Patent Application
20210179997
The present invention relates to a cell compression device for applying dynamic compression stress to cells. The invention also relates to a kit for cell compression comprising a cell compression device and a mounting tool. In addition, the invention relates to a method for applying dynamic compression stress to cells.
Cells are constantly exposed to wide range of mechanical stimuli (such as stretching and/or compression) in various organ movement, development and functions. These mechanical stimuli are essential to proper cell functions, such as for example migration, proliferation, differentiation, . . . [Tse, Cheng et al. “Mechanical compression drives cancer cells toward invasive phenotype” Proc Natl Acad Sci USA 109(3): 911-6; Engler, Sen et al. “Matrix Elasticity Directs Stem Cell Lineage Specification” Cell 126(4): 677-689 (2006); Vogel and Sheetz “Local force and geometry sensing regulate cell functions” Nat Rev Mol Cell Biol 7(4): 265-275 (2006); Wozniak and Chen “Mechanotransduction in development: a growing role for contractility” Nat Rev Mol Cell Biol 10(1): 34-43 (2009)]. As confirmed in literature [Tse, Cheng et al. “Mechanical compression drives cancer cells toward invasive phenotype” Proc Natl Acad Sci USA 109(3): 911-6; Wang, Tytell et al. “Mechanotransduction at a distance: mechanically coupling the extracellular matrix with the nucleus” Nat Rev Mol Cell Biol 10(1): 75-82. (2009); Shivashankar “Mechanosignaling to the cell nucleus and gene regulation” Annu Rev Biophys 40: 361-78 (2011)], cells sense such mechanical stimuli through a process called mechanotransduction. Abnormal mechanotransduction leads to several patho-genesis, such as cancer, asthma, or heart disease [Tse, Cheng et al. “Mechanical compression drives cancer cells toward invasive phenotype” Proc Natl Acad Sci USA 109(3): 911-6; Jaalouk and Lammerding “Mechanotransduction gone awry” Nat Rev Mol Cell Biol 10(1): 63-73 (2009); DuFort, Paszek et al. “Balancing forces: architectural control of mechanotransduction” Nat Rev Mol Cell Biol 12(5): 308-19 (2011)]. In Mechanobiology studies, it is vital to advance technologies for the application of physiologically mimicking mechanical force to cultured cells and tissue due to the complex in vivo biological system.
In particular, cell compression devices have been developed by applying a weighted piston on cells, these devices showing that mechanical compression drives cancer cells towards invasive phenotype [Tse, Cheng et al. “Mechanical compression drives cancer cells toward invasive phenotype” Proc Natl Acad Sci USA 109(3): 911-6]. The other compression technique made use of microfabricated PDMS spacers to define a vertical confinement to cells, which induces fast Amoeboid migration of slow mesenchymal cells [Liu, Le Berre et al. “Confinement and low adhesion induce fast amoeboid migration of slow mesenchymal cells” Cell 160(4): 659-72]. US 20110076758A1 describes another cell compression device using pressurized fluid to elastically deform a membrane and move pressurizing member downwardly to compress cells.
However, all the reported cell compression techniques available are limited in precision and dynamic control of force.
It is therefore an object of the present invention to provide a device that solves, at least in part, the above mentioned problems. In particular, to provide a cell compression device that is efficient, simple to use (compatible with high resolution live cell imaging) and able to perform high precision dynamic compression stress.
This object is achieved by the cell compression device, the kit and the method according to the independent claims. Further advantageous combinations and designs are given in the dependent claims therefrom.
The cell compression device according to the present invention comprises a main body having a central internal cavity with a symmetry longitudinal axis A and a lower opening aligned with said axis A. Also, the device comprises a deformable membrane having a piston attached thereto, wherein the piston comprises a main portion with a symmetry longitudinal axis B. In particular, the membrane can assumes different configurations between a relaxed configuration, wherein the membrane is not under tension, and a stretched configuration, wherein the membrane is under tension.
In addition, the compression device comprises clamping means to clamp the membrane to the main body at the lower opening to close the central internal cavity so that the closed central internal cavity forms a variable pressure chamber. In this clamped or assembled configuration, the attached piston is located outside the variable pressure chamber and the membrane is clamped to the main body in correspondence of clamping points.
Specifically, in an assembled configuration, that is when the deformable membrane is clamped to the main body, the membrane is in the relaxed configuration, the distance of two opposing clamping points being lower than the length of the membrane between said to opposing clamping points, and the symmetry longitudinal axis B of the main portion of the piston coincides with the symmetry longitudinal axis A of the central cavity.
