Patent Application
20230221299
The present disclosure relates broadly to a device and a method for studying interactions of a first cell type with a second cell type.
Cancer is a leading cause of death and expensive to treat. The health care costs to society associated with treating cancer, which affects older adults most often, will only increase in the future considering humans are now living longer. Today, medical treatments, even for cancer, are often not focused on a personalized patient care approach. Where feasible, a more personalized approach to treating cancer can lead to potentially more accurate prediction of how a therapy will affect an individual. Better predictions of how individual patients will respond to treatment benefits not only health care providers, but can also lower costs for insurers and give patients a better peace of mind by knowing that therapies are individually prescribed.
For a certain class of blood-derived cancers such as acute lymphoblastic leukemia (ALL), lymphomas and multiple myelomas, the cutting-edge of cancer treatment lies in a relatively new therapy called adopted cell transfer (ACT). This “living” treatment uses the patient's own immune cells, modifies them, and reintroduces them into the body, using the patient's own immune system to destroy the cancer. These immune-cell mediated therapies can be combined with previously established chemotherapeutic standard-of-care drugs or combinations thereof, to form completely new types of cancer therapies. There is now an opportunity to apply these immunotherapy breakthroughs not only to haematological cancers but to solid tumor cancers. However, there remain both scientific and practical challenges to doing this, namely the difficulty in predicting how immune cells will interact with solid tumor bodies as well as the high cost, risks, and patient-sensitive nature of this type of research. Therefore, it is increasingly difficult to conduct studies in this area and progress in developing new types of cancer treatments is slowed.
In view of the above, there is a need to address at least ameliorate the above-mentioned problems. In particular, there is a need to provide a device and method for studying interactions between different cells (e.g., immune cells and tumor cells) that address or at least ameliorate the above-mentioned problems.
In one aspect, there is provided a microfluidic device comprising a first region configured to hold tumor cells; a second region configured to hold immune cells; and an array of microstructures disposed between the first and second regions, wherein the first region is in fluid communication with the second region, and wherein the array of microstructures is configured to selectively allow movement of immune cells, from the second region to an interaction zone that is at least partially disposed within the first region, for interaction with tumor cells in the interaction zone.
In one embodiment, the first and second regions are symmetrical about a same line of symmetry.
In one embodiment, the device further comprises one or more third regions, the third region being in fluid communication with the first and second region, wherein the array of microstructures comprises microstructures disposed between the third region and the first region.
In one embodiment, the array of microstructures comprises microstructures disposed between the third region and the second region.
In one embodiment, the third region substantially surrounds the first region.
In one embodiment, the first, second and third regions are symmetrical about a same line of symmetry.
In one embodiment, the array of microstructures comprises microstructures organised in a radial manner or a grid-like manner.
In one embodiment, the array of microstructures comprises microstructures organised as substantially concentric rows of microstructures.
In one embodiment, the distance between the microstructures in the row closest to the first region is smaller than the distance between the microstructures in the row furthest from the first region.
herein the size of the microstructures in the row closest to the first region is smaller than the size of the microstructures in the row furthest from the first region.
In one embodiment, each of said regions comprises a shape defined by tapering of a bigger area to a smaller area.
In one embodiment, the device further comprises ports corresponding to each of said regions for providing access to each of the regions.
In one embodiment, the device comprises a seeding layer and a support layer, wherein the ports are disposed on the seeding layer and the corresponding regions are disposed on the support layer.
In one embodiment, the first region has a larger depth than the second region.
In one embodiment, the second region has substantially the same depth as the third region.
In one aspect, there is provided a chip comprising a plurality of the device disclosed herein.
In one aspect, there is provided a method of studying interactions of a first cell type with a second cell type, the method comprising: providing a device comprising a first region configured to hold a first cell type; a second region configured to a second cell type; and an array of microstructures disposed between the first and second regions, wherein the first region is in fluid communication with the second region, and wherein the array of microstructures is configured to selectively allow movement of the second cell type from the second region to an interaction zone that is at least partially disposed within the first region, to allow interaction of the first cell type and the second cell type in the interaction zone; seeding the first cell type in the first region of the device; applying a first external force to direct the first cell type to the interaction zone; seeding the second cell type in the second region of the device; allowing the second cell type to migrate from the second region to the interaction zone for interaction with the first cell type in the interaction zone; and monitoring migration of the second cell type and interaction of the second cell type with the first cell type.
