WO2009043014A2

WO2009043014A2 - Devices for studying cancer invasion and migration

Info

Publication number: WO2009043014A2
Application number: PCT/US2008/078140
Authority: WO
Inventors: Wei Li; Biaoyang Lin; Hai Wang
Original assignee: University Of Washington
Priority date: 2007-09-27
Filing date: 2008-09-29
Publication date: 2009-04-02
Also published as: WO2009043014A3

Abstract

A medical testing device is formed from a polymeric chip. The device comprises a first well in the chip; a second well in the chip; and a microcellular porous foam in the chip, wherein the first well is connected to the second well through the microcellular porous foam and the microcellular porous foam is formed from the material of the polymeric chip. The device is useful for studying cancer cell migration and invasion. Methods for using the device for cell migration, invasion assays, and methods for making the device are also disclosed.

Description

DEVICES FOR STUDYING CANCER INVASION AND MIGRATION

STATEMENT OF GOVERNMENT LICENSE RIGHTS

This invention was made with Government support under contract No. 0348767 awarded by the National Science Foundation. The Government has certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application claims the benefit of the U.S. Provisional Application No. 60/975,746, filed September 27, 2007, which is incorporated herein by reference in its entirety.

BACKGROUND

Chemotaxis describes the directed cell locomotion in concentration gradients of soluble extracellular agents. Substances that induce a chemotactic response are known as chemo tactic factors. Chemotactic behavior of cells has been linked to cancer cell migration and invasion, which is an important process in tumor progression. It is believed that disruption of cancer cell migration and invasion may be an effective way to control tumor progression. Therefore, a compound that can slow down or stop cell migration and invasion is likely to be useful in cancer treatment.

Cell migration and invasion assays are commonly used to identify a potential drug that can slow down or stop cell migration and invasion. A Boyden chamber is the most commonly used device for cell migration and invasion assays. FIGURE 1 is a schematic illustration of a cell migration assay in a Boyden chamber.

Referring to FIGURE 1, a Boyden chamber 100 has an upper chamber 110 and a lower chamber 120. The two chambers are separated by a porous membrane 130 having pores through which cells 125 can pass. A cancer cell suspension 140 containing cancer cells 125 suspended in serum- free medium is put into the upper chamber 110 while a specimen solution 150 containing a chemotactic factor 155 is put into the lower chamber 120. After incubating the cells for a period of time, cells are fixed. Cells migrating toward the chemotactic factor 155 through the porous membrane 130 or cells appearing on the back face of the porous membrane 130 are counted.

Chemoinvasion is the migration of cells across an extracellular matrix barrier. A cell invasion assay is similar to the migration assay, except that the top of the porous membrane is coated with a layer of extracellular matrix material, MATRIGEL™, and the cells are starved before placed onto the MATRIGEL™-coated membrane.

Using conventional Boyden chambers for cell migration and invasion assays has several potential disadvantages. Currently available Boyden chambers for studying cancer migration and invasion involve vertical cell culture inserts and wells. After migrating through the porous membrane on the bottom of the cell culture insert, some cells will attach to the bottom of the membrane, some cells will stay suspended in the medium, and some cells will fall onto the bottom of the chamber. Often, only the cells attaching to the bottom of the membrane are counted, resulting in inaccuracy for assays involving slow migrating cancer cells. Furthermore, when using a conventional Boyden chamber for an assay, each of the chambers has to be counted individually. It is time-consuming to complete a study when multiple Boyden chambers are involved.

Therefore, there is a need for an improved device to provide increased accuracy for cell migration and invasion assays involving slow migrating cancer cells and improved efficiency for cell migration and invasion studies using multiple chambers.

SUMMARY

This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

In one aspect, a medical testing device formed from a polymeric chip is disclosed.

The device includes a first well in the chip; a second well in the chip; and a microcellular porous foam in the chip, wherein the first well is connected to the second well through the microcellular porous foam and the microcellular porous foam is formed from the material of the polymeric chip.

The medical testing device may have a first micro channel from the first well to the microcellular porous foam and a second micro channel from the second well to the microcellular porous foam.

The polymeric chip is biocompatible or biodegradable. The polymeric chip may be made from any suitable thermoplastic material, including, for example, poly(methyl methacrylate), poly(lactic acid), poly(lactic-co-glycolic acid), or polystyrene.

