WO2018234163A1 - Method for determining cell migration and invasion - Google Patents

Method for determining cell migration and invasion Download PDF

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Publication number
WO2018234163A1
WO2018234163A1 PCT/EP2018/065876 EP2018065876W WO2018234163A1 WO 2018234163 A1 WO2018234163 A1 WO 2018234163A1 EP 2018065876 W EP2018065876 W EP 2018065876W WO 2018234163 A1 WO2018234163 A1 WO 2018234163A1
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Prior art keywords
cells
microfluidic device
invasion
channel
combination
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PCT/EP2018/065876
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French (fr)
Inventor
Devrim Pesen Okvur
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İni̇ti̇o Biyomedikal Mühendislik Danişmanlik Sanayi Ve Ticaret Limited Şirketi
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Publication of WO2018234163A1 publication Critical patent/WO2018234163A1/en

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    • 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
    • G01N33/5029—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 cell motility
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • 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
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability

Definitions

  • the invention relates to a method for determining cell migration and invasion.
  • Microfluidic technology provides precise spatial and temporal control, high-throughput analysis, low fabrication costs ve portability. Used material and waste volumes can be as low as picoliters. Using small volumes of unknown or toxic materials provides safe experimental study. Moreover, microfluidic technology can provide means to mimic physiological microenvironments. This feature can help us more realistically study cells in both health and disease states and improve drug testing approaches. It can also help reduce animal testing.
  • Microfluidic device-based devices and methods are present for chemotactic tropism of cells.
  • a microfluidic-based method which is able to imitate the Boyden Chamber method, is more useful than that, is able to better mimic the physiological environment and ensure obtaining more data, is not present.
  • the purpose of the invention is to examine cell migration and invasion in time at intervals or continuously by using a microfluidic device.
  • Matrix (3) is loaded into the central channel (2).
  • Agent (5) is loaded to the test channel (4).
  • Cells (7) are ensured to settle down at the interface between the central channel (2) and the control channel (6) by keeping the microfluidic device (1) upright.
  • the invention is a method for determining cell migration and invasion.
  • This method comprises the stages of adding a matrix (3) to the central channel (2) of the microfluidic device (1), adding an agent (5) to the test channel (4) of the microfluidic device (1), adding cells (7) to the control channel (6) of the microfluidic device (1), by positioning the microfluidic device (1) such that the control channel (6) remains at the top, under suitable culture conditions, ensuring cells (7) to settle down at the interface of the test channel (4) and the central channel (2) and culture of them, determining migration and invasion of cells (7) in a matrix channel.
  • Matrix (3) loaded to central channel can consist of matrigel, collagen, laminin, agarose, polyacrylamide, biocompatible matrices, puramatrix, alginate, fibrin or a combination thereof. Necessary environment is provided after the loading for polymerization in accordance with the type of the matrix (3), if necessary.
  • matrigel is loaded to the central channel (2) in a cold state, then the matrigel is ensured to polymerize by maintaining the microfluidic device (1) at 37 C.
  • puramatrix polymerizes rapidly in salt solution, therefore its polymerization can be completed at the moment it is loaded to the central channel (2) when added to a salt solution.
  • Agent (5) can be cells, cell-laden or cell-free matrix, polymer, culture medium, physiological buffer solution, cell conditioned culture medium, one or more biological or chemical molecules or a combination thereof.
