangement may then be in a hexagonal or linear pattern. Since the neurite outgrowth of each cell is monitored and analyzed, but not the resulting compounds, this analysis method is very complex.
  • the object of the invention is to create a simple but effective possibility for analyzing neurite formation or neurite outgrowth.
  • This object is achieved by a method for analyzing neurite outgrowth in which a substrate is provided with a raster pattern having first regions of standardized size and position on which neurons and neuron-like cells can deposit, the first regions respectively of second regions on which neurons and neuron-like cells can not attach, where neurons or neuron-like cells are deposited on the first regions of the substrate and then the neurons or neuron-like cells are exposed to one or more or no treatments and / or during this and / or or thereafter analyzing the neurite outgrowths from the neurons or neuron-like cells by recognizing and quantifying the compounds formed by the neurite outgrowths between the first regions.
  • the invention is based on the use of cell pattering techniques to provide a spatially standardized assay for the measurement of neurite outgrowth during the onset of treatment (e.g., test substances).
  • substrates which have a raster pattern with first and second regions, wherein the first regions form attachment sites for the neurons or neuron-like cells and the second regions surrounding these first regions are designed so that attachment of neurons or neuron-like cells is not possible ,
  • a substrate which has first areas of standardized size and position, resulting in two major advantages:
  • the connections or their lengths arising between the neurite outgrowths between the first regions are likewise standardized.
  • This standardized arrangement also standardizes the neurite outgrowth lengths, making length measurements superfluous.
  • the spacing of the first regions is chosen so as to satisfy standard neurite classification criteria.
  • neurite outgrowth is defined as a process of length greater than or equal to one or more cell body diameters. This criterion is ensured by the substrate design.
  • the neurite outgrowths are not analyzed as such, but rather the rite outgrowths between the first areas, and quantified.
  • the number of compounds formed is a much higher functional indicator than the analysis of neurite outgrowth alone.
  • quantitative measurements of neurite outgrowths are also valuable, but they require detailed length measurements so that an "outgrowth" can be classified as neurite.
  • the method allows for standardized quantization of the simple quantification of the neurites, since the classification criterion is automatically met when a connection between first areas exists. This can be done both manually, but more preferably automatically by automatic image processing techniques and analysis. This enables high-content screening of neuroactive substances.
  • test substance for the treatment of the neurons or neuron-like cells, these are exposed to one or more test substance (s) and / or test condition (s).
  • test conditions are also physical influences, such as different rays (eg gamma rays (radiotherapy), electromagnetic rays (WLAN, UMTS, etc.) 131 iodine radiation (beta emitters)), other treatments, such as pH changes or other interventions in the milieu as well as temperature changes and the like. to understand.
  • rays eg gamma rays (radiotherapy), electromagnetic rays (WLAN, UMTS, etc.) 131 iodine radiation (beta emitters)
  • other treatments such as pH changes or other interventions in the milieu as well as temperature changes and the like.
  • the change in the occupancy of the substrate matrix can be easily determined by counting the number of occupied first regions before and after a treatment (e.g., exposure to a test substance).
  • the first regions are equidistant from each other and / or arranged in a hexagonal pattern to provide equal distances between all adjacent cell attachment sites (first regions).
  • the first regions may also be arranged in a linear pattern.
  • the first regions are preferably round and have a diameter between 2 to 200 .mu.m, preferably 5 to 200 .mu.m, more preferably 10 to 200 .mu.m.
  • the first regions are preferably separated from one another by a distance of between 5 and 1000 ⁇ m, preferably between 10 and 1000 ⁇ m.
  • Substrates are preferably used in which adjacent first regions are connected to one another by paths along which neurites can form, the width of these paths preferably being between 100 nm and 10 ⁇ m.
  • the invention provides a new protocol for the measurement of neurite outgrowth:
  • the cells are first arrayed in a raster pattern on a substrate, and after a time the number of viable cells in first regions and neurite outgrowths are measured by a selection of possible methods including the number of neurite outgrowths per viable cell, number of neurite outgrowths, and / or neurite outgrowth rates ,
  • Cell patterns can be treated with or without one or more test substances. If the cell patterns are not exposed to a test substance, this provides an experimental control for determining the relative effect of one or more substances of interest.
  • substrates are used in which the first regions contain one or more cell adhesion promoting materials, preferably cell adhesion proteins, such as laminin, fibronectin, collagen or lemon citrin, peptide sequences, poly-lysine, or other cell adhesion molecules or cell adhesion materials, such as hydrophilic polystyrene, glass, aminated surfaces, hydrophilic poly-dimethylsiloxane (PDMS).