The first well and the second well are horizontally configured. For example, the first well and the second well may be at the same elevation in the chip. The device may include a plurality of pairs of wells, each pair comprising oppositely placed wells connected to each other through the microcellular porous foam, wherein each microcellular porous foam is formed from the material of the polymeric chip. In one embodiment, the device may include a single microcellular porous foam section, wherein each pair of wells is connected at a different length along the single foam section.

For studying cell migration and invasion, the first well of the device may contain a cell suspension and the second well contains a chemotactic factor. In one embodiment, the cell suspension comprises a plurality of cancer cells and the chemotactic factor is a chemotactic factor for the cancer cell.

In another embodiment, the first well of the device may contain a cell suspension and the second well contains a drug. The drug is active in modulating the behavior of the cell. Representative drugs useful in the present invention include a small organic molecule, a protein such as an antibody, or a nucleic acid. The microcellular porous foam may be coated or filled with a hydrogel. In one embodiment, the microcellular porous foam may be filled with MATRIGEL™ for studying cell invasion. Alternatively, tissue cells can be grown in the microcellular porous foam.

The medical testing device may include a plurality of individual microcellular porous foams and wells, each individual microcellular porous foam connecting one well to another well through the microcellular porous foam and each of the microcellular porous foams is formed from the material of the polymeric chip.

In another aspect, methods for studying cells migration and/or invasion are disclosed. In one embodiment, the method includes the steps of placing a cell suspension including a plurality of cells in a first well of a medical testing device formed from a polymeric chip; placing a chemotactic factor in a second well of the medical testing device, wherein the first well is connected to the second well through a microcellular porous foam comprising pores in the range of 50 μm to about less than 80 μm and the microcellular porous foam is formed from the material of the polymeric chip; and studying whether the cells migrated from the first well to the second well.

In one embodiment, the method includes the steps of placing a cell suspension including a plurality of cells in a first well of a medical testing device formed from a polymeric chip; placing a chemo tactic factor in a second well of the medical testing device, wherein the first well is connected to the second well through a microcellular porous foam filled with a hydrogel and the microcellular porous foam is formed from the material of the polymeric chip; and studying whether the cells migrated from the first well to the second well.

In one embodiment, the method includes the steps of placing a cell suspension including a plurality of cells in a first well of a medical testing device formed from a polymeric chip; placing a chemo tactic factor in a second well of the medical testing device, wherein the first well is connected to the second well through a microcellular porous foam filled with MATRIGEL™ and the microcellular porous foam is formed from the material of the polymeric chip; and studying whether the cells invaded from the first well to the second well.

In one embodiment, the method includes the steps of placing a cell suspension including a plurality of cancer cells in a first well of a medical testing device formed from a polymeric chip; placing a chemo tactic factor in a second well of the medical testing device, wherein the first well is connected to the second well through a microcellular porous foam and the first well is horizontally configured with respect to the second well; and studying whether the cancer cells migrated from the first well to the second well.

In another aspect, methods for making a medical testing device are disclosed. In one embodiment, the method includes the steps of providing a polymeric chip; impregnating the polymeric chip with a gas to produce a gas-impregnated polymeric chip; applying ultrasonic energy to a region of the gas-impregnated polymeric chip to produce a localized region of microcellular porous foam; and creating a first well and a second well on the polymeric chip, wherein the first well is connected to the second well through the microcellular porous foam and the first well is horizontal to the second well.

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

FIGURE 1 is a schematic illustration of a traditional Boyden chamber for studying cell migration and invasion; FIGURE 2 shows a representative medical testing device of the present invention; FIGURE 3 is a photograph showing a representative medical testing device; FIGURE 4 is a schematic illustration of a system for selectively producing microcellular porous foam for a medical testing device of FIGURES 2 and 3 with high intensity ultrasound energy;

FIGURE 5 is a schematic illustration of a focused ultrasound device; FIGURE 6 is a photograph showing the microcellular foam region of a medical testing device; and

FIGURE 7 is a flow diagram of cell migration assay using a medical testing device.

