WO2010036913A2

WO2010036913A2 - Quantifying cell migration in wound healing assays

Info

Publication number: WO2010036913A2
Application number: PCT/US2009/058410
Authority: WO
Inventors: Daniel Irimia; Flavia Nery; Xandra Breakfield; Mehmet Toner
Original assignee: The General Hospital Corporation
Priority date: 2008-09-26
Filing date: 2009-09-25
Publication date: 2010-04-01
Also published as: WO2010036913A3

Abstract

Methods and systems that can be used for quantifying cell migration in wound healing assays can include: an optically transmissive substrate; a covering removably attached to the optically transmissive substrate such that the covering extends across a first portion of a face of the optically transmissive substrate and a second portion of the face of the optically transmissive substrate is free of the covering; and a layer of material having optical properties that are different than the optical properties of the optically transmissive substrate, the layer of material applied to the face of the optically transmissive substrate.

Description

Quantifying Cell Migration in Wound Healing Assays

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Serial No. 61/194,490, filed on September 26, 2008 which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

This invention relates to wound healing assays.

BACKGROUND

The wound-healing assay is a simple, inexpensive method to study directional cell migration in vitro. This method mimics cell migration during wound healing in vivo. The basic steps involve creating a "wound" in a cell monolayer, capturing the images at the beginning and at regular intervals during cell migration to close the wound, and comparing the images to quantify the migration rate of the cells. It is particularly suitable for studies on the effects of cell-matrix and cell-cell interactions on cell migration. (Rodriguez LG, Wu X, Guan JL. Wound-healing assay. Methods MoI Biol. 2005; 294:23-9.).

SUMMARY

The experimental systems and methods described below can provide precise quantitative measurement of cell migration in the classical "wound healing assays". The systems and methods can provide an independent and extremely precise (on the order of few microns) physical reference along the defined original position of the wound edge. The fixed reference systems allow for repeated microscopic measurements of cell movement during long term observations, even when the cells are moved back and forth between the incubator and the microscope stage. The system allows the investigator to induce a crisp edge on the "wound" in a cultured monolayer of cells in a reproducible fashion and a means to return to the exact point on the wound over repeat measurements, independent of the operator skills or previous experience. In one aspect, devices for quantifying cell migration in wound healing assays include: an optically transmissive substrate; a covering removably attached to the optically transmissive substrate such that the covering extends across a first portion of a face of the optically transmissive substrate and a second portion of the face of the optically transmissive substrate is free of the covering; and a layer of material having optical properties that are different than the optical properties of the optically transmissive substrate, the layer of material applied to the face of the optically transmissive substrate.

In one aspect, methods of preparing an experimental system for quantifying cell migration in wound healing assays include: removably attaching a polymeric membrane to a glass substrate such that a portion of the face of the optically transmissive substrate is free of the polymeric membrane; and applying a metallic layer to at least a portion of the face of the glass substrate.

In one aspect, methods of performing a wound healing assay include: growing a layer of cells extending across a substantially planar covering removably attached to a face of an optically transmissive substrate and a portion of the face of the optically transmissive substrate that is free of the covering; and removing the covering and cells growing on the covering from the optically transmissive substrate; and observing a migration of cells into a gap in the layer of cells created by removal of the substantially planar covering.

Embodiments can include one or more of the following features alone or in combination.

In some embodiments, the layer of material extends across the covering and the second portion of the optically transmissive substrate. In some cases, the optically transmissive substrate include glass. The covering can include a polydimethyl siloxane membrane reversibly bonded to the glass by hydrophobic interactions.

In some embodiments, the covering includes a polymeric membrane (e.g., a membrane containing or made of polydimethyl siloxane). The polymeric membrane can be between about 2 μm and about 5000 μm in thickness (e.g., between about 175 μm and 225 μm) In some embodiments, the layer of the material includes a material that can be applied as a thin film and is biocompatible. In some embodiments, the layer of the material includes a material selected from a group consisting of gold, platinum, and chrome. In some embodiments, the layer of material is disposed between the optically transmissive substrate and the covering. In some cases, the second portion of the face of the optically transmissive substrate is substantially free of the layer of material.

In some embodiments, removably attaching the polymeric membrane to the glass substrate includes reversibly bonding a polydimethyl siloxane membrane to the glass substrate by hydrophobic interactions.

