Patent Application
20230374430
This application claims priority from a Chinese patent application number 202210557883.0 filed May 19, 2022, and the disclosure of which is incorporated herein by reference in its entirety.
The present invention relates to medical devices, in particular, to a microfluidic chip for cell culture and drug screening, and more particularly, to a confined migration microfluidic device for cell culture and drug screening.
Cancer is one of the leading causes of death today and it causes about 9 million deaths each year worldwide. The mortality rate of cancer is low if it is found at an early stage, but the mortality rate will be greatly increased once the cancer metastasizes. Metastasis of cancer cells is the leading cause of cancer-induced deaths.
Currently, metastasis involves several sequential steps, specifically invasion, penetration into the vessels, circulation, and penetration out of the vessels. However, the 4 steps described above are extremely complex, and metastasis is the least well-known process in tumor progression. Cell migration is a key process of cancer metastasis, and because the hardness and mechanical properties of tumor tissues are significantly different from those of normal tissues, migration of cancer cells is faced with more severe migration conditions. In particular, during metastasis, cancer cells are stimulated by a variety of mechanical forces, including stromal and hydrodynamic forces. A large amount of extracellular matrix builds up in the solid tumor microenvironment, forming many restrictive micropores or microchannels for cell migration. For example, the arrangement and binding of collagen fibers around tumors provide clues for directed migration; cells may also migrate through unbound extracellular matrix (ECM), such as fibrillar collagen, which presents porous migration spaces; micro-tracking also occurs within and around blood vessels; cells may also migrate between epithelial and endothelial surfaces. Therefore, the confined migration of cancer cells is one of the key steps of tumor metastasis. However, there is no effective condition for simulating the confined migration of cancer cells at present, let alone knowing which drugs or cells can effectively block the confined migration of cancer cells and thereby prevent the metastasis of cancer cells, and therefore it is particularly difficult to find effective drugs or associated cells or tissues that restrict metastasis of cancer cells.
In view of the prior arts, there is an urgent need to establish an device capable of simulating the confined migration of cancer cells and a screening device capable of screening drugs or cells for restricting the migration of cancer cells on a platform.
Accordingly, to solve the problems described hereinabove, the present invention provides a confined migration microfluidic device for cell culture and drug screening.
In one aspect, there is provided a confined migration microfluidic device for cell culture and drug screening including:
In certain embodiments, the disposition of the second inlet and second outlet at the two opposite ends of each of the second channels allows different potential drug candidates or cells are introduced through different channels for different assays and screening.
In certain embodiments, each microfluidic chip further includes converging channels each disposed at two opposite ends of each of the first channels corresponding to an inlet converging channel and an outlet converging channel, respectively, and channelizing all the first channels such that the introduced cells or drug candidates to different first channels are identical to each other.
In certain embodiments, the microfluidic chip further includes an outlet connection channel for connecting the first outlet and the outlet converging channel.
In certain embodiments, the microfluidic chip further includes a pyramid-like flow diverging structure, where the number of flow channels at the most bottom (last) gradient level of the pyramid-like flow diverging structure is equal to the number of the first channels.
In certain embodiments, the pyramid-like flow diverging structure is a gradient flow structure, where one flow channel is added to each subsequent gradient level with respect to a preceding gradient level, so as to diverge the flow more evenly.
In certain embodiments, the pyramid-like flow diverging structure includes multiple gradient flow channels, where each gradient level includes a lateral channel and a longitudinal channel; the longitudinal channel of a preceding gradient level channelizes the lateral channel of a subsequent gradient level; and each of the lateral channels of the last gradient level channelizes each of the first channels.
In certain embodiments, the top gradient level of the pyramid-like flow diverging structure includes only a first diverging channel channelizing the first inlet and the lateral channel at a second gradient level.
In certain embodiments, the microfluidic chip includes an upper chip and a lower chip, where the upper chip is provided therethrough the first inlet, the first outlet, the second inlet, and the second outlet; the upper and lower chips are associated with each other to form the microfluidic chip, where the first channel, second channel, first extension channel, second extension channel and confined migration channels are formed between the upper and lower chips.
In certain embodiments, the upper chip includes an upper channel section; the lower chip includes a lower channel section; combination of the upper channel section and the lower channel section forms all types of channels of the microfluidic chip.
In certain embodiments, one or more of the chips is/are polygonal or circular.
In certain embodiments, one or more of the chips is/are octagonal.
In certain embodiments, a cell adhesion reinforcement agent is added onto the interior surface of the channels.
Preferably, the cell adhesion reinforcement agent is fibronectin.
In certain embodiments, the microfluidic device further includes an accommodation structure for accommodating multiple chips, where the accommodation structure includes a plurality of slots/holes.
In certain embodiments, the slots or holes capable of accommodating the chips are circular slots or holes for ease of placement and removal.