DETAILED DESCRIPTION OF THE INVENTION

This application discloses medical testing devices for cell migration and invasion assays, methods for using the devices, and methods for making the devices.

A diagrammatical illustration of a representative medical testing device 200 is shown in FIGURE 2. The device is formed from a polymeric chip 210. The polymeric chip can be made of any biocompatible or biodegradable material known in the art. In addition, the polymeric chip may be made of polymeric materials that can be sterilized.

For example, the polymeric chip can be made of polymethyl methacrylate (PMMA), a biocompatible polymeric material, which has been widely used in bio-related studies such as cell culture devices. Other polymeric material useful in the present invention includes, but is not limited to, poly(lactic acid), poly(lactic-co-glycolic acid), and polystyrene.

The device 200 has a first well 220 and a second well 230 in the chip 210. The first well 220 and the second well 230 are connected through microcellular porous foam

240. The top view of the device 200 is being illustrated, however, in use, the device 200 will be placed so that the wells 220 and 230 will lie in a plane paralleled to the ground surface.

To this end, the first well 220 and the second well 230 are horizontally configured, meaning that the device is made to lie flat when used such that the first well and the second well will be at generally same elevation. This is in contrast to the conventional Boyden chamber of FIGURE 1.

A microcellular porous foam 255 is formed from the material of the polymeric chip at a localized area in the chip 210. The pores of the microcellular porous foam may vary from tens of micrometers to a few hundred micrometers depending upon the type of cells under study. The pores are interconnected so as to provide fluid movement therethrough. In one embodiment, the pores of the microcellular porous foam have an average diameter from about 50 μm to about 200 μm. In one embodiment, the pores of the microcellular porous foam have an average diameter from about 50 μm to about less than 80 μm. In one embodiment, the pores of the microcellular porous foam have an average diameter from about 80 μm to about 200 μm.

A first micro channel 240 connects the first well 220 to a thin slab of about 100 μm in length of the microcellular porous foam structure 260 derived from the microcellular porous foam 255. A second micro channel 250 connects the second well 230 to the thin slab of microcellular porous foam structure 260. Therefore, the first well 220 is connected to the second well 230 through the first micro channel 240, the thin slab of microcellular porous foam structure 260, and the second micro channel 250.

For cell migration and invasion assays, the first well may contain a cell suspension containing a plurality of cells under study, and the second well may contain a chemotactic factor. In one embodiment, the cell suspension may comprise a plurality of cancer cells and the chemotactic factor is a chemotactic factor for the cancer cell. Alternatively, the first well may contain a cell suspension containing a plurality of cells under study, and the second well contains nutrients.

The device can be used to study chemotactic behavior of varieties of cells including, for example, epithelia cells, endothelial cells, smooth muscle cells, leucocytes, neurons, fibroblasts, and monocytes. Particularly, the device can be used for studying the migration and invasion of cancer cells including, but not limited to, breast cancer cells, liver cancer cells, colon cancer cells, bone cancer cells, ovarian cancer cells, pancreatic cancer cells, prostate cancer cells, renal cancer cells, lung cancer cells, and thyroid cancer cells.

Chemotactic factors are substances possessing chemotactic activity including, but not limited to, various leukotactic factors and tumor necrosis factors. Chemotactic factors can either attract or repulse the cells, i.e. the cells may move towards the source of chemotactic factors (towards an increasing concentration gradient) or in the opposite direction. For example, CXCLl 6 is a known chemotactic factor for prostate cancer cells; and CXCL 12 is a known chemotactic factor for breast cancer cells. In studying cancer cell migration and/or invasion behavior, a person skilled in the art would recognize that the type of chemotactic factors to be placed in the second well 230 is dependent upon the type of cells in the first well 220.

The microcellular porous foam thin slab 260 may be filled or coated with a hydrogel. Not wanting to be limited by the theory, it is hypothesized that loading the pores of the microcellular porous foam with a hydrogel would help to establish a concentration gradient of the chemotactic factor or nutrients across the slab of the microcellular porous foam structure 260 between the first well 220 and the second well 230, so that the concentration will not equilibrate too fast between the two wells. In addition to filling the pores with hydrogel, the pore size of the thin slab 260 can be made progressively smaller until the desired concentration gradient is established.

