WO2014118311A1 - Dispositif et procédé pour la régulation de la distribution spatiale de cellules et de leur descendance - Google Patents

Dispositif et procédé pour la régulation de la distribution spatiale de cellules et de leur descendance Download PDF

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WO2014118311A1
WO2014118311A1 PCT/EP2014/051873 EP2014051873W WO2014118311A1 WO 2014118311 A1 WO2014118311 A1 WO 2014118311A1 EP 2014051873 W EP2014051873 W EP 2014051873W WO 2014118311 A1 WO2014118311 A1 WO 2014118311A1
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cell
patterns
pattern
network
adhesive
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PCT/EP2014/051873
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English (en)
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Yoran MARGARON
Sébastien DEGOT
Violaine CHAPUIS-PERROT
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Cytoo
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    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/52—Fibronectin; Laminin
    • 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
    • C12N2535/10—Patterned coating

Definitions

  • the invention relates to a device and a method for controlling the spatial distribution of adherent cells and their progeny.
  • tissue culture plastic or microcarriers which may be coated with extracellular matrix components to increase adhesion properties and provide signaling pathways supervising cell growth and differentiation.
  • cells When grown in an appropriate cell medium in a cell incubator with controlled temperature and gas mixture, cells can divide and daughter cells are randomly disposed in the neighboring area.
  • cells can be detached by known techniques such as a mixture of trypsin-EDTA and a small amount of these cells can be recycled to seed a new culture.
  • cells at the periphery of the colony are fully proliferating compared to cells located at the center that are physically constrained by surrounding cells and usually poorly proliferating.
  • adherent culture is an organotypic culture, which involves growing cells in a three-dimensional (3D) environment as opposed to the classical two-dimensional culture dishes.
  • This 3D culture system is biochemically and physiologically more relevant to in vivo tissue, but is technically challenging to maintain.
  • document EP 1 664 266 proposes a culture device comprising an adhesive surface having a determined shape onto which only one cell can adhere. Once cells are attached to such surfaces, they use the adhesive clues provided to adopt the overall shape of the surface while cell's organelles are disposed in a controlled organization.
  • Cells may then be normalized from their spatial configuration that adopts the geometry of the adhesive surface.
  • Another drawback of this technique is that detailed analysis of organized cell compartments must be performed before the first cell division (i.e. within about 18 to 30 hours from seeding, depending on the cell line). Indeed, once two cells (daughter cells from the initial mother cell) are on the same pattern, the stability of each cell shape cannot be controlled, except for a few specific motifs.
  • a culture device may comprise several patterns that are separated by a cytophobic surface, the distance between adjacent patterns being large enough to prevent spreading of a cell over two patterns or more.
  • Document US 5,776,748 discloses a device for adhering cells on a plurality of rectangular patterns arranged according to a grid array.
  • the cytophobic regions separating the patterns are sufficiently wide so as to confine cell spreading to the respective pattern and prevent any contact between cells adhering on adjacent patterns.
  • the cell shape is not controlled.
  • the control of cell shape is lost as soon as the first cell division occurs, since two or more progeny cells are constrained to adhere to the same pattern.
  • Document EP 2 180 042 discloses a device to constrain multicellular arrangements in stable and reproducible spatial configuration.
  • Such a device comprises a plane surface onto which a set of two or more adhesive patterns are formed, wherein adjacent patterns are separated from each other by an essentially non-adhesive surface, the distance between said patterns being large enough to prevent a cell on a pattern to reach another pattern.
  • the area covered by the set i.e. the patterns and the essentially non-adhesive surface, is chosen so as to adhere a determined number of at least two cells.
  • the patterns belonging to the same set may be connected by an adhesive zone.
  • the device may comprise several sets of patterns, each set being spatially isolated from the others by a cytophobic surrounding surface, such that cells seeded on a set cannot reach another set.
  • pattern designs are mainly organized for interacting cell lines such as epithelial cells or cells building tight cell-cell junctions.
  • the set geometries are designed to accommodate a defined number of cells, preventing the spatial stabilization to occur before the exact number of cells is reached.
  • cells positioned at the center are surrounded by cells. As they cannot access to free adhesive area, such cells do not receive structural clues and do not stabilize properly.
  • T Before division ( Figure 1A), cells C are stably spread (in light grey) on "T” motifs (referred to as T).
  • a goal of the invention is to provide device and method for controlling the spatial distribution of a cell population from their initial seeding to several generations of daughter cells.