In some embodiments, removably attaching the polymeric membrane to the glass substrate includes applying the polymeric layer to the glass substrate before applying the metallic layer extending over the polymeric layer and the portion of the face of the optically transmissive substrate that is free of the polymeric membrane. In some embodiments, removably attaching the polymeric membrane to the glass substrate includes attaching the polymeric membrane to the glass substrate by applying the polymeric layer to the metallic layer after the metallic layer is applied to the face of the glass substrate and subsequently removing portions of the metallic layer that are not covered by the polymeric layer. In some embodiments, removably attaching the polymeric membrane to the glass substrate includes attaching multiple membranes to the substrate.

In some embodiments, observing a migration of cells includes observing a migration of cells relative to a layer of material applied to the face of the optically transmissive substrate, the layer of material having optical properties that are different than the optical properties of the optically transmissive substrate. In some cases, the layer of the material includes a material selected from a group consisting of gold, platinum, and chrome and the substantially planar covering includes a polymeric membrane.

In some embodiments, removing the covering and cells growing on the covering from the optically transmissive substrate includes peeling the polymeric membrane from the optically transmissive substrate. In some cases, methods include peeling the polymeric membrane from the optically transmissive substrate in a single step. In some cases, methods include peeling the polymeric membrane from the optically transmissive substrate in multiple, successive steps.

These systems and methods provide several advantages. In some embodiments, the systems and methods described can provide very precise measurement of cell migration relative to "wound healing assays" in which a monolayer of cells is scratched with the tip of a pipette and the time it takes to the cells to "close the wound" is measured. Ln such assays, the wound itself provides the reference for cell migration, however the measurements cannot be very precise because the edge for the wound is usually irregular. In contrast, the systems and methods described herein provide a clear reference created at the precise location of the edge of the wound. This precise quantification of cell migration this enables can be used in evaluating the potential of different drugs to promote or inhibit cell migration.

In some embodiments, the systems and methods described can provide enable movement of a slide between a microscope stage and an incubator. The fixed reference system described allows for repeated microscopic measurements of cell movement during long term observations, even when the cells are moved back and forth between the incubator and the microscope stage. This can reduce the infrastructure requirements (temperature and humidity control on the stage), associated with approaches that track the advancement of only one edge of the "wound", which usually allow for more precise measurements but also require holding the substrate in fixed position on the microscope stage. The movement of a slide between a microscope stage and an incubation chamber can also provide increased throughput of observations.

These systems and methods can also substantially avoid leaving damaged cells on the substrate surface, where they interfere with the migration of other cells as can occur when performing automated assays of this sort using electric cell substrate impedance sensing ("ECIS").

Potential applications of the systems and methods described include drug screening (e.g., for promoters of cell migration in wound healing and/or tissue regeneration, for inhibitors of cell migration in cancer). These systems and methods can also be used for basic research (e.g., to assess signaling pathways and proteins involved in molecular mechanisms of cell migration using fluorescent reporter proteins to tag critical molecules, and expressing high levels or mutant forms of specific proteins or using siRNA or cells from knock-out animals cells to reduce levels).

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. IA- IE are a series of schematic views illustrating the formation and use of an experimental system.

FIG. 2 is a schematic view of an experimental system. FIG. 3A includes a temporal series of micrographs showing cell migration during the use of an embodiment of the experimental system shown in FIGS. 1 A-IE. FIG. 3B illustrates the results of the experiment shown in FIG. 3A. FIGS. 4A-4D illustrate a micropatterned mold for forming a membrane of an experimental system. Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

The experimental systems and methods illustrated in FIG 1 can provide precise quantitative measurement of cell migration in the classical "wound healing assays". The systems and methods can provide an independent and extremely precise (on the order of few microns) physical reference along the defined original position of the wound edge. The fixed reference systems allow for repeated microscopic measurements of cell movement during long term observations, even when the cells are moved back and forth between the incubator and the microscope stage. The system allows the investigator to induce a crisp edge on the "wound" in a cultured monolayer of cells in a reproducible fashion and a means to return to the exact point on the wound over repeat measurements, independent of the operator skills or previous experience.

The system 100 of FIG. IA includes a substrate 110 and a covering 112 extending over part of one face of the substrate 110. The substrate can be optically transmissive such that standard and fluorescence microscopy can be used to image cells through the substrate. The substrate 110 should also be able to support the growth of attached cells (e.g., be biocompatible) and be strong enough to provide structural integrity. For example, in some embodiments, the substrate can be a glass coverslip (Fisher Scientific, Pittsburg, PA). Other materials including, for example, optically transmissive plastics also can be used as the substrate 110.