  • cell adhesion proteins such as laminin, fibronectin, collagen or lemon citrin, peptide sequences, poly-lysine, or other cell adhesion molecules or cell adhesion materials, such as hydrophilic polystyrene, glass, aminated surfaces, hydrophilic poly-dimethylsiloxane (PDMS).
  • PDMS hydrophilic poly-dimethylsiloxane
  • substrates are used in which the second regions contain one or more cell adhesion-preventing materials, preferably polyethylene glycol, polyethylene oxide, agarose, albumin, poly-dimethylsiloxane (PDMS). , Polystyrenes and polyacrylamide.
  • cell adhesion-preventing materials preferably polyethylene glycol, polyethylene oxide, agarose, albumin, poly-dimethylsiloxane (PDMS). , Polystyrenes and polyacrylamide.
  • the first regions of the respective substrate are preferably hydrophilic and the second regions are preferably hydrophobic.
  • the paths of the same materials as the first areas or from others There are materials that support neurite outgrowth and either promote or not promote cell adhesion.
  • the first regions may be an exposed surface of the subject substrate, which itself is a standard tissue culture substrate, including polystyrene, polypropylene, and glass.
  • cell-repelling PDMS can be printed or embossed as a thin film pattern on standard tissue culture substrates including polystyrene, polypropylene and glass.
  • the neurons or neuron-like cells are deposited on the substrate without fixation and labeling and subsequently analyzed.
  • This procedure provides a particularly simple but effective way to determine neurite outgrowth.
  • the development of the network can be tracked periodically or even continuously. It is possible to use the functionality of the network (s) between the neurons or neuron-like cells with live imaging techniques, e.g. Calcium imaging, analyze. Changes in the functionality of the network can be used as a further indicator of the effect of a test substance or other treatments.
  • the neurons or neuron-like cells are fixed to the substrate by standard methods and / or that the neurons or neuron-like cells are completely or partially labeled.
  • standard fixing methods include, for example, formaldehyde and glutaraldehyde methods
  • the labeling can be carried out, for example, with Giemsa or other total cell dyes. In doing so, parts of the new cells or neuron-like cells, including their cell nuclei, their cytoskeleton or other cellular compartments, their neurite outgrowths, or parts of their neurite outgrowths.
  • the labels can be fluorescent, with DAPI for nuclear staining, phalloidin for actin staining, and fluorescent molecules or particles bound to antibodies or aptamers.
  • Neurite outgrowth can be analyzed by one or more of the following measurements:
  • Measurements of neurite outgrowth may be made continuously, periodically, or at the end of the exposure period of treatment of the neurons or neuron-like cells.
  • the particular substrate may be placed in a standard tissue culture or molecular analysis chamber including 6, 12, 24, 96, 384, 1536 well plates.
  • the chamber itself can be provided with first regions and second regions corresponding to the substrate for attachment or non-attachment of neurons or neuron-like cells.
  • the supply or removal of liquids, and in particular test substances, can be carried out by means of pipettes, automated systems for handling liquids or microfluidic systems.
  • microchips can be used as substrate carriers.
  • a plurality of substrates with raster patterns may be arranged, e.g. 5 x 5 substrates, each with 367 first areas.
  • FIG. 1 shows a substrate with a hexagonal arrangement of the cells suitable for the attachment of cells first areas
  • FIG. 2 shows a substrate similar to FIG. 1 with first regions interconnected by paths, FIG.
  • 3a to 3c show a method sequence for producing a substrate by microcontact printing
  • each first region contains a neuron cell
  • FIG. 6 shows the substrate according to FIG. 5 with an exemplary representation of neurite growth
  • Fig. 8 is an illustration of the number of connections per first area after 24, 48 and 72 hours.
  • FIGS. 1 and 2 show two different types of substrate, which have been found to be particularly suitable for carrying out the method according to the invention.
  • a substrate which has designated with 1 first regions which are arranged hexagonally. These first regions 1 are such that neurons and neuron-like cells can attach to them. Due to the hexagonal arrangement, these first regions 1 have an equidistant spacing and are preferably round. They have, for example, a diameter between 5 to 200 microns and are arranged at a distance of 10 to 1,000 microns from each other. The first regions 1 are surrounded by second regions 2, which form the background or the substrate base surface. These second regions 2 are such that neurons and neuron-like cells can not accumulate on them.
  • FIG. 2 shows a substrate which in principle corresponds to that according to FIG. 1, ie hexagonally arranged first regions 1 are provided which are surrounded by second regions 2, ie the substrate base surface.
  • first regions 1 are interconnected by paths 3 along which neurites can form.
  • the width of these paths 3 is between 100 nm and 10 ⁇ m.