  • the invention provides a method for controlling the spatial distribution of adherent cells and their progeny over time, comprising the steps of:
  • a device comprising a surface with a plurality of cell-adhesive patterns on said surface, said cell-adhesive patterns being disposed so as to form at least one network consisting of a plurality of recurring elementary arrangements, each elementary arrangement comprising at least one cell-adhesive pattern, wherein:
  • the distance between adjacent cell-adhesive patterns of said network is chosen so as to allow a cell seeded on a pattern to move to at least one adjacent pattern
  • the shape of the cell-adhesive patterns and distance between adjacent patterns of said network is selected so as to promote conformation of one such cell to a pattern and migration of daughter cells to adjacent patterns of said network;
  • a pattern means a cell-adhesive surface whose dimension is sufficient to anchor a single and having a determined shape enabling controlling the spatial organization of said cell.
  • Such a pattern may be continuous, i.e. formed of a single adhesive surface, or discontinuous, i.e. formed of a plurality of discrete adhesive surfaces that together define the shape and size of the pattern.
  • a network means a plurality of patterns that are disposed according to recurring elementary arrangements and with a distance between patterns that allows spreading or migration of a cell from a pattern to an adjacent pattern.
  • An elementary arrangement may consist of a single pattern or of at least two patterns. In the latter case, the patterns may be identical or have different shapes.
  • said recurring elementary arrangements may be disposed in parallel rows and/or columns, in staggered rows, in fractal figures, or in any regular geometric disposition, etc.
  • the distance between adjacent patterns is defined as the minimal distance between adjacent patterns. If adjacent patterns are in contact, said distance is null.
  • the disposition of the cell-adhesive patterns and/or of the elementary arrangements within a network is selected so as to control the flow of cell division on said network.
  • the size and/or shape of the cell-adhesive patterns is preferably selected so as to allow stabilization of the shape of a single cell seeded on said pattern.
  • Adjacent cell-adhesive patterns of said network may be at least partially separated by a cytophobic surface.
  • the shape of said at least one cell-adhesive pattern may be approximately a disc, a crossbow, an L, a Y, a rectangle, or any combination thereof.
  • Each elementary arrangement may consist of a single pattern. Alternatively, each elementary arrangement may consist of at least two identical patterns.
  • each elementary arrangement may consist of at least two patterns having different shapes and/or sizes and/or orientations, wherein at least a one pattern, called normalizing pattern, has a shape chosen so as to promote conformation of one cell to said pattern and at least one other pattern, called intermediate pattern, has a shape chosen so as to promote cell displacement from one normalizing pattern to another normalizing pattern.
  • normalizing pattern has a shape chosen so as to promote conformation of one cell to said pattern
  • intermediate pattern has a shape chosen so as to promote cell displacement from one normalizing pattern to another normalizing pattern.
  • Said recurring elementary arrangements may be disposed in parallel rows and/or columns, in staggered rows, according to a regular geometric array or according to a fractal figure.
  • the rate of cells conformed to one respective cell-adhesive pattern of the network is greater than or equal to 20%, in particular comprised between 20% and 35%.
  • the distance between adjacent patterns is comprised between 4 ⁇ and 30 ⁇ .
  • Said method advantageously further comprises acquiring images of the cells during said division and migration and determining cell shape and/or motility parameters from said acquired images.
  • the method comprises a preliminary step of manufacturing said device by the following steps:
  • the invention relates to a cell culture system comprising a device having a surface with a plurality of cell-adhesive patterns on said surface and at least one cell normalized on at least one of said cell-adhesive pattern, said cell-adhesive patterns being disposed so as to form at least one network consisting of a plurality of recurring elementary arrangements, each elementary arrangement comprising at least one cell-adhesive pattern, wherein:
  • the distance between adjacent cell-adhesive patterns of said network is chosen so as to allow a cell seeded on a pattern to move to at least one adjacent pattern
  • said cell culture system comprises at least one cell and progeny, wherein said cell and progeny are normalized on adjacent cell-adhesive patterns of the network.
  • Another aspect of the invention is a device for carrying out the method described above.
  • Said device comprises a surface and a plurality of cell-adhesive patterns on said surface, wherein said cell-adhesive patterns are disposed so as to form at least one network, each network consisting of a plurality of recurring elementary arrangements, each elementary arrangement comprising at least one cell-adhesive pattern, wherein the distance between adjacent cell-adhesive patterns of said network is chosen so as to allow a cell seeded on a pattern to move to at least one adjacent pattern.