The covering 112 is removably attached to the substrate 110. The covering 112 can be positioned such that the covering 112 extends across a first portion 114 of a face 116 of the optically transmissive substrate 110 and a second portion 118 of the face 116 of the optically transmissive substrate 110 is free of the covering 112. The covering 112 should be readily attachable to the substrate 110 such that the covering tends to remain attached to the substrate in the absence of the application of a removing force. However, the covering 112 also be readily removable from the substrate such that the removing force can readily be manually applied by a technician gripping the edge of the covering 112 and peeling the covering 112 away from the substrate 110. The covering 112 should also be sturdy enough to remain intact while being removed from the substrate 110.

In some embodiments, the covering 112 is a thin membrane that is removably bonded to a glass coverslip (substrate 110). For example, the covering 112 can be a thin polydimethyl siloxane membrane (PDMS, Dow Coming, Midland, MI) between about 2 and about 5000 μm in thickness (e.g., between about 175 μm and 225 μm). Other polymeric membranes including, for example, elastomers and plastics can also be used in some embodiments. The membrane can be complete or have holes extending through the membrane.

In some embodiments, multiple membranes are applied to the substrate. These membranes can be spaced apart from each other or can be overlapping. In some embodiments, the covering 112 is a membrane that has a linear edge with micropattemed marks to identify positions along the edge. For example, the membrane can be formed with indexing indentations and protrusions to help identify the location along the edge during microscopy. The edges of the membrane can be cut vertically or at an angle (positive or negative), or built in several steps. Such micropattemed membranes can be manufactured by casting small amounts of PDMS between a micropattemed mold and a flat Mylar film. The mold can be produced using standard micro fabrication techniques including photopatterning a 200μm layer of expoy-photoresist (SU8, MicroChem, Newton, MA) on a silicon wafer through a photolithography mask (Fineline Imaging, Colorado Springs, CO). In embodiments with a glass coverslip substrate 110 and a PDMS membrane covering 112, the PDMS membrane can be reversibly bonded on the glass surface by hydrophobic interaction between the membrane and the glass surface. Other attachment mechanisms including, for example, adhesives that are biocompatible and/or dissolvable (e.g., a sugar layer which dissolves during cell culturing) can be used to removably attach the covering 112 to the substrate 110.

A layer of material 120, having optical properties that are different than the optical properties of the optically transmissive substrate, can be applied to the face of the optically transmissive substrate. As shown in FIG. IB, the layer of material can extend across the covering 112 and the second portion 118 of the face 116 of the optically transmissive substrate 110. The layer of the material 120 can be a material that can be applied as a thin film and is biocompatible (e.g., gold, platinum, and chrome). Other materials which provide that the desired optical properties and upon which cells can be grown can also be used. For example, in the experiment described in more detail below, a thin (50A) layer of chrome was deposited on a glass substrate and polymeric membrane using a sputtering machine (Kurt J Lesker, Pittsburgh, PA). This provides the layer of material attached to the substrate in a pattern that complements the shape of the covering (e.g., the covering acts a mask blocking the layer of material from attaching to covered portions of the substrate).

Alternatively, in a system 200 as shown in FIG. 2, the optically transmissive substrate 110 can be coated with the layer of material 120 before the covering 112 is applied on top of the layer of material 120. Portions of the layer of material 120 not between the covering 112 and the substrate 1 10 can then be removed. For example, portions of a chrome layer on a glass slide can be covered by a PDMS membrane. Portions of the chrome layer not covered by the PDMS membrane can then be removed by, for example, chemical etching. This approach can be used to provide a chrome layer in the same shape as the PDMS layer. The resulting layer of material 120 (e.g., chrome) is disposed between the optically transmissive substrate 110 (e.g., the glass slide) and the covering 112 (e.g., the PDMS membrane) and the second portion 118 of the face 116 of the optically transmissive substrate 110 is substantially free of the layer of material. After cell culture and PDMS membrane removal, the monolayer of cells will be on transparent glass, and the migrating cells will move on the semitransparent layer.