Provided herein also is a method for preparing the microfluidic chip, where the method includes:
In certain embodiments, the cells are normal cells or abnormal (disease) cells, or both.
In certain embodiments, the normal cells include kidney cells, lung cells, gastrointestinal cells, brain cells, liver cells, fibroblasts, endothelial cells, immune cells and macrophages.
In certain embodiments, the disease cells include tumor cells, tumor-associated macrophages or tumor-associated fibroblasts, and a model of the disease cells is derived from a model of the normal cells with certain modifications.
In certain embodiments, the tumor cells include gastric cancer cells, pancreatic cancer cells, colorectal cancer cells, liver cancer cells, bone cancer cells, lung cancer cells, kidney cancer cells, prostate cancer cells, breast cancer cells, brain cancer cells, neuroendocrine tumor (cancer) cells and all other tumor cells associated thereto.
The present invention further provides a method of using the microfluidic chip or device described herein for screening substances with potentials of inhibiting confined migration of tumor cells.
The present invention further provides a method of using the microfluidic chip or device described herein for screening drug candidates with potentials of inhibiting confined migration of tumor cells.
The present invention further provides a method of using the microfluidic chip or device described herein for screening cells with potentials of inhibiting confined migration of tumor cells.
The present invention further provides a method of using the microfluidic chip or device described herein in preparing a medicament for inhibiting confined migration of tumor cells.
Some of the beneficial effects of the present invention are summarized, as follows: the chip described herein can be used for well observing the confined migration of a type of cancer cells. It can also be used to observe the interference effect of different drugs on the cell migration after the different drugs are used on the other side of the confined channel, and thus effective drug candidates of inhibiting the cell migration can be determined for potential cancer treatment. Effects of different concentrations of the same drug on cell migration can also be evaluated, where a therapeutically effective amount of migration-inhibiting drug can also be determined, or the effect of the same drug at the same concentration on the migration of different cells can be evaluated. It can also be used to evaluate any combined effect of co-culture and drug on cell migration. The provision of pyramid-like flow diverging channel configuration on a single chip ensures consistency in concentration and quantity of the introduced drugs or cells in each channel, reducing errors to the greatest extent, and improving operation and evaluation efficiency due to the presence of sufficient number of drug screening channels. In addition, the plural confined migration channels extending out of each drug screening channel can further be used to evaluate the possibility of multiple confined migrations and migration inhibition by potential drug candidates, which may be realized under the same condition, and thus multiple confined migrations are possible to be evaluated, and errors arising from such evaluation can be significantly reduced due to high specificity. The present microfluidic chip or device is simple to use, and is able to deliver more evaluation data with higher accuracy, that is, high throughput assay under a consistent testing condition can be achieved by the present chip or device.
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 or essential 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. Other aspects of the present invention are disclosed as illustrated by the embodiments hereinafter.
The appended drawings, where like reference numerals refer to identical or functionally similar elements, contain figures of certain embodiments to further illustrate and clarify the above and other aspects, advantages and features of the present invention. It will be appreciated that these drawings depict embodiments of the invention and are not intended to limit its scope. The invention will be described and explained with additional specificity and detail through the use of the accompanying drawings in which:
Skilled artisans will appreciate that elements in the figures are illustrated for simplicity and clarity and have not necessarily been depicted to scale.
It will be apparent to those skilled in the art that modifications, including additions and/or substitutions, may be made without departing from the scope and spirit of the invention. Specific details may be omitted so as not to obscure the invention; however, the disclosure is written to enable one skilled in the art to practice the teachings herein without undue experimentation.
Turning to
In certain embodiments, four to ten identical confined migration channels 3 are provided between the first channel 1 and the second channel 2; there may be four, six, eight and ten confined migration channels.
In certain embodiments, two extension channels, namely a first extension channel 13 and a second extension channel 23, are provided at two different (opposite) ends of each of the confined migration channels 3, respectively, where the first extension channel 13 connects the first channel 1 and the confined migration channel 3; the second extension channel 23 connects the second channel 2 and the confined migration channel 3; and a depth of the first extension channel 13 and a depth of the second extension channel 23 are each greater than that of each of the confined migration channels 3. This configuration ensures that the cells can be completely subject to confined migration after entering the extension channels, which is more similar to the real migration condition and can avoid the situation that cells will grow along the interior surface of the channels and thereby no migration will occur, in turn, the actual effect and efficiency can not be exerted. Reference is made to
In certain embodiments, the first extension channel 13 has the same depth as that of the first channel 1; the second extension channel 23 has the same depth as that of the second channel 2, such that it can simplify the fabrication process and ensure that cells and the drugs or cells that inhibit cell migration flow into corresponding extension channels effectively.