  • Cells in the control channel (7) and cells used as agent (5) can be the same or different.
  • Cells (7) can be cell lines, primary culture cells, biopsy cells, stem cells or a combination thereof.
  • cancerous or normal cell lines can be used, tumor biopsies obtained from patients can be used.
  • the microfluidic device (1) can be continued to be positioned upright or can be maintained horizontally under suitable culture conditions.
  • matrigel is loaded to the central channel (2).
  • an agent (5) estimated to increase cell migration and invasion for example a growth agent
  • cells (7) for example cancer cells
  • cancerous cells are ensured to settle down at the interface of the central channel (2) and the control channel (6) by positioning the microfluidic device (1) upright such that the control channel is at the top, under culture conditions.
  • Migration and invasion of cancer cells in the central channel (2) can be examined in time continuously or at intervals. If the growth agent is efficient as anticipated, cells will show more migration and invasion compared to the condition without the growth agent.
  • Cell migration and invasion can be examined by microscopy or spectroscopy.
  • Cells (7) can be altered genetically as to give fluorescent signal or can be labeled with chemical dyes. Cells can also be examined by standard light microscope techniques in the event that they do not give fluorescent signal.
  • Cell migration and invasion can be determined by counting cells (7) that have entered the central channel, by measuring the distance covered by the cells (7) in the central channel (2) in one or more time points, by measuring shapes, speed, persistence of the cells (7) in the central channel (2).
  • a matrix (3) showing an increase or decrease in fluorescent signal when degraded by cells can be loaded to the central channel (2).
  • the matrix (3) degradation by cells (7) can be examined.
  • Cells (7) entering the central channel (2) can be extracted from the microfluidic device (1) and used for out-of-device assays such as polymerase chain reaction, ELISA. Cells (7) entering the central channel (2) can also be fixed and sectioned, separately or within the matrix (5) they are in, for out-of-device assays such as electron microscopy, histological staining.
  • Microfluidic device (1) used for this method can comprise glass, polydimethylsiloxane (PDMS), polystyrene (PS), polymethylmethacrylate (PMMA), cyclic olefin copolymer (COC) or a combination thereof. Molds to be used for the manufacturing of the microfluidic device (1) can be produced with a method comprising UV lithography, three-dimensional printing, metal casting or a combination thereof.
  • PDMS polydimethylsiloxane
  • PS polystyrene
  • PMMA polymethylmethacrylate
  • COC cyclic olefin copolymer
  • Central channel (2) in the microfluidic device (1) used for the method for determining cell migration and invasion is between the test channel (4) and the control channel (6).
  • Channels (2, 4, 6) are separated from one another by more than one column (8).
  • cells (7) and for example other agents (5) being able to change places with diffusion can switch between channels (2, 4, 6).
  • Channels (2, 4, 6) can be at height and width between 50 micrometers and 1 centimeter. Channels (2, 4, 6) can be at length between 500 micrometers and 20 centimeters.
  • Cross sections of the columns (8) can be in different shapes like triangle, trapezoid, circle, ellipse. Longest horizontal axis of columns (8) can be at length between 50 micrometers and 1 centimeter.
  • the invention can be used in the pharmaceutical industry for determining agents that can affect cell migration and invasion.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Hematology (AREA)
  • General Engineering & Computer Science (AREA)
  • Urology & Nephrology (AREA)
  • Analytical Chemistry (AREA)
  • Molecular Biology (AREA)
  • Sustainable Development (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Dispersion Chemistry (AREA)
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  • General Physics & Mathematics (AREA)
  • Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Toxicology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