  • said device comprises at least two networks that differ by at least one of the size, shape of the cell-adhesive patterns and the distance between adjacent cell-adhesive patterns.
  • Another object of the invention is a cell culture kit comprising such a device and instructions for controlling cells and their progeny according to the present invention.
  • This invention can be useful in target validation, drug screening, in vitro toxicology, diagnostics, biochips, and cellular or gene therapy.
  • FIGS. 2A and 2B show respectively a network according to an embodiment of the invention and the corresponding elementary arrangement of cell-adhesive patterns
  • FIG. 3 shows a network according to another embodiment of the invention.
  • - Figure 4 shows a network according to another embodiment of the invention
  • - Figures 5A to 5D show networks consisting of similar elementary arrangements of patterns, but with increasing distances between patterns;
  • FIG. 6A to 6C show networks based on identical patterns, but wherein the recurring elementary arrangements of said patterns (each elementary arrangement consisting in this case of a single pattern) are disposed respectively in rows and columns, in staggered rows and in fractal figures;
  • Figure 8 illustrates a top view of a 96-well plate according to the invention
  • Figures 9A to 9C relate to the characterization of the most suitable pattern(s) for normalizing HeLa cell shape and polarity:
  • Figure 9A is an image after 72 hrs incubation on a honeycomb network consisting of Y patterns having a surface of 1600 ⁇ 2 with 12 ⁇ distance between adjacent patterns;
  • Figure 9B shows the amount of cell shape normalization on one pattern and
  • Figure 9C is a cartography of the normalization of HeLa cells on a plate as shown on Figure 8;
  • Figure 10 shows the mitochondria status of different cell types after 72 hrs incubation on a network according to the invention;
  • Figure 1 1 shows the quantification of cells with a spindle orientation axis normalized within a cell population cultured on a network according to the invention
  • Figures 12A to 12G show respectively a "stairs" network, one elementary arrangement that forms this network and images of the cells cultured on said network acquired at different times after seeding;
  • Figures 13A to 13G show respectively a "honeycomb” network, one elementary arrangement that forms this network and images of the HeLa Kyoto cells cultured on said network acquired at different times after seeding;
  • Figures 14A to 14F show respectively a "honeycomb” network, one elementary arrangement that forms this network and images of the A549 cells cultured on said network acquired at different times after seeding;
  • Figures 15A to 15D show results of cell tracking using H2B-mCherry HeLa Kyoto cell fluorescence for different networks.
  • the method uses a network of cell-adhesive patterns that, allows cell shape normalization while preserving the cell physiology, said normalization being also applicable to progeny.
  • Figure 2A shows an example of a network 100 formed of a plurality of Y-shaped cell- adhesive patterns 1 .
  • Said network 100 is in fact formed of a plurality of recurring elementary arrangements 10 of said cell-adhesive patterns 1 .
  • Figure 2B shows the elementary arrangement 10 of Y-shaped cell-adhesive patterns 1 that form said network.
  • the elementary arrangement 10 comprises six identical cell- adhesive patterns 1 that are disposed with defined orientations with respect to one another, in order to form a snowflake shape.
  • the distance between adjacent cell-adhesive patterns 1 is chosen so as to allow a cell seeded on a pattern to move to at least one adjacent pattern
  • the distances a, b and c are of 14.5 ⁇ , 36.2 ⁇ and 21 .5 ⁇ , respectively.
  • the surface 2 that separates adjacent patterns 1 is preferably a cytophobic surface.
  • cells that adhered to and adopted the shape of a pattern can move to adjacent pattern(s) and re-adopt their shape before the next cell division.
  • daughter cells When cell division occurs, daughter cells can move to adjacent patterns and adopt an equivalent stable morphology.
  • this cell shape stability can be maintained over several cell divisions, thus allowing both short and long term experiment.
  • Cell spreading properties are specific to each cell line and depend on many parameters, including cell size and its ability to contract on matrix substratum.
  • adhesive patterns named "Disc”, “Y”, “L”, and “Crossbow” offered by CYTOO may be used, which induce cells area of 700 ⁇ 2 , 1 ,100 ⁇ 2 , 1 ,600 ⁇ 2 , 2,200 ⁇ 2 and 3,000 ⁇ 2 .
  • FIGS 3 and 4 shows networks made of different cell-adhesive patterns.