A person skilled in the art would recognize that any biocompatible or biomimetic hydrogel would be useful in the present invention. Representative hydrogels include hydrogels derived from the natural source including alginate, collagen, hyaluronic acid, agarose, hydrogels derived from the synthetic sources including poly(ethylene glycol) (PEG)-based hydrogels such as poly(ethylene glycol) diacrylate (PEGDA) hydrogels, and extracellular matrix (ECM)-like hydrogels such as MATRIGEL™.

In one embodiment, the microcellular porous foam may be filled with MATRIGEL™ for cell invasion assays. MATRIGEL™ is the trade name for a gelatinous protein mixture secreted by mouse tumor cells and marketed by BD Biosciences. This mixture resembles the complex extracellular environment found in many tissues and is often used as a substrate for cell culture.

Alternatively, tissue cells may be grown in the pores of the microcellular porous foam thin slab 260. Not wanting to be limited by the theory, it is believed that growing tissues cells in the pores of the microcellular porous foam thin slab 260 would create a barrier to slow down the equilibrating process of the chemotactic factor or nutrients between the two wells and therefore, help to establish a concentration gradient across the slab of the microcellular porous foam between two wells. In addition to growing tissue cells in the thin slab 260, the pore size can be made progressively smaller until the desired concentration gradient is established. The geometrical size of the device may be determined based on the type and number of the cells to be studied. The representative device photographed in FIGURE 3 has 6 mm diameter wells and 500 μm wide micro channels. To use the device 300, the cancer cells under study can be loaded in the first well 310. Various chemicals such as chemotactic factors can be loaded in the second well 320. For migration assays, the slab of microcellular porous foam 350 separating the first well 310 from the second well 320 can be untreated or coated or filled with hydrogels or tissue cells. For invasion assays, the slab of microcellular porous foam 350 can be coated or filled with extracellular matrix (ECM)-like hydrogels, such as MATRIGEL™.

As shown in FIGURE 3, a plurality of pairs of wells may be packed on one polymeric chip to increase the efficiency of an assay. In one embodiment, the medical testing device may include a plurality of pairs of wells, each pair including oppositely placed wells connected to each other through the microcellular porous foam, and the microcellular porous foam is formed from the material of the polymeric chip. In a further embodiment, the device may include a single microcellular porous foam section, and each pair of wells is connected at a different length along the single foam section.

To make the representative device 300 shown in FIGURE 3, an original poly(methyl methacrylate) sample was cut into a polymeric chip 305 having a rectangular shape of 40 mm by 60 mm. The thickness of the poly(methyl methacrylate) samples was 2.5 mm.

The poly(methyl methacrylate) sample is first foamed using a selective high intensity ultrasonic foaming method described below to provide a region of microcellular porous foams 307. The pore size of the foamed region 307 can be controlled by controlling the gas saturation process of the poly(methyl methacrylate) sample and the ultrasound insonation parameters.

As shown in FIGURE 3, the ultrasonic foaming method can be used to create a single long section of microcellular foam 307. A plurality of wells are then created on each side of the microcellular porous foam 307, and channels are created from each well to the microcellular porous foam 307 in a manner that connects one well to an oppositely placed well. In this instance, the single long section of microcellular porous foam 307 is used to create individual thin foam slab that connects each pair of wells. The microchannels that connect each well to the single long section of microcellular porous foam will be perpendicular to the elongated foam 307. Alternatively, an individual microcellular foam can be created for each pair of wells with the use of the x-y translation stage described below.

The selective high intensity ultrasonic foaming method employs high intensity focused ultrasound to foam gas -impregnated polymers. The method uses inert gases including carbon dioxide and nitrogen as blowing agents so as not to introduce any organic solvent or other harmful substances. The method is capable of creating interconnected open-celled microcellular porous foams with varying topographical features at selected locations on a polymeric chip. The effects of major process variables, including ultrasound power, scanning speed, and gas concentration, affect both the pore size and interconnectivity of the microcellular porous foam.