The invention relates to a method for determining cell migration and invasion continuously or at intervals using a microfluidic device (1).

Description

DESCRIPTION METHOD FOR DETERMINING CELL MIGRATION AND INVASION
Technical field
The invention relates to a method for determining cell migration and invasion. Prior art
Microfluidic technology provides precise spatial and temporal control, high-throughput analysis, low fabrication costs ve portability. Used material and waste volumes can be as low as picoliters. Using small volumes of unknown or toxic materials provides safe experimental study. Moreover, microfluidic technology can provide means to mimic physiological microenvironments. This feature can help us more realistically study cells in both health and disease states and improve drug testing approaches. It can also help reduce animal testing.
Cell migration and invasion is most commonly analyzed with the method named Boyden Chamber. However, this method is an end-point method. It does not ensure continuous investigation of cell migration and invasion. There are membranes with certain hole sizes in the mechanism used. These hole sizes are needed to be chosen according to different cells. What is more, using a membrane negatively affects providing a physiological environment.
Microfluidic device-based devices and methods are present for chemotactic tropism of cells. However, a microfluidic-based method which is able to imitate the Boyden Chamber method, is more useful than that, is able to better mimic the physiological environment and ensure obtaining more data, is not present.
Technical problems to be solved by the invention
The purpose of the invention is to examine cell migration and invasion in time at intervals or continuously by using a microfluidic device.
Method steps having the specified features are shown in the drawing below.
Drawings are not to scale, unless necessary. Description of drawings
Figure 1: Method steps.
a) Top view of microfluidic device (1).
b) Matrix (3) is loaded into the central channel (2).
c) Agent (5) is loaded to the test channel (4).
d) Cells (7) are loaded to the control channel (6).
e) Cells (7) are ensured to settle down at the interface between the central channel (2) and the control channel (6) by keeping the microfluidic device (1) upright.
f) Cell migration and invasion is determined in the setup maintained in suitable culture conditions.
Description of the references in drawings
1. Microfluidic device.
2. Central channel
3. Matrix
4. Test channel
5. Agent
6. Control channel
7. Cell
8. Column
9. Hole
Description of the invention
The invention is a method for determining cell migration and invasion. This method comprises the stages of adding a matrix (3) to the central channel (2) of the microfluidic device (1), adding an agent (5) to the test channel (4) of the microfluidic device (1), adding cells (7) to the control channel (6) of the microfluidic device (1), by positioning the microfluidic device (1) such that the control channel (6) remains at the top, under suitable culture conditions, ensuring cells (7) to settle down at the interface of the test channel (4) and the central channel (2) and culture of them, determining migration and invasion of cells (7) in a matrix channel.
Matrix (3) loaded to central channel can consist of matrigel, collagen, laminin, agarose, polyacrylamide, biocompatible matrices, puramatrix, alginate, fibrin or a combination thereof. Necessary environment is provided after the loading for polymerization in accordance with the type of the matrix (3), if necessary.
For example, matrigel is loaded to the central channel (2) in a cold state, then the matrigel is ensured to polymerize by maintaining the microfluidic device (1) at 37 C. However, puramatrix polymerizes rapidly in salt solution, therefore its polymerization can be completed at the moment it is loaded to the central channel (2) when added to a salt solution.
Agent (5) can be cells, cell-laden or cell-free matrix, polymer, culture medium, physiological buffer solution, cell conditioned culture medium, one or more biological or chemical molecules or a combination thereof. Cells in the control channel (7) and cells used as agent (5) can be the same or different.
Cells (7) can be cell lines, primary culture cells, biopsy cells, stem cells or a combination thereof. For example, cancerous or normal cell lines can be used, tumor biopsies obtained from patients can be used. After the cells (7) settled down at the interface of the central channel (2) and the control channel (6), the microfluidic device (1) can be continued to be positioned upright or can be maintained horizontally under suitable culture conditions.
In an exemplary practice, matrigel is loaded to the central channel (2). After the matrigel polymerizes, an agent (5) estimated to increase cell migration and invasion, for example a growth agent, is added to the test channel (4). Then cells (7), for example cancer cells, are loaded to the control channel (6). Cancerous cells are ensured to settle down at the interface of the central channel (2) and the control channel (6) by positioning the microfluidic device (1) upright such that the control channel is at the top, under culture conditions. Migration and invasion of cancer cells in the central channel (2) can be examined in time continuously or at intervals. If the growth agent is efficient as anticipated, cells will show more migration and invasion compared to the condition without the growth agent.
Cell migration and invasion can be examined by microscopy or spectroscopy. Cells (7) can be altered genetically as to give fluorescent signal or can be labeled with chemical dyes. Cells can also be examined by standard light microscope techniques in the event that they do not give fluorescent signal.
Cell migration and invasion can be determined by counting cells (7) that have entered the central channel, by measuring the distance covered by the cells (7) in the central channel (2) in one or more time points, by measuring shapes, speed, persistence of the cells (7) in the central channel (2).
A matrix (3) showing an increase or decrease in fluorescent signal when degraded by cells can be loaded to the central channel (2). Thus, the matrix (3) degradation by cells (7) can be examined.
Cells (7) entering the central channel (2) can be extracted from the microfluidic device (1) and used for out-of-device assays such as polymerase chain reaction, ELISA. Cells (7) entering the central channel (2) can also be fixed and sectioned, separately or within the matrix (5) they are in, for out-of-device assays such as electron microscopy, histological staining.
Microfluidic device (1) used for this method can comprise glass, polydimethylsiloxane (PDMS), polystyrene (PS), polymethylmethacrylate (PMMA), cyclic olefin copolymer (COC) or a combination thereof. Molds to be used for the manufacturing of the microfluidic device (1) can be produced with a method comprising UV lithography, three-dimensional printing, metal casting or a combination thereof.
Central channel (2) in the microfluidic device (1) used for the method for determining cell migration and invasion is between the test channel (4) and the control channel (6). Channels (2, 4, 6) are separated from one another by more than one column (8). Thus cells (7) and for example other agents (5) being able to change places with diffusion can switch between channels (2, 4, 6).
Channels (2, 4, 6) can be at height and width between 50 micrometers and 1 centimeter. Channels (2, 4, 6) can be at length between 500 micrometers and 20 centimeters. Cross sections of the columns (8) can be in different shapes like triangle, trapezoid, circle, ellipse. Longest horizontal axis of columns (8) can be at length between 50 micrometers and 1 centimeter.
There is at least one hole (9) in each of the channels (2, 4, 6) such that loading can be realized. Industrial applicability of the invention
The invention can be used in the pharmaceutical industry for determining agents that can affect cell migration and invasion.