In certain embodiments, the first extension channel 13 and the second extension channel 23 have identical configuration, such that it can ensure a symmetrical migration and interference to the greatest extent and mitigate errors.
In certain embodiments, a plurality of the first channels 1 and the second channels 2 are provided on one microfluidic chip, and by this configuration, confined migration culturing of different cell types and co-culture screening assays of different drugs and cells on inhibiting the confined migration of target cells can be performed on the same microfluidic chip, so that unnecessary operations are avoided, and multiple results can be observed on one single chip.
In certain embodiments, two opposite ends of each of the second channels 2 are provided with the second inlet 11 and the second outlet 22, respectively, such that it can ensure that different drugs or cells are introduced through the channels for different processes and screenings.
In certain embodiments, two opposite ends of each of the first channels 1 are provided with the first inlet 11 and the first outlet 12, respectively. In other embodiments, converging channels are further provided on the same chip at two opposite ends of the first channels 1 which channelize all the first channels 1. Turning to
In certain embodiments, the chip further includes an outlet connection channel 16 provided in a central part of the outlet converging channel 15; the outlet connection channel 16 connects the first outlet 12 and the outlet converging channel 15.
In certain embodiments, to ensure that liquid flowing into each of the first channels 1 is completely the same with each other in terms of their content and volume, a pyramid-like flow diverging structure 17 is further provided on the chip, and the number of flow channels at the most bottom (last stage) of the pyramid-like flow diverging structure 17 is equal to the number of the first channels 1; a top end of the pyramid-like flow diverging structure 17 is connected to the first inlet 11. A good flow diverging effect among the first channels 1 is thereby achieved by providing the pyramid-like flow diverging structure 17, thereby avoiding occurrence of uneven flow distribution. In certain embodiments, the pyramid-like flow diverging structure 17 is a gradient flow diverging structure, and each subsequent layer (stage) is added with one flow channel with respect to the number of flow channels of its preceding layer, such that a more even flow distribution is achieved. The pyramid-like flow diverging structure 17 includes multiple layers (stages) of flow diverging channels, and each level (stage) of flow diverging channels includes a lateral channel 171 and a longitudinal diverging channel 172; the longitudinal diverging channel 172 of a preceding stage connects to the lateral channel 171 of its subsequent stage; the lateral channels 171 of the last stage connect to all the first channels 1; a top layer of the pyramid-like flow diverging structure 17 only includes one diverging channel 173, which is a first diverging channel in longitudinal flow direction, and the first diverging channel 173 connects the first inlet 11 and a second-stage lateral channel 171. Reference is also made to
In certain embodiments, each of the longitudinal channel 172 and the lateral channel 171 has a depth identical to that of the first channel 1.
Turning to
In certain embodiments, the upper chip 41 includes an upper channel section; the lower chip 42 includes a lower channel section; and the upper channel section and the lower channel section are combined to form all the channels.
In certain embodiments, the upper chip 41 includes an upper set of the first channels 1, an upper set of the second channels 2 and an upper set of the confined migration channels 3; the lower chip 42 includes a lower set of the first channels 1, a lower set of the second channels 2 and a lower set of the confined migration channels 3; a sum of the depth of the upper first channels 1 and that of the lower first channels 1 is equal to the sum of the depth of the first channels 1; a sum of the depth of the upper second channels 2 and that of the lower second channels 2 is equal to the sum of the depth of the second channels 2; a sum of the depth of the upper confined migration channels 3 and that of the lower confined migration channels 3 is equal to the sum of the depth of the confined migration channels 3.
In certain embodiments, the channel portions of the upper chip 41 and those of the lower chip 42 are identical, and the confined migration channels 3 is centered between the first extension channel 13 and the second extension channel 23.
In another embodiment, a depth of all the channel portions of the upper chip 41 or the lower chip 42 is equal to that of the confined migration channel 3, and no channel is provided in a corresponding portion of the lower chip 42 or in a corresponding portion of the upper chip 41 with respect to the confined migration channel 3.
In certain embodiments, the depth of all the channel portions of the lower chip 42 is equal to that of the confined migration channel 3, and no channel is provided in a corresponding portion of the upper chip 41 with respect to the confined migration channel 3, forming the confined migration channel 3. Exclusive of the corresponding portion with respect to the confined migration channel 3, a depth of the other channel portions of the upper chip 41 is equal to the difference between the total channel depth (i.e., the sum of the depth of other channel portions of the upper chip 41 and the depth of all the channel portions of the lower chip 42) and the depth of the confined migration channel 3. Reference is made to
In certain embodiments, the depth of the confined migration channel 3 is in a range of 4-10 microns; the depth of the other channels excluding the confined migration channel 3 is in a range of 20-40 microns. By this configuration, it can effectively enable the cell culture and establishment of a confined migration environment.