  • first regions 1 and 2 are provided on a substrate.
  • substrates which have a multiplicity of first regions 1, for example 367 first regions.
  • Such substrates can be arranged on microchips, for example " 5 ⁇ 5 substrates can be arranged on a customary microchip.
  • a first process sequence is shown, namely the process of microcontact printing for the arrangement of thin PDMS patterns.
  • a stamp 4 is contacted with a thin PDMS liquid film 5 which is placed on a flat substrate, e.g. Glass substrate 6, is arranged (Fig. 3a).
  • the stamper 4 which is subsequently wetted with the PDMS liquid film 5, is lifted off and contacted with a cell culture substrate 7 to transfer the PDMS liquid film 5 to the substrate 7 (Fig. 3b).
  • the punch 4 is removed, and the pattern thus formed on the cell culture substrate 7 (second portions of PDMS) is thermally cured (Fig. 3c).
  • cell-repulsive hydrophobic materials are printed on hydrophilic substrates, such as glass or polyesters, on which cells can usually anneal and grow.
  • hydrophilic substrates such as glass or polyesters
  • liquid PDMS was dissolved in chloroform (1: 0, m / m), a volume of 500 ul the glass substrate 6 is added and spin-coated at 6,000 rpm for 30 seconds. After evaporation of the chloroform, a uniform PDMS film layer 5 remains. The use of the punch 4 with the liquid PDMS film 5 was achieved by contact of the punch 4 with the thin film 5 for a maximum of 10 seconds.
  • the wetted die 4 was then used to print the PDMS film 5 on glass substrates 7 or grade / tissue culture polystyres for a maximum of 10 seconds. This was followed by a curing step at 70 ° C for about 30 minutes to produce the thin film pattern (substrate).
  • superfluous PDMS is removed by a first contact pressure for a maximum of 10 seconds on a so-called “sacrificial" glass slide, followed by a second printing step, again for a maximum of 10 seconds on the cell culture substrate 7.
  • a hexagonal array of first areas 1 on the substrate with evenly distributed round first areas with typical diameters of 70 microns and distances of 100 microns between each digit can be achieved.
  • an embossing technique as shown in FIGS. 4a to c may be used.
  • a thin film of PDMS 8 is spin-deposited or the like onto a substrate 9 of glass or poly-styrene.
  • the substrate is then placed on a hot plate at about 70 ° C and a plunger 10 is pressed against the substrate for about 1 minute pressed (pressure 0.15 Nmm "2) and then removed (Fig. 4b).
  • a further step for thermal curing about 10 minutes
  • the cells were grown on a substrate according to FIG. 1. is either inoculated by the method according to FIG. 3 or according to FIG. 4 in a medium containing serum protein and incubated overnight. After exchange of the medium, adherent cells remain as a pattern attached to the exposed regions of the underlying substrate.
  • human SH-SY5Y neuroblastoma cells were used and differentiated into neurone-like cells by culturing in trans -retinoic acid for three days prior to attachment to the substrate. As a result, an additional advantage was achieved by synchronizing the cells for the experiment.
  • the neuronal patterns were cultured during which it came to neurite growth, which in turn led to connections between the neurons of adjacent first regions (attachment sites).
  • FIG. 5 shows the arrangement or attachment of the neuron-like cells on a substrate with a hexagonal arrangement of the first regions 1.
  • the neuron-like cells are denoted by Z.
  • FIG. 6 the neurite outgrowths or connections connecting the neuron-like cells Z are shown and designated V.
  • the standardized arrangement of the first regions 1 as attachment parts and the standardized defined (eg constant) length of the connections V forming between the neurite outgrowths between adjacent first regions 1 are recognizable. This simplifies the use of automated image processing techniques and neurite recognition, in particular their quantification. This enables "high-content” screening of new substances.
  • the neurons or neuron-like cells thus arranged on a substrate can be used to study the effects of substance on the growth of neurites. Model substances can be used to assess the suitability of neuronal patterns for the measurement of increased and decreased growth rates. For example, nerve growth factor (NGF) enhances neurite outgrowth, while acrylamide suppresses it.
  • NGF nerve growth factor
  • the experiments involve the patterning of neurons followed by cultivation in the presence of substances of interest.
  • neuronal patterns were cultured in the normal medium without these substances to provide control. After a cultivation period of, for example, two days, the neurite outgrowths were measured (eg number of neurites having neurites and / or the number of neurites per neuron). The measurements were carried out by means of phase contrast microscopy, for example with an inverse microscope IX 71 from Olympus. By comparison with the control sample, the toxic or promoting effects can be determined. In addition, neuronal patterns may be exposed to desired concentrations of the toxic or promoting model substances that serve as negative and / or positive controls.
  • FIGS. 7a to 7c show the development of an SH-SY5Y neural network over three days (after 24 hours, FIG. 7a, after 48 hours, FIG. 7b and after 72 hours, FIG. 7c).
  • Fig. 7c shows the state after 72 hours, seven neurite connections are recognizable.