Illustrated in FIGURE 4 is a high intensity focused ultrasound (HIFU) system 400 that may be used to create the microcellular porous structures described herein. The system 400 includes a high intensity focusing ultrasound transducer 404 connected to a power amplifier 402. Referring to FIGURE 5, a schematic close-up illustration of the high intensity focusing ultrasound transducer 404 is illustrated. The high intensity focusing ultrasound transducer 404 includes a focusing substrate 522 having a concave surface that focuses ultrasound energy into a concentrated focal zone 526. The focal length of the high intensity focusing ultrasound transducer 404 is denoted by "f." The focal plane is denoted by "r." Preferably, when a polymeric chip is insonated with ultrasound energy, the polymeric chip is located anywhere in the focal zone 526. The high intensity focusing ultrasound transducer 404 produces high intensity ultrasound waves or energy that can be focused so that the focal plane can be targeted on a translation stage of a positioning system 406. The positioning system 406 includes means to move the translation stage in three directions. Those directions being the x and y direction in the focal plane and in the z direction forwards and rearwards of the focal plane. A gas impregnated polymeric chip 408b is placed at or on the translation stage at or near to the focal plane of the transducer 404. The polymeric chip 408b is in the target area of the high intensity focusing ultrasound transducer 404. As can be appreciated, the polymeric chip 408b can be moved in the x, y, and z directions so that any location on the polymeric chip 408b can be exposed to the ultrasound energy produced by the high intensity focusing ultrasound transducer 404. The high intensity focused ultrasound energy can be focused on the surface of the polymeric chip 408b or internally in the polymeric chip 408b and at any thickness within the chip. The high intensity focusing ultrasound transducer 404 and the polymeric chip 408b are located in a tank 410 of distilled water for ultrasound wave propagation. The high intensity focusing ultrasound transducer 404 is stabilized by a support arm 412 connected to the high intensity focusing ultrasound transducer 404. The power amplifier 402 is connected to, and thereby, controlled by a computer 414. Computer 414 may be any one of a variety of devices including, but not limited to, personal computing devices, server-based computing devices, mini and mainframe computers, laptops, or other electronic devices having a type of memory. The computer 414 may include a processor, memory, computer-readable medium drive (e.g., disk drive, a hard drive, CD-ROM/DVD-ROM, etc.) that are all communicatively connected to each other by a communication bus. The computer 414 may also include a display and one or more user input devices, such as a mouse, keyboard, etc. Applications for running the system 400 may be stored in memory in the computer 414. Applications may be described in the context of computer-executable instructions, such as program modules being executed by the computer 414. Such applications may be used to control the amount of power from the power amplifier 402 passed to the high intensity focusing ultrasound transducer 404, and also to control the position of the polymeric chip 408b in relation to the focal plane or focal point of the high intensity focusing ultrasound transducer 404 by controlling the movement of the positioning system 406. To this end, the computer 414 is also connected to, and thereby communicates with, the positioning system 406. The computer 414 can issue commands to the positioning system 406 that permit the polymeric chip 408b to be moved in any direction in the xy plane and at any speed. Additionally, the computer 414 can issue commands to the positioning system 406 to move the polymeric chip 408b in the z direction so as to move the polymeric chip 408b to be within the focal plane or forward or rearward of the focal plane of the high intensity focusing ultrasound transducer 404.