Differently from prior art compression devices that mechanically move a pressurizing member to compress cells, the cell compression device according to the present application is based on the direct transfer of precise air pressure to piston and then cells through a relaxed or suspended thin membrane. In this way, it is possible to reach a compression to single cells at the resolution of nano newtons. Also, the cell compression device according to the present application can apply precise and computer program controlled dynamic compressive load to cells growing in any cell culture dish. The piston, that is in direct contact with cells, can be made from variety of materials and can apply a uniform or localized compressive load (indentation) to cells. In particular, the device can be adapted to any cell culture dish and can be compatible with any inverted microscope for high resolution real time live cell imaging.
The main body can have any suitable form to comprise a central internal cavity with a symmetry longitudinal axis. Preferably, the main body has a cylindrical or ring form and the central internal cavity can assume in turn a cylindrical or ring form aligned with the main body. In other words, the main body and the central internal cavity can be represented by two coaxial elements. Furthermore, the central internal cavity, has a lower opening that is in turn aligned with the symmetry longitudinal axis of the cavity and then with the main body. Preferably, the lower opening has a circular form.
The membrane is made of a deformable or resilient material such as silicone and can have any suitable form. Preferably, the membrane is a surface having a center of symmetry, such as a square-shaped surface, a circular-shaped surface, or the like. The piston can be represented by any element able to press on the cells and is attached to the membrane through appropriate connecting means, such as plasma treat bonding (for PDMS) or glue or the like. Alternatively, the piston can be integrated in the membrane so that piston and membrane can form a single structure. The piston is preferably attached at the center of symmetry of the surface of the membrane. In particular, the piston comprises a main portion having a symmetric shape and therefore having its own symmetry longitudinal axis. Preferably, the piston has the form of a cylinder or of a cube.
The deformability of the membrane allows the membrane to assume different configurations. According to the present application, it is intended that the membrane assumes a relaxed or suspended configuration when it is not under tension. In the present case, maintaining the membrane in a relaxed configuration after the clamping to the main body means that the distance between two clamping points on the main body is always lower than the length of the membrane between these two clamping points, wherein a clamping point is intended as the point where the membrane is in contact (and then clamped to) the main body. Accordingly, the membrane results in a suspended configuration, that is without tension, between these two points. With the expression “two opposing clamping points”, it is intended two points—where the membrane is clamped to the main body—located at the maximum distance one from the other.
In case for example the clamping points are distributed on a circular clamping region, the distance of two opposing clamping points represents the diameter of this circular clamping region.
The clamping means is any suitable means for fixing the membrane to the main body and specifically to a lower portion of the main body. Preferably, the clamping means comprises at least a projection element to be inserted in a recess element present in the main body. In a preferential embodiment, the clamping means has a ring shape having an upper edge acting as projecting element whereas the lower portion of the main body comprises a continuous annulus-shaped recess to receive the edge of the ring (clamping means). In a clamping configuration, the membrane is placed and fixed at clamping points between the recess element and the projecting element.
It is noted that clamping the membrane to the lower portion of the main body at the lower opening determines the closure of the central internal cavity. Preferably, the closure is airtight. This means that the closed central internal cavity can function as a chamber in which the internal pressure can be varied by means of appropriate devices, such as a pressure regulator. As the membrane is relaxed/suspended, the increase in applied air pressure is not used to tense and move the elastic membrane (as in prior art), but is directly counter balanced by the compressed cells underneath. In this dynamic balanced way, it is possible to precisely and dynamically transfer air pressure to compress cells.
It is furthermore noted that clamping the membrane to the lower portion of the main body at the lower opening determines an automatic alignment of the piston with the center of the variable pressure chamber. In fact, the symmetry longitudinal axis B of the main portion of the piston coincides with the symmetry longitudinal axis A of the central internal cavity.
In order to vary the internal pressure of the closed central internal cavity (or variable pressure chamber), the central internal cavity is connectable to pressure regulator means. In particular, the pressure regulator means can comprise a pump and/or one or more valves to finely regulate the air pressure inside the chamber. Preferably, the pressure regulator means can determine a pressure variation between 0 mbar to 25 mbar.
The pressure regulator means is connected to the cell compression device through suitable pressure connecting means, such as a flexible tube or a hose. Accordingly, the central internal cavity is provided with a dedicated opening couplable to said connecting means.
According to one embodiment of the present application, the main body further comprises connection means to directly interlock the device to a cell culture dish. In particular, said connection means are located in the lower portion of the main body. Preferably, the connection means comprises mechanical ribs that abut against the internal edges of a Petri dish when the cell compression device is interlocked to the dish. In this way, the cell compression device is firmly fixed relative to the dish and the piston is in the position to carry out a load to the cells distributed on the (internal) bottom of the dish.
In particular, the device can have a circular shape that can be scaled down a diameter around 3.5 cm. In this way, the device according to the present invention is reduceable to the size of a cell culture dish. In particular, the device can be configured to be interlocked with a 3.5 cm cell culture Petri dish. Of course, the device can also be configured to be interlocked with cell culture dishes or culture wells of different diameters.