  • the cell-adhesive patterns are of "crossbow” type and arranged in staggered rows.
  • the cell-adhesive patterns are of "L" type and arranged in staggered rows.
  • minimal distances between adjacent cell-adhesive patterns may be variable to adapt to cell capabilities.
  • the minimal distance may depend on the contractility of the cells.
  • An array of minimal distances were defined as 0 ⁇ , 2 ⁇ , 4 ⁇ , 6 ⁇ , 7.5 ⁇ , 8 ⁇ , 12 ⁇ , 14.5 ⁇ 15 ⁇ , 20 ⁇ , 30 ⁇ , 40 ⁇ and 50 ⁇ .
  • the minimal distance between adjacent patterns may be comprised between 4 ⁇ (for poorly contractile cells, such as MDA MB231 ) and 30 ⁇ (for contractile cells, such as A549 or fibroblasts NIH3T3).
  • Figures 5A-5D displays a variety of minimal distances between patterns within a network type.
  • the cell-adhesive patterns are of "Y" type and are disposed as snowflakes networks as explained above.
  • the distance between the patterns is of 0 ⁇ , meaning that the patterns are in contact with one another; in Figures 5B-5D, the distance is of 4 ⁇ , 8 ⁇ and 12 ⁇ , respectively.
  • the respective orientation of the cell-adhesive patterns may also be defined in order to control the flow of cell division on the network.
  • the angular range between adjacent patterns is from 0° to 360°.
  • crossbows patterns can be disposed as networks so as to form parallel rows and columns, staggered rows or and fractal figure such as Cayley's trees, respectively, depending on the angle between adjacent patterns.
  • an elementary arrangement may be formed of several identical patterns.
  • an elementary arrangement may be formed of several different patterns with variable sizes and shapes.
  • some of them may have a shape and surface sufficient for allowing conformation and stabilization of a cell thereon, whereas other patterns (so-called intermediate patterns) may have a smaller surface and are only intended for promoting spreading of a cell from one normalizing pattern to another normalizing pattern.
  • the shape and orientation of the intermediate patterns with respect to the normalizing patterns are chosen so as to help cell displacement.
  • Figure 7A shows an example of a network where normalizing "crossbow” patterns are combined with intermediate rectangle patterns arranged between the "crossbow” patterns to help cell displacement.
  • Figure 7B shows an example of an elementary arrangement where a "disc” pattern is combined with "Y" patterns arranged around the disc.
  • the disc may be used as a cell reservoir while the Y patterns may be normalizing patterns.
  • Figure 7C shows an example of a network combining rectangular patterns of different shapes arranged alternately so as to form dashed lines.
  • the largest rectangular patterns may be normalizing patterns wherein the smallest ones may be intermediate patterns.
  • the above-described network may be formed on a surface of any device usually intended for cell culture.
  • the device may be a glass, plastic or silicon oxide substrate, a cell culture plate or dish (this list not being intended to be limitative).
  • a device comprising such a network can be made by known technologies.
  • a method for manufacturing a device comprising at least one network may comprise the following steps:
  • the master template is prepared from a silicon wafer coated with a photoresist layer illuminated with UV through a mask on which the adhesive patterns have been designed.
  • the stamp is preferably made of PDMS (poly(dimethylsiloxane)) or of another siloxane-based polymer.
  • the non-printed surface of the device is made cytophobic by incubation with an inert material such as polyethyleneglycol.
  • EP 1 664 266 For more details about such a method, one can refer to EP 1 664 266.
  • the device may comprise at least two networks, wherein the distance between adjacent networks is chosen so as to prevent any movement of cells from a network to another one.
  • the device is a multi-well cell culture plate, e.g. a 96-well plate such as the CYTOOplateTM plates offered by CYTOO.
  • a network of cell-adhesive patterns is formed in each well of the plate.
  • the networks can be identical (in terms of shape, size of the cell-adhesive patterns and of distance between adjacent patents) in each well.
  • At least two wells comprise different networks, said networks differing by at least one of the cell-adhesive pattern shape, size, or distance between adjacent patterns.
  • This configuration allows testing simultaneously, on a same plate, a plurality of network geometries in order to determine the most suitable design(s) that may be used to define the behavior of a new cell line.
  • a device comprising at least two different networks may be a useful tool to design a suitable pattern to long-lasting study of a determined type of cell.
  • dynamic cell shape model can be determined from individual eel- adhesive patterns.