In certain embodiments, the depth of the confined migration channel 3 is 6 microns; the depth of the other channels is 30 microns.
In certain embodiments, excluding the corresponding portion with respect to the confined migration channel 3 that has no depth, the depth of the other channel portions of the upper chip 41 is 24 microns; the depth of all the channels of the lower chip 42 is 6 microns.
In certain embodiments, a width of all the channels is in a range of 40-60 microns;
In certain embodiments, a length of the first extension channel 13 or a length of the second extension channel 23 is in a range of 60-80 microns;
In certain embodiments, a length of the confined migration channel 3 is in a range of 40-60 microns;
In certain embodiments, each of the confined migration channels 3 has a uniform square cross-section and identical dimension (length and width) with each other; the length and the width of each of the confined migration channels are 50 microns each.
In certain embodiments, the microfluidic chip or chips is/are made of polydimethylsiloxane (PDMS).
In certain embodiments, the microfluidic chip or chips is/are polygonal or circular.
In certain embodiments, the microfluidic chip or chips is/are octagonal, or in a shape according to the embodiments depicted in any of
In certain embodiments, a material that enhances cell adhesion is incorporated into the channels, wherein the material is selected from fibronectin.
In certain embodiments, the present device further includes an accommodation structure 5 capable of accommodating a plurality of chips, and the accommodation structure 5 is provided with a plurality of chip placement holes or slots 51 each for accommodating one chip. By this configuration, a plurality of chips can be placed on the accommodation structure 5 and analyzed simultaneously, such that image analyses of a plurality of chips can be performed in one time, and it is not necessary to repeatedly place and remove the chips. In case where some studies require analyzing multiple chips, a one-time analysis of multiple chips is feasible by the present invention, and the results are allowed to be directly presented on the same image.
In certain embodiments, the chip placement holes or slots 51 are circular holes capable of accommodating the chip(s). This configuration as circular holes eases placing and removal of the chips from the device. Reference is made to
Provided herein is a method for preparing the microfluidic chip or chips of the present invention, which includes:
Provided herein are different scenarios of applying the present invention according to various embodiments of providing a plurality of the first channels 1 and second channels 2 on a single chip and further providing the pyramid-like flow diverging structure 17:
(1) Method for Studying the Effect of Different Drugs or Different Concentrations of Various Drugs on Migration of a Single Type of Cells
After the chip treatment is completed, a cell suspension is loaded into the first inlet 11 of the pyramid-like flow diverging structure 17. A pipette tip is used to slightly suction at the first outlet 12 to let the cells flow into the first channels 1 evenly, and then enter the first extension channels 13 evenly. Different drugs are loaded into the second inlets 11 of different second channels 2, respectively. The channels are photographed and observed for 48 hours in real time by using an etaluma LS720 fluorescence microscope, and a cell migration image is taken every half an hour. The effect of different drugs or different concentrations of various drugs on the migration of the same type of cells is thereby evaluated.
(2) Method for Studying the Effect of a Drug on Migration of Different Cell Types
After the chip treatment is completed, different cell suspensions are loaded into inlets of the second channels 2 of the chip, respectively, and allowed to wait for 1 minute until liquid in the channels is equalized and the cells completely flow into the second channels 2 and then enter the second extension channels 23. A drug-containing liquid is added into the first inlet 11 with respect to the pyramid-like flow diverging structure 17. A pipette tip is used to slightly suction to let the drug-containing liquid flow into the first channels 1 evenly and enter the first extension channels 13 evenly. The channels are photographed and observed for 48 hours in real time by using an etaluma LS720 fluorescence microscope, and a cell migration image is taken every half an hour. The effect of a drug on the migration of different cells is thereby evaluated.
(3) Method for Studying the Effect of Cell-Cell Interaction in Co-Culture and Cell-Drug Interaction on Cell Migration
After the chip treatment is completed, a co-culture cell medium, such as liquid containing tumor-associated fibroblasts, is added into the first inlet 11 of the pyramid-like flow diverging structure. A pipette tip is used to slightly suction to let the tumor fibroblasts-containing liquid flow into the first channels 1 evenly and enter the first extension channels 13 evenly; tumor cells and drug liquid are added into the second inlet 11 of the second channels 2, so that the tumor cells and the drug liquid enter the second extension channels 23. The channels are photographed and observed for 48 hours in real time by using an etaluma LS720 fluorescence microscope, and a cell migration image is taken every half hour. The effect of the fibroblasts and the drug on the cell migration in real time is thereby evaluated.
Although the invention has been described in terms of certain embodiments, other embodiments apparent to those of ordinary skill in the art are also within the scope of this invention. Accordingly, the scope of the invention is intended to be defined only by the claims which follow.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202210557883.0 | May 2022 | CN | national |