In a preferable embodiment of the present invention, all the components of the main body are made of a plastic or other material suitable to be printed by means of a 3D-printer. This implies that the device can be mass produced easily by other industrial process and using different material.
The components of the device can also be made of different materials which are not necessary apt to be printed with a 3D printer but are suitable for the purpose of the present intention.
In a further embodiment of the present application, the piston can comprise a plurality of micropillars to apply localized compression (indentation) to the nucleus of the cells. The micropillars can be located at the bottom of the main portion of the piston and preferably have a diameter of 2 μm. Alternatively, the pillars can be circular with a diameter of 200 μm in order to apply a uniform compression stress to individual cells.
The piston can be made of a polymeric compound, such as polydimethylsiloxane (PDMS) in order to easily form microfabricated structures. Alternatively, the piston can be made of gel or other material suitable for the purpose of applying a compression stress to underlying cells.
According to another aspect of the present invention, the deformable membrane has a thickness comprised between 100 μm and 140 μm, preferably 120 μm. Such a thin membrane allows a finely controlled pressure transfer from the air inside the chamber to the piston and then to the cells.
In an embodiment of the present application, the central internal cavity further comprises an upper opening aligned with the lower opening and sealable with a transparent coverslip for allowing transmitted light to pass through the upper and the lower opening.
This is extremely useful when the cell compression device is used in combination with an inverted light microscope located at the bottom of the device, for example at the bottom of the cell culture dish interlocked with the main body of the device.
The kit for cell compression according to the present invention comprises a cell compression device according to any embodiment described above and a mounting tool insertable in a lower portion of the cell compression device for pre-stressing the deformable membrane before said membrane is clamped to the main body and left in the relaxed configuration. In particular, the main body comprises recess means and the mounting tool comprises projecting means to be inserted into said recess means.
The mounting tool is a critical element to first pre-stress the membrane and then relax the membrane in a suspended configuration when the mounting tool is removed.
The mounting tool can have a circular profile having an upper round edge apt to be inserted in an annulus recess in the lower portion of the main body of the cell compression device. It is appreciated that the mounting tool can further comprise an upper portion that is insertable, at least in part, into the central internal cavity of the main body when the mounting tool is inserted in the main body. Preferably, the shape of this upper portion is similar to the shape of the central internal cavity. In case the central internal cavity is cylindrical, the upper portion of the mounting tool can have a circular shape with a diameter lower that the diameter of the central internal cavity. Due to this particular configuration of the mounting tool, when the mounting tool is inserted into the lower portion of the main body, the interposed membrane, that follows the profile of the mounting tool, is partially pushed inside the central internal cavity of the main body. As a consequence, after clamping the membrane using the clamping means, the length of the membrane between two clamping points is lower than the distance between these two clamping points at the main body. This length difference corresponds to the length of the membrane inserted in the central internal cavity due to the push of the upper portion of the mounting tool. Therefore, when the mounting tool is removed, the membrane will relax in a suspended state.
In order to automatically align the piston with the center of the variable pressure chamber, the mounting tool further comprises a central recess to receive the piston attached to the membrane when said mounting tool is inserted in the lower portion of the cell compression device. In particular, the central recess is located in the upper portion of the mounting tool.
The method for applying dynamic compression stress to cells according to the present application comprises the steps of providing a main body having a central cavity with a symmetry longitudinal axis A and a lower opening aligned with said axis A and of providing a relaxed membrane having a piston attached thereto, the membrane assuming a relaxed configuration, wherein the membrane is not under tension and the piston comprising a main portion with a symmetry longitudinal axis B.
The method furthermore comprises the step of clamping the membrane to the main body at the lower opening to close the central cavity and forming a variable pressure chamber, the attached piston being located outside said variable pressure chamber and the membrane being clamped to the main body in correspondence of clamping points, wherein in an assembled configuration, when the deformable membrane is clamped to the main body, said membrane is in the relaxed configuration, the distance of two opposing clamping points being lower than the length of the membrane between said to opposing clamping points, and the symmetry longitudinal axis B of the main portion of the piston coincides with the symmetry longitudinal axis A of the central cavity.
Also, the method comprises locking a lower portion of the main body to a cell culture dish, varying the air pressure inside the variable pressure chamber and applying a dynamic compression stress to the cells in the cell culture dish based on the direct transfer of air pressure to the piston and then to the cells through the membrane in the relaxed configuration.
According to the present application, before the step of clamping the membrane to the main body, the method further comprises the step of inserting a mounting tool in a lower portion of the main body and pre-stressing the deformable membrane and wherein after the step of clamping the membrane to the main body, the method comprises the step of removing the mounting tool thereby leaving the membrane in the relaxed configuration.