In use, systems can be placed in a device or environment for culturing cells. As shown in Fig. 1C, the system 100 can be placed, for example, in a Petri dish 122 and covered with a suspension of cells 124. The systems is left in place while a layer of cells 124 grows extending across the substantially planar covering 112 removably attached to a face 116 of an optically transmissive substrate 110 and a portion 118 of the face 116 of the optically transmissive substrate 110 that is free of the covering 112 but includes the layer of material 120. After cells 124 adhere, they will form a monolayer 124 on the glass/chrome 110/120 and the thin PDMS membrane 112 as shown in Fig. ID. The wound assay starts by removing the PDMS membrane 112, which will also remove the cell monolayer 124 attached to the PDMS membrane 112 while leaving the cell monolayer 124 on the chrome layer 120 as shown in Fig. IE. The edge of the wound is the same as the edge of the chrome layer 120.

The user can observe a migration of cells into a gap 128 in the layer of cells created by removal of the substantially planar covering (e.g., the PDMS membrane). In system 100, the cells 120 are visible through the semi-transparent chrome layer 120 and the migrating cells 120 can be imaged through the clear glass using standard and fluorescence microscopy. The reverse is true in the system 200 in which the cells 124 on the glass substrate 110 are left in place and the cells above the PDMS membrane overlying the chrome layer 120 are removed.

The membrane(s) 112 could be removed all at once or in successive steps. A control experiment with cells cultured on a chrome patterned slide showed that the chrome did not alter the adhesion of cells to a glass slide.

The systems and methods described herein provide a clear reference created at the precise location of the edge of the wound. This precise quantification of cell migration this enables can be used in evaluating the potential of different drugs to promote or inhibit cell migration.

These systems and methods were employed in a study (discussed in more detail in "TorsinA binds the KASH domain of nesprins and participates in linkage between nuclear envelope and cytoskeleton", Nery et al., J. Cell Sd., 121, 3476-3486 (2008)) assessing torsinA +/+ and torsinA -/- MEFs effects on cell migration. Glass coverslips (Fisher Scientific, Pittsburg, PA) were partially covered by a thin

(2-300μm) PDMS membrane (Dow Corning, Midland, MI). The membrane had one edge linear with micropatterned marks to identify positions along the edge. The micropatterned membranes were manufactured by casting small amounts of PDMS between a micropatterned mold (see FIGS. 4A-4D) and a flat Mylar film. The mold was produced using standard microfabrication techniques and involve photopatterning a

200μm layer of expoy-photoresist (SU8, MicroChem, Newton, MA) on a silicon wafer through a photolithography mask (Fineline Imaging, Colorado Springs, CO). After the PDMS membrane was reversibly bonded on the glass surface by hydrophobic interaction, a thin (50A) layer of chrome was deposited on the glass using a sputtering machine (Kurt J Lesker, Pittsburgh, PA).

Cells were plated on the coverslips and allowed to form a confluent monolayer on the surface of the coverslips. A wound was initiated by removal of the PDMS membrane, made visible microscopically by the absence of chrome in the wound region. The cells were visible through the semi-transparent chrome layer and the migrating cells were imaged using standard and fluorescence microscopy. Migration of cells into the cleared space was monitored at different time points. Photographs (see Fig 3A) of fibroblast monolayers were taken under phase contrast microscopy at 0, 4, 8 and 26 h after "wounding" (representative region shown - magnification 1 OX (magnification bar 150 mm). The migration of the leading edge of cells was measured in mm from fixed points on the wound edge with the results presented in Fig. 3B. Values represent mean + S. D. of measurements in 6 regions per time point for each genotype from 7 experiments. Differences between torsinA +/+ and -/- cells were at all time points significant (p< O.OOOlat all time points).

A significant delay in the migration of torsinA-/- cells was noted, with only 23±7% of the distance covered by torsinA+/+ cells at 4 hours post- wounding, 31±4% at 8 hours and 47±3% at 26 hours (n=7; PO.0001 at all time points, two-way ANOVA)

The semi-transparent coating of the slide and PMDS allowed cells to attach on both surfaces (i.e., the glass and PDMS membrane). After removing the PDMS membrane, the edge of the semitransparent layer was used as reference for the original position of the wound edge. Both the cells in the original monolayer and the migrating cells could be observed using standard/fluorescence microscopy techniques. The additional micropatterns on the PDMS edge were helpful in establishing a reference system along the wound edge for repeated observations in the same location over extended periods of time. A PMDS membrane of only 100-200um thick was successfully removed for wound initiation. The edge of membrane was formed at an 80 degree angle to improve cell adhesion and formation of a continuous monolayer on the slide and PDMS.