  • the selected networks are formed on the device and cells are cultured on each of said networks to study their behavior.
  • FIG. 8 An embodiment of a 96-well plate comprising a plurality of networks is shown in Figure 8.
  • the wells (which are here represented as discs) are disposed according to 8 rows (R1 to R8) and 12 columns (C1 to C12).
  • the cell-adhesive patterns In the 4 upper rows (R1 to R4), the cell-adhesive patterns have a "Y" shape, with a surface of the pattern varying from 700 ⁇ 2 to 2200 ⁇ 2 from the first to the fourth row.
  • the cell-adhesive patterns of these networks are arranged according to a honeycomb layout, as shown in Figure 13A.
  • the cell-adhesive patterns In the 4 lower rows (R4 to R8), the cell-adhesive patterns have a "L" shape, with a surface of the pattern varying from 700 ⁇ 2 to 2200 ⁇ 2 from the first to the fourth row.
  • the cell-adhesive patterns of these networks are arranged according to a stairs layout, as shown in Figure 12A.
  • the distance between the cell- adhesive patterns varies from 0 ⁇ to 20 ⁇ .
  • the wells comprised in both rows R1 -R4 and columns C1 -C2 are devoid of any network: the surface of the well is formed of a uniform cell-adhesive surface like in conventional plates.
  • Cells are cultured in their classical growing media (Gibco) supplemented with 10% FBS (Sigma) and 0.5% penicilin and streptomycine (Gibco).
  • Cells are centrifuged 4 minutes at 300 g and the pellet is resuspended in fresh culture media.
  • 500 ⁇ are seeded on a CYTOOchip presenting a panel of networks and mounted into a CYTOOchamber.
  • Living cells are imaged every 30 minutes during 72 hours using a videomicroscope (Ti Eclipse, Nikon) with controlled environment (humidified atmosphere, 7% C02, 37°C).
  • a videomicroscope Ti Eclipse, Nikon
  • controlled environment humidity, 7% C02, 37°C.
  • Epifluorescence channels can be used to image fluorescent probes, exogenous proteins with fluorescent tags as well as fluorescent networks.
  • Cell motility can be analyzed using the tracking function of Metamorph software (Molecular Devices).
  • a device as described above - in particular a multiwall plate as shown on Figure 8 - allows characterizing the normalization of cell shape and polarity of a specific cell line.
  • Figure 9A shows the characterization of normalization of HeLa cells on a plate as shown in Figure 8.
  • HeLa cells were deposited and incubated in each well of the plate during 72 hours.
  • nucleus/actin/focal adhesions left picture
  • nucleus/golgi/centrosome right picture
  • the dashed line represents the nucleus/golgi axis.
  • Figure 9B shows the amount of cell shape normalization on one pattern of each of the 96 wells.
  • the wells can thus be classified with different levels of cell shape normalization, e.g.:
  • Figure 9C shows four relative levels of normalization that have been attributed to each well based on the characterization of the normalization of HeLa cells: high (1 ), good (" ⁇ ), medium (2), low (3).
  • the wells belonging to both rows R1 -R4 and columns C1 -C2 do not comprise any network (level 4) and are thus not considered in the evaluation of normalization.
  • the inventors have also shown that the above-described networks respect cell physiology.
  • the table below shows the comparison of cell doubling time of different cell lines described in the literature (the cells being incubated on a uniform cell-adhesive surface in this case) and measured on networks according to the invention in similar incubation conditions.
  • Figure 10 shows the evaluation of cell health after 72 hours incubation on a network by using mitochondria staining (Mitotracker Green).
  • Figure 1 1 shows the quantification of cells with a spindle orientation axis normalized within a cell population cultured on a network.
  • the network is either a honeycomb network formed of "Y" patterns with different surfaces or a stairs network formed of linked "L” patterns with different surfaces. In both cases the distance between adjacent patterns is of 12 ⁇ .
  • HeLa kyoto cells were seeded on a network composed of continuous "L” patterns coated with fibronectin (adhesion protein).
  • Figure 12A shows said network.
  • Figure 12B shows an elementary arrangement of said network, consisting of two "L” patterns that are linked so as to form a "stairs" shape.
  • the distance a was in this case of 42.8 ⁇ .
  • Figures 12C-12G show images acquired at different times, i.e. 0 hour (cell deposition); 25.5 hours; 30.5 hours; 43 hours and 66 hours, respectively.