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Toxicology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Food Science & Technology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

L'invention concerne un procédé d'analyse de la croissance neuritique selon lequel un substrat est préparé avec un motif de grille, lequel présente des premières régions sur lesquelles des neurones et des cellules analogues aux neurones peuvent se fixer, les premières régions étant respectivement entourées par des deuxièmes régions sur lesquelles des neurones et des cellules analogues aux neurones ne peuvent pas se fixer, des neurones ou des cellules analogues aux neurones étant fixées sur les premières régions du substrat et les neurones ou cellules analogues aux neurones étant ou n'étant pas ensuite exposés à un ou plusieurs traitements et les excroissances neuritiques des neurones ou des cellules analogues aux neurones étant analysées pendant ce temps ou par la suite. À cet effet, les liaisons se formant entre les premières régions en raison des excroissances neuritiques sont détectées et quantifiées.
EP10720130A 2009-05-19 2010-05-07 Procédé d'analyse de la croissance neuritique Withdrawn EP2433123A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP10720130A EP2433123A1 (fr) 2009-05-19 2010-05-07 Procédé d'analyse de la croissance neuritique

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DE200910021876 DE102009021876A1 (de) 2009-05-19 2009-05-19 Verfahren zur Analyse des Neuritenwachstums
EP09012960 2009-10-14
EP10720130A EP2433123A1 (fr) 2009-05-19 2010-05-07 Procédé d'analyse de la croissance neuritique
PCT/EP2010/002811 WO2010133301A1 (fr) 2009-05-19 2010-05-07 Procédé d'analyse de la croissance neuritique

Publications (1)

Publication Number Publication Date
EP2433123A1 true EP2433123A1 (fr) 2012-03-28

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EP10720130A Withdrawn EP2433123A1 (fr) 2009-05-19 2010-05-07 Procédé d'analyse de la croissance neuritique

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US (1) US20120065102A1 (fr)
EP (1) EP2433123A1 (fr)
CA (1) CA2762322A1 (fr)
WO (1) WO2010133301A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2019161048A1 (fr) * 2018-02-14 2019-08-22 The Trustees Of Columbia University In The City Of New York Système microphysiologique neuronal hiérarchique pour la fonction cérébrale et les troubles cérébraux
DE102020202610B4 (de) 2020-02-28 2021-09-23 Gottfried Wilhelm Leibniz Universität Hannover Verfahren und Vorrichtung zur automatisierten mikroskopischen Analyse von nervenzellhaltigen Proben

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Publication number Priority date Publication date Assignee Title
DE60023905T2 (de) * 1999-08-05 2006-07-27 Cellomics, Inc. Optische Systemanalyse von Zellen
GB2381535A (en) * 2001-10-30 2003-05-07 Qinetiq Ltd Device for forming a cellular network

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
TOUROVSKAIA A ET AL: "Micropatterns of chemisorbed cell adhesion-repellent films using oxygen plasma etching and elastomeric masks", LANGMUIR, AMERICAN CHEMICAL SOCIETY, NEW YORK, NY; US, vol. 19, no. 11, 27 May 2003 (2003-05-27), pages 4754 - 4764, XP002367767, ISSN: 0743-7463, DOI: 10.1021/LA0267948 *

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CA2762322A1 (fr) 2010-11-25
US20120065102A1 (en) 2012-03-15

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