A gas-saturation system 416 includes a gas cylinder 418 connected to a pressure vessel 420. The pressure vessel 420 receives gas and can include pressure regulating means to control the gas pressure within the interior of the pressure vessel 420. The pressure vessel 420 may also include timing means to keep track of the time at a given pressure. The pressure vessel 420 can be used to hold a polymeric chip 408a for a given time and at a given pressure. The gas-saturation system 416 is used to impregnate the polymeric chip 408a with the gas. In one embodiment, the pressure used to impregnate polymeric chip 408a may be in the range of 2 MPa to 10 MPa at room temperature. Additionally, the pressure vessel 420 may be opened to the atmosphere in order to allow gas to desorb from the polymeric chip 408a. Once the polymeric chip 408a has been impregnated with gas from gas cylinder 418, the polymeric chip 408a may be fully saturated with gas or partially saturated with gas. The polymeric chip 408a may additionally undergo desorption of gas for a given period of time at atmospheric pressure. This allows for a quicker method of achieving a desired gas concentration for partial saturation of the polymeric chip 408a. For example, the polymeric chip 408a may be impregnated with gas to full saturation at a high pressure. Thereafter, the pressure vessel 420 may be opened to atmosphere to allow the polymeric chip 408a to desorb gas to bring the saturation level to less than full saturation. This achieves a quicker low gas concentration level in the polymeric chip 408a as compared to initially impregnating the polymeric chip 408a with gas at a lower pressure. From the pressure vessel 420, the polymeric chip 408a is transferred to the arm of the positioning system 406 and may be insonated with high intensity focused ultrasound energy to create a localized microcellular foam within the polymeric chip 408b. One or more areas of the polymeric chip 408b may be insonated with high intensity focused ultrasound energy to create one or more localized and separated areas of microcellular porous foam. The microcellular porous foams are formed from and are the same material as the polymeric chip 408b and can be interior to or on the surface of the polymeric chip 408b. Furthermore, one or more regions of microcellular porous foams can be arranged on the chip in any configuration desirable by controlling the positioning system 406. This allows the creation of regions of microcellular porous foam that can be separated and distinct from each other within the same polymeric chip 408b. This is possible because of the polymeric chip 408b being mounted to the translation stage of the positioning system 406, and further, the computer 414 can control the start and stop of insonation of high intensity ultrasound energy to permit selective foaming in any desired location on the polymeric chip 408b. It is further possible to control one or more variables that influence the pore size diameter of the pores in the microcellular porous foam and also to control whether the pores are interconnected open-celled pores or close-celled pores. These variables include but are not limited to controlling the gas pressure in the pressure vessel 420, controlling the time that the polymeric chip 408a is exposed to gas under pressure, controlling the time that the polymeric chip 408a is allowed to desorb gas after gas impregnation, controlling the power of the high intensity focused ultrasound transducer 404, controlling the speed that the polymeric chip 408b is moved with respect to the focused beam of ultrasound energy, and controlling the distance of the polymeric chip 408 with respect to the focal plane or focal point. For producing interconnected open-celled pores, the method includes providing a polymeric chip having a gas concentration of 3-5% by weight. This concentration may be obtained by removing the polymeric chip 408a from the pressure vessel 420 before equilibrium is reached. Or alternatively, the polymeric chip 408a may be fully saturated, and then allowed to desorb gas to achieve the desired partial saturation and gas concentration.

A method of making microcellular porous foams on a polymeric chip in accordance with one embodiment of the invention includes impregnating a polymeric chip 408 with gas, followed by applying high intensity focused ultrasound energy onto the gas-impregnated chip. In the gas impregnation step, the polymeric chip 408a is placed into the high-pressure vessel 420 filled with an inert gas, such as nitrogen or carbon dioxide. Over time, the gas molecules dissolve into the polymeric chip 408a so that the chip 408a becomes gas-impregnated. Depending on the gas pressure and the impregnation time (the time that the chip 408a remains in the pressure vessel 420), the final gas concentration in the impregnated polymeric chip 408a can be controlled. In a subsequent step, the gas-impregnated polymeric chip 408a is retrieved from the pressure vessel and mounted on a computer controlled XYZ stage for ultrasonic insonation with the high intensity focusing ultrasound transducer 404. Because of the heating and implosion effects induced by ultrasound waves or energy, the gas-impregnated polymeric chip 408b becomes thermodynamic ally unstable and undergoes phase separation to generate micro structure porous foam.

The high intensity focused ultrasound polymer foaming effect happens based on two ultrasound related processes: high intensity focused ultrasound heating and high intensity focused ultrasound cavitation. When the polymeric chip is under ultrasound insonation, part of the acoustic energy will be deposited into (or absorbed by) the polymer matrix during the sound wave propagation. The amount of the acoustic energy dissipation depends on the properties of material and the sound wave such as the attenuation coefficient and the ultrasound frequency, and in turn causes the ultrasound heating effect. Besides the heating effect, high intensity focused ultrasound has a cavitation effect in a viscous fluid. High intensity focused ultrasound cavitation happens during the negative cycle of sound pressure, under the conditions that a) the local acoustic pressure is beyond a certain pressure threshold, and b), the existence of tiny cavities in the medium which serve as cavitation nuclei. FIGURE 6 shows a SEM image of the cross-section of the region of microcellular porous foams 307 after the high intensity focused ultrasound (HIFU) insonation. It can be seen that a 3D microcellular porous foam is generated from the material of the polymer, which does not require a different or foreign material to be used for the microcellular porous foam structure. There are many small holes on the pore walls, indicating that the pores are interconnected.