  • This method thus induces the control of the cell spatial distribution.
  • HeLa kyoto cells were seeded on a "honeycomb" network (see Figure 13A) composed of "Y” patterns coated with fibronectin (adhesion protein) that were separated by a cytophobic area of at least 7.5 ⁇ (distance b) (see Figure 13B).
  • each branch of the "Y" patterns was of 18 ⁇ (distance a) and the height of a honeycomb was of 76 ⁇ (distance c).
  • Figures 13C-13G show images acquired at different successive times.
  • a cytophobic area separates two adjacent patterns and stabilizes cells on their initial position.
  • A549 cells are seeded on a "honeycomb” network (see Figure 14A) composed of "Y" fibronectin patterns that are separated by a cytophobic area of at least 30 ⁇ (distance b) (see Figure 14B).
  • the length of each branch of the "Y" patterns was of 18 ⁇ (distance a) and the height of a honeycomb was of 128 ⁇ (distance c).
  • Figures 14C-14F show images acquired at different successive times.
  • Example 2 In contrast to Example 1 but similarly to Example 2, the cytophobic area that separates two adjacent patterns maintains the cells on their initial pattern in the network.
  • the daughter cells when cells divide, the daughter cells can migrate entirely to the adjacent pattern where they completely individualize from the mother cells and adopt a stable triangular shape.
  • This type of networks allows therefore a tight control of cell distribution and shape normalization over time.
  • This example is intended to characterize cell behavior by tracking of cell movements using H2B-mCherry cell fluorescence of HeLa Kyoto cells.
  • the network of cell-adhesive patterns as described above have multiple advantages in view of controlling the spatial distribution of cells and of their progeny.
  • cells shapes are stabilized on the adhesive pattern after the first cell division, for at least three cell divisions and potentially extendable without time and surface constraint.
  • the network is thus fully customizable depending on the cell to be tested.
  • the network is suited to provide a spatial and temporal homogeneity of the cell population distribution.
  • the network allows optimization of the cell population distribution, increasing the number of measurements for each condition.
  • the network allows a better individualization of daughter cells shapes from one mitosis and direct comparison between them, as well as a comparison of cell shape and migratory behavior within and between cells generations.
  • the network provides an increase of the SCORE, i.e. the rate of single cell on an adhesive pattern, which is an important parameter in High Content Analysis (HCA).
  • HCA High Content Analysis

Abstract

L'invention concerne un procédé pour la régulation de la distribution spatiale de cellules adhérentes et de leur descendance, comprenant les étapes de : - fourniture d'un dispositif comprenant une surface ayant une pluralité de motifs d'adhésion cellulaire sur ladite surface, lesdits motifs d'adhésion cellulaire étant disposés de sorte qu'ils forment au moins un réseau, dans lequel : (i) la distance entre des motifs d'adhésion cellulaire adjacents dudit réseau est choisie de telle sorte qu'elle permet à une cellule ensemencée sur un motif de se déplacer sur au moins un motif adjacent, et (ii) la forme des motifs d'adhésion cellulaire et la distance entre des motifs adjacents dudit réseau étant choisies de sorte qu'elles favorisent la conformation d'une telle cellule en un motif et la migration de cellules filles sur des motifs adjacents dudit réseau; - l'ensemencement d'au moins une cellule sur au moins un motif; - la culture de ladite cellule de telle sorte qu'elle se divise et que sa descendance se déplace sur d'autres motifs dudit réseau, au moins une partie desdites cellules en culture étant conforme à un motif d'adhésion cellulaire respectif du réseau au cours du temps, pendant plusieurs générations cellulaires.
PCT/EP2014/051873 2013-02-01 2014-01-31 Dispositif et procédé pour la régulation de la distribution spatiale de cellules et de leur descendance WO2014118311A1 (fr)

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EP3536402A1 (fr) 2018-03-09 2019-09-11 Ibidi Gmbh Chambre d'essai
US11674952B2 (en) 2016-02-24 2023-06-13 The Rockefeller University Embryonic cell-based therapeutic candidate screening systems, models for Huntington's Disease and uses thereof

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11674952B2 (en) 2016-02-24 2023-06-13 The Rockefeller University Embryonic cell-based therapeutic candidate screening systems, models for Huntington's Disease and uses thereof
EP3536402A1 (fr) 2018-03-09 2019-09-11 Ibidi Gmbh Chambre d'essai

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