Other Embodiments

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A device for quantifying cell migration in wound healing assays, the device comprising: an optically transmissive substrate; a covering removably attached to the optically transmissive substrate such that the covering extends across a first portion of a face of the optically transmissive substrate and a second portion of the face of the optically transmissive substrate is free of the covering; and a layer of material having optical properties that are different than the optical properties of the optically transmissive substrate, the layer of material applied to the face of the optically transmissive substrate.

2. The device of claim 1, wherein the layer of material extends across the covering and the second portion of the optically transmissive substrate.

3. The device of claim 2, wherein the optically transmissive substrate comprises glass.

4. The device of claim 3, wherein the covering comprises a polydimethyl siloxane membrane reversibly bonded to the glass by hydrophobic interactions.

5. The device of claim 2, wherein the covering comprises a polymeric membrane.

6. The device of claim 4, wherein the polymeric membrane comprises polydimethyl siloxane.

7. The device of claim 4, wherein the polymeric membrane is between about 2 μm and about 5000 μm in thickness.

8. The device of claim 7, wherein the polymeric membrane is between about 175 μm and 225 μm.

9. The device of claim 1 , wherein the layer of the material comprises a material that can be applied as a thin film and is biocompatible.

10. The device of claim 1 , wherein the layer of the material comprises a material selected from a group consisting of gold, platinum, and chrome.

1 1. The device of claim 1 , wherein the layer of material is disposed between the optically transmissive substrate and the covering.

12. The device of claim 11, wherein the second portion of the face of the optically transmissive substrate is substantially free of the layer of material.

13. The device of claim 1 , wherein the covering has an edge with micropatterned marks to identify positions along the edge.

14. A method of preparing an experimental system for quantifying cell migration in wound healing assays, the method comprising removably attaching a polymeric membrane to a glass substrate such that a portion of the face of the optically transmissive substrate is free of the polymeric membrane; and applying a metallic layer to at least a portion of the face of the glass substrate.

15. The method of claim 14, wherein removably attaching the polymeric membrane to the glass substrate comprises reversibly bonding a polydimethyl siloxane membrane to the glass substrate by hydrophobic interactions.

16. The method of claim 14, wherein removably attaching the polymeric membrane to the glass substrate comprises applying the polymeric layer to the glass substrate before applying the metallic layer extending over the polymeric layer and the portion of the face of the optically transmissive substrate that is free of the polymeric membrane.

17. The method of claim 14, wherein removably attaching the polymeric membrane to the glass substrate comprises attaching the polymeric membrane to the glass substrate by applying the polymeric layer to the metallic layer after the metallic layer is applied to the face of the glass substrate and subsequently removing portions of the metallic layer that are not covered by the polymeric layer.

18. The method of claim 14, wherein removably attaching the polymeric membrane to the glass substrate comprises attaching multiple membranes to the substrate.

19. A method of performing a wound healing assay, the method comprising: growing a layer of cells extending across a substantially planar covering removably attached to a face of an optically transmissive substrate and a portion of the face of the optically transmissive substrate that is free of the covering; and removing the covering and cells growing on the covering from the optically transmissive substrate; and observing a migration of cells into a gap in the layer of cells created by removal of the substantially planar covering.

20. The method of claim 19, wherein observing a migration of cells comprises observing a migration of cells relative to a layer of material applied to the face of the optically transmissive substrate, the layer of material having optical properties that are different than the optical properties of the optically transmissive substrate.

21. The method of claim 20, wherein the layer of the material comprises a material selected from a group consisting of gold, platinum, and chrome and the substantially planar covering comprises a polymeric membrane.

22. The method of claim 21 , wherein removing the covering and cells growing on the covering from the optically transmissive substrate comprises peeling the polymeric membrane from the optically transmissive substrate.

23. The method of claim 22, comprising peeling the polymeric membrane from the optically transmissive substrate in a single step.

24. The method of claim 22, comprising peeling the polymeric membrane from the optically transmissive substrate in multiple, successive steps.

25. The method of claim 20, wherein, after removing of the covering, the layer of material has an edge with micropatterned marks to identify positions along the

80 edge.

26. The method of claim 25, wherein observing the migration of cells into the gap in the layer of cells created by removal of the substantially planar covering comprises repeatedly observing the location of cells on an imaging device and transferring the optically transmissive substrate from an imaging device to an incubator between at least

85 some observations.

27. The method of claim 25, wherein observing the migration of cells into the gap in the layer of cells created by removal of the substantially planar covering comprises repeatedly observing the location of cells on an imaging device and using the micropatterned marks to identify relative positions of the cells.