In particular, the dynamic compression stress applied to single cells is at the precision of nano newtons.
Also, the lower movement of the piston in contact with cells is at the precision of microns.
It is noted that all the features characterizing the device and the kit described above also apply for the method according to the present application. Therefore, similar features are not hereby repeated.
In summary, the cell compression device, the kit and the corresponding method for applying dynamic compression stress to cells are characterized by following advantages.
The feature according to which a relaxed or/suspended membrane is used to directly transfer air pressure to cells is fundamental to achieve high precision (nano newtons) compression stress to single cells. Differently from prior art methods, the membrane is not elastically deformed/tensed and moved by liquid pressure, where the membrane tension would interfere with the amount and uniform distribution of compression force on the cells.
By using the cell compression device according to the present application, it is possible to apply ramp pressure as well as cyclic compression at nano newton precision to mimic physiological condition.
Furthermore, the piston of the cell compression device according to the present application is versatile. The piston can be made of PDMS, which provide its great potential in using microfabrication to create variety of micron sized geometry, such as micropillars for localized cell compression. The piston can also be made of gels to mimic physiological condition. The versatility of piston material as well as its potential for surface modification can provide novel applications. The piston surface can be coated with extra cellular matrix (ECM) protein to provide additional signaling to cells, such as 3D or sandwich cell culture. The ECM protein coated piston can also be used to adhere to the top of the cells followed by vertical cell stretching experiment.
In addition, the cell compression device according to the present application can be mounted on any cell culture dish so as to compress cells as well as tissue samples. It can be adapted to any microscope for high resolution imaging.
The device, the kit and the method according to the present invention are designed and configured to be utilized for different applications and under different conditions. In particular, the cell compression device is designed for high resolution, live cell as well as fixed cell immunofluorescence imaging for variety of cell experiments requiring mechanical stimuli. Specifically, to study cell morphology/cytoskeleton/nucleus transcription factor/gene expression under mechanical compression condition.
Preferred embodiments of a cell compression device, the kit and the method in accordance with the invention will be explained herein below in greater detail with reference to the accompanying drawings, in which:
The method 100 comprises the steps of providing 102 a main body 12, providing 104 a deformable membrane 18 having a piston 20 attached thereto, and clamping 106 the membrane 18 to the main body 12 at the lower opening 16 to close the central cavity 14 and forming a variable pressure chamber 26, wherein in the assembled configuration, the membrane 18 is in the relaxed configuration. The method 100 furthermore comprises the step of locking 108 a lower portion of the main body 12 to a cell culture dish D, varying 110 the air pressure inside the variable pressure chamber 26, and applying 112 a dynamic compression stress to the cells C in the cell culture dish D based on the direct transfer of air pressure to the piston 20 and then to the cells C through the membrane 18 in the relaxed configuration.
The method furthermore comprises the step of inserting 114 a mounting tool 36 in a lower portion of the main body 12 and pre-stressing the deformable membrane 18 before the step 106 of clamping the membrane 18 to the main body 12. Also comprises the step of removing 116 the mounting tool 36 from the main body 12 thereby leaving the membrane 18 in the relaxed configuration. It is noted that the membrane 18 can be pre-stressed in other different ways than using exactly the mounting tool 36 as described in the present application. Therefore, the method 100 can also be performed without the steps 114 and 116 assuming however that the membrane 18 is maintained in a relaxed configuration as intended in the present application after performing the clamping step 106.
Materials and Methods
The cell compression device 10 according to the present application is designed using Solidworks, and the components are 3D printed (Form 2, Formlabs) and assembled. The cell compression device components were printed using Dental SG resin (Formlabs) for its biocompatibility. All the components were printed and then washed with IPA for 20 minutes, followed by post processing in UV chamber as suggested by Formlabs. As shown in
Cells C were plated on the glass bottom of a Petri dish D and maintained in cell incubator. Before any experiment, the cell compression device 10 was capped and locked on the cell culture dish 10. Cell imaging was captured using 40× oil lens (NA=1.3) in Perkin Elmer spinning disk microscope.
RESULTS
It was found that performing three repeated cell compressions using the device of the invention induced actin polymerization at the perinuclear region.
Whilst features have been presented in combination of the above description, this is intended solely as an advantageous combination. The above description is not intended to show required combinations of features, rather it represents each of the aspects of the disclosure. Accordingly, it is not intended that any described specific combination of features is necessary for the functioning of the cell compression device and the corresponding method and/or the kit for cell compression.
| Number | Date | Country | Kind |
|---|---|---|---|
| 17200056.4 | Nov 2017 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/080304 | 11/6/2018 | WO | 00 |