Referring back to FIGURE 3, upon completion of the HIFU foaming process, micro channels 330 and 340 were machined on the foamed poly(methyl methacrylate) chip by Computer Numerically Controlled (CNC) micro milling. The channels 330 and 340 cross-section were in a rectangular shape with a height of 125 μm and width of 575 μm. The channels 330 and 340 were cut to connect to the region of microcellular porous foam 307, leaving a thin slab of microcellular foam 350 connecting the micro channel 330 and micro channel 340.

In another aspect, methods for using the medical testing devices are disclosed. The medical testing device described above may be used in assays studying the chemo tactic behavior of cells. For example, the medical testing device described above may be used to study cancer cells migration and/or invasion.

As shown in FIGURE 7, a cell migration assay 500 can be carried out with a medical testing device having a first well connected to a second well through a microcellular porous foam 510. There are four alternatives for setting up a concentration gradient. In the first alternative, the pores of the thin slab (260 in FIGURE 1) may be untreated, as in block 512. In this alternative, the pores of the thin slab are reduced in size to achieve the desired concentration gradient. In the second and third alternatives, the microcellular porous foam thin slab 260 may be coated or filled with hydrogel or MATRIGEL™, as shown in blocks 514 and 516, respectively. In the fourth alternative, tissue cells may be grown in the microcellular porous foam, in which case, the microcellular porous foam essentially functions as a tissue scaffold, as shown in block 518. The second, third, and fourth alternatives may also be coupled with pore size reduction of the thin slab 260 as a means of further achieving the desired concentration gradient. In block 520, a cell suspension including a plurality of cancer cells, such as breast cancer cells, were seeded in the first well for a period of time. After that, in block 530, a solution containing nutrients and/or a chemotactic factor was added to the second well. The cells are then incubated such as in a humidified atmosphere of 5% CO₂, for example, as shown in block 540. The cells are fixed at the end of the study and migrated cells are counted under a light microscope to study whether the cells migrated from the first well to the second well, as shown in block 550.

Compared to the traditional devices for studying cell migration and invasion such as a Boyden chamber, the medical testing device of the present invention provide several advantages. First, the horizontal design of the wells allows counting the cells at both sides of the porous structure accurately. Second, the horizontal design of the wells also allows for frequent monitoring of the cell culture process. For slow migration cells, all of them can be observed. Third, the device improves the throughput and the efficiency of an assay because multiple chambers can be fabricated on the same chip and observed by simply moving the microscope slide. Finally, the manufacturing of the device involves the integration of selective foaming and fabrication of horizontal cell culture wells and channels on polymeric chips.

COMPARATIVE EXAMPLE A cell migration assay was conducted with a polymer chip as shown in

FIGURE 2. In this example, the average diameter of the pores of the microcellular foam was around 80 um. The thickness of the microcellular foam slab between the first and the second channels was about 125 um. Breast cancer cells, for example, the MCF-7 cells having a size of about 10 μm, were seeded in the first well for 24 hours. After that, nutrient was added to the second well. Cell migration was not observed in this case. The reason was that the pore size of the microcellular foam was believed to be too large to create a nutrient gradient needed for the cancer cells to migrate.

This comparative example suggests that certain hydrogel permeable materials, for example, MATRIGEL™, need to be loaded in the microcellular porous foam slab 260 so that the nutrient concentration will not equilibrate too fast between the two wells. Alternatively, tissue cells may be grown in the porous foam. This will not only create a natural barrier to slow down the equilibrating process of the nutrient concentration, but also provide a more realistic environment to study cancer migration. Another alternative would be to progressively reduce the pore size and increase the thickness of the microcellular porous foam slab 260 until the desired concentration gradient is achieved.

While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

Claims

CLAIMSThe embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A medical testing device formed from a polymeric chip, comprising: a first well in the chip; a second well in the chip; and a microcellular porous foam in the chip, wherein the first well is connected to the second well through the microcellular porous foam and the microcellular porous foam is formed from the material of the polymeric chip.