Claims

CLAIMS METHOD FOR DETERMINING CELL MIGRATION AND INVASION
1. A method for determining cell migration and invasion, comprising;
i. Loading matrix (3) to the central channel (2) of a microfluidic device (1), ii. Loading an agent (5) to the test channel (4) of the same microfluidic device
Figure imgf000008_0001
2. The method of claim 1, wherein the matrix (3) comprises matrigel, collagen, laminin, agarose, fibrin, puramatrix, alginate, biocompatible matrices or a combination thereof.
3. The method of claim 1, wherein the agent (5) comprises cells, cell-laden or cell-free matrices, polymer, culture medium, physiological buffer solution, cell conditioned culture medium, one or more biological or chemical molecules or a combination thereof.
4. The method of claim 1, wherein the cells (7) comprises cell lines, primary culture cells, biopsy cells, stem cells or a combination thereof.
5. The method of claim 1, wherein cell migration and invasion in the central channel (2) are examined by microscopy or spectroscopy.
6. The microfluidic device (1) of claim 1, wherein it is made of glass, polydimethylsiloxane (PDMS), polystyrene (PS), polymethylmethacrylate (PMMA), cyclic olefin copolymer (COC) or a combination thereof.
7. The microfluidic device (1) of claim 1, wherein the central channel (2) is between the test channel (4) and the control channel (6).
8. The microfluidic device (1) of claim 1, wherein the interfaces of the central channel (2) with the test channel (4) and with the control channel (6) comprise more than one column
(8).
9. The channels (2, 4, 6) of claim 1, wherein each one of them has at least one hole (9) for loadings.
10. The channels (2, 4, 6) of claim 1, wherein they have a height between 50 micrometers and 5 centimeters.
11. The channels (2, 4, 6) of claim 1, wherein they have a width between 50 micrometers and 5 centimeters.
12. The channels (2, 4, 6) of claim 1, wherein they have a length between 500 micrometers and 20 centimeters.
13. The columns (8) of claim 8, wherein their longest horizontal axis has a length between 50 micrometers and 1 centimeter.
14. The microfluidic device (1) of claim 1, wherein the mold used for its manufacturing is realized by UV lithography, three dimensional printing, metal casting or a combination thereof.
PCT/EP2018/065876 2017-06-16 2018-06-14 Method for determining cell migration and invasion WO2018234163A1 (en)

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TR201708905 2017-06-16
TR2017/08905 2017-06-16

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009126524A2 (en) * 2008-04-08 2009-10-15 Massachusetts Institute Of Technology Three-dimensional microfluidic platforms and methods of use thereof
WO2013151616A1 (en) * 2012-04-01 2013-10-10 Emd Millipore Corporation Cell culture and gradient migration assay methods and devices

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009126524A2 (en) * 2008-04-08 2009-10-15 Massachusetts Institute Of Technology Three-dimensional microfluidic platforms and methods of use thereof
WO2013151616A1 (en) * 2012-04-01 2013-10-10 Emd Millipore Corporation Cell culture and gradient migration assay methods and devices

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CHEN ZHANG ET AL: "A Sensitive Chemotaxis Assay Using a Novel Microfluidic Device", BIOMED RESEARCH INTERNATIONAL, vol. 2013, 1 January 2013 (2013-01-01), pages 1 - 8, XP055520764, ISSN: 2314-6133, DOI: 10.1155/2013/373569 *
DAYOUNG YOON ET AL: "Study on chemotaxis and chemokinesis of bone marrow-derived mesenchymal stem cells in hydrogel-based 3D microfluidic devices", BIOMATERIALS RESEARCH, vol. 20, no. 1, 2 August 2016 (2016-08-02), XP055520778, DOI: 10.1186/s40824-016-0070-6 *
HSIEH-FU TSAI ET AL: "Tumour-on-a-chip: microfluidic models of tumour morphology, growth and microenvironment", JOURNAL OF THE ROYAL SOCIETY. INTERFACE, vol. 14, no. 131, 1 June 2017 (2017-06-01), GB, pages 20170137, XP055520849, ISSN: 1742-5689, DOI: 10.1098/rsif.2017.0137 *
JING LI ET AL: "Microfluidic device for studying cell migration in single or co-existing chemical gradients and electric fields", BIOMICROFLUIDICS, vol. 6, no. 2, 1 June 2012 (2012-06-01), pages 024121, XP055520668, DOI: 10.1063/1.4718721 *
KE YANG ET AL: "A dual-docking microfluidic cell migration assay (D 2 -Chip) for testing neutrophil chemotaxis and the memory effect", INTEGRATIVE BIOLOGY, vol. 9, no. 4, 18 April 2017 (2017-04-18), Cambridge, pages 303 - 312, XP055520727, ISSN: 1757-9694, DOI: 10.1039/C7IB00037E *

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