In one embodiment, the method further comprises, subsequent to the monitoring step, applying a second external force to direct cells present within the interaction zone away from the interaction zone for retrieval and analysis.
In one embodiment, the monitoring step comprises monitoring the migration and interaction of the cells with an image capturing apparatus.
In one embodiment, the device further comprises one or more third regions, and the method further comprises, prior to the step of applying the first external force, seeding microenvironment materials into the one or more third regions, wherein the third region is in fluid communication with the first and second regions, and wherein the array of microstructures comprises microstructures disposed between the third region and the first region.
The term “micro” as used herein is to be interpreted broadly to include dimensions from about 1 micron to about 1000 microns and from about 1 micron to about 100 microns.
The terms “coupled” or “connected” as used in this description are intended to cover both directly connected or connected through one or more intermediate means, unless otherwise stated.
The term “associated with”, used herein when referring to two elements refers to a broad relationship between the two elements. The relationship includes, but is not limited to a physical, a chemical or a biological relationship. For example, when element A is associated with element B, elements A and B may be directly or indirectly attached to each other or element A may contain element B or vice versa.
The term “adjacent” used herein when referring to two elements refers to one element being in close proximity to another element and may be but is not limited to the elements contacting each other or may further include the elements being separated by one or more further elements disposed therebetween.
The term “and/or”, e.g., “X and/or Y” is understood to mean either “X and Y” or “X or Y” and should be taken to provide explicit support for both meanings or for either meaning.
Further, in the description herein, the word “substantially” whenever used is understood to include, but not restricted to, “entirely” or “completely” and the like. In addition, terms such as “comprising”, “comprise”, and the like whenever used, are intended to be non-restricting descriptive language in that they broadly include elements/components recited after such terms, in addition to other components not explicitly recited. For example, when “comprising” is used, reference to a “one” feature is also intended to be a reference to “at least one” of that feature. Terms such as “consisting”, “consist”, and the like, may in the appropriate context, be considered as a subset of terms such as “comprising”, “comprise”, and the like. Therefore, in embodiments disclosed herein using the terms such as “comprising”, “comprise”, and the like, it will be appreciated that these embodiments provide teaching for corresponding embodiments using terms such as “consisting”, “consist”, and the like. Further, terms such as “about”, “approximately” and the like whenever used, typically means a reasonable variation, for example a variation of +/−5% of the disclosed value, or a variance of 4% of the disclosed value, or a variance of 3% of the disclosed value, a variance of 2% of the disclosed value or a variance of 1% of the disclosed value.
Furthermore, in the description herein, certain values may be disclosed in a range. The values showing the end points of a range are intended to illustrate a preferred range. Whenever a range has been described, it is intended that the range covers and teaches all possible sub-ranges as well as individual numerical values within that range. That is, the end points of a range should not be interpreted as inflexible limitations. For example, a description of a range of 1% to 5% is intended to have specifically disclosed sub-ranges 1% to 2%, 1% to 3%, 1% to 4%, 2% to 3% etc., as well as individually, values within that range such as 1%, 2%, 3%, 4% and 5%. It is to be appreciated that the individual numerical values within the range also include integers, fractions and decimals.
Furthermore, whenever a range has been described, it is also intended that the range covers and teaches values of up to 2 additional decimal places or significant figures (where appropriate) from the shown numerical end points. For example, a description of a range of 1% to 5% is intended to have specifically disclosed the ranges 1.00% to 5.00% and also 1.0% to 5.0% and all their intermediate values (such as 1.01%, 1.02% . . . 4.98%, 4.99%, 5.00% and 1.1%, 1.2% . . . 4.8%, 4.9%, 5.0% etc.,) spanning the ranges. The intention of the above specific disclosure is applicable to any depth/breadth of a range.
Additionally, when describing some embodiments, the disclosure may have disclosed a method and/or process as a particular sequence of steps. However, unless otherwise required, it will be appreciated that the method or process should not be limited to the particular sequence of steps disclosed. Other sequences of steps may be possible. The particular order of the steps disclosed herein should not be construed as undue limitations. Unless otherwise required, a method and/or process disclosed herein should not be limited to the steps being carried out in the order written. The sequence of steps may be varied and still remain within the scope of the disclosure.
Furthermore, it will be appreciated that while the present disclosure provides embodiments having one or more