2. The medical testing device of Claim 1, comprising a first micro channel from the first well to the microcellular porous foam and a second micro channel from the second well to the microcellular porous foam.

3. The medical testing device of Claim 1, wherein the first well and the second well are horizontally configured.

4. The medical testing device of Claim 1, wherein the first well and the second well are at the same elevation in the chip.

5. The medical testing device of Claim 1, wherein the first well contains a cell suspension and the second well contains a chemotactic factor.

6. The medical testing device of Claim 5, wherein the cell suspension comprises a plurality of cancer cells and the chemotactic factor is a chemotactic factor for the cancer cell.

7. The medical testing device of Claim 1, wherein the first well contains a cell suspension and the second well contains a drug.

8. The medical testing device of Claim 1, wherein the pores of the microcellular porous foam have a size from about 50 μm to about less than 80μm.

9. The medical testing device of Claim 1, wherein the microcellular porous foam is filled or coated with a hydrogel.

10. The medical testing device of Claim 1, wherein the microcellular porous foam is filled or coated with MATRIGEL™.

11. The medical testing device of Claim 1, wherein the microcellular porous foam comprises tissue cells.

12. The medical testing device of Claim 1, wherein the polymeric chip is biocompatible or biodegradable.

13. The medical testing device of Claim 1, wherein the polymeric chip is made from poly(methyl methacrylate), poly(lactic acid), poly(lactic-co-glycolic acid), or polystyrene.

14. The medical testing device of Claim 1, comprising a plurality of pairs of wells, each pair comprising oppositely placed wells connected to each other through the microcellular porous foam, wherein the microcellular porous foam is formed from the material of the polymeric chip.

15. The medical device of Claim 14, comprising a single microcellular porous foam section, wherein each pair of wells is connected at a different length along the single foam section.

16. A method for studying cancer migration, comprising: placing a cell suspension including a plurality of cells in a first well of a medical testing device formed from a polymeric chip; placing a chemotactic factor in a second well of the medical testing device, wherein the first well is connected to the second well through a microcellular porous foam comprising pores in the range of 50 μm to about less than 80 μm and the microcellular porous foam is formed from the material of the polymeric chip; and studying whether the cells migrated from the first well to the second well.

17. A method for studying cancer migration, comprising: placing a cell suspension including a plurality of cells in a first well of a medical testing device formed from a polymeric chip; placing a chemotactic factor in a second well of the medical testing device, wherein the first well is connected to the second well through a microcellular porous foam filled with a hydrogel and the microcellular porous foam is formed from the material of the polymeric chip; and studying whether the cells migrated from the first well to the second well.

18. A method for studying cancer invasion, comprising: placing a cell suspension including a plurality of cells in a first well of a medical testing device formed from a polymeric chip; placing a chemotactic factor in a second well of the medical testing device, wherein the first well is connected to the second well through a microcellular porous foam filled with MATRIGEL™ and the microcellular porous foam is formed from the material of the polymeric chip; and studying whether the cells migrated from the first well to the second well.

19. A method for studying cancer invasion, comprising: placing a cell suspension including a plurality of cells in a first well of a medical testing device formed from a polymeric chip; placing a chemotactic factor in a second well of the medical testing device, wherein the first well is connected to the second well through a microcellular porous foam comprising tissue cells and the microcellular porous foam is formed from the material of the polymeric chip; and studying whether the cells migrated from the first well to the second well.

20. A method for studying cancer invasion or migration, comprising: placing a cell suspension including a plurality of cancer cells in a first well of a medical testing device formed from a polymeric chip; placing a chemotactic factor in a second well of the medical testing device, wherein the first well is connected to the second well through a microcellular porous foam and the first well is horizontally configured with respect to the second well; and studying whether the cancer cells migrated from the first well to the second well.

21. A method for making a medical testing device, comprising: providing a polymeric chip; impregnating the polymeric chip with a gas to produce a gas-impregnated polymeric chip; applying ultrasonic energy to a region of the gas-impregnated polymeric chip to produce a localized region of microcellular porous foam; and creating a first well and a second well on the polymeric chip, wherein the first well is connected to the second well through the microcellular porous foam and the first well is horizontal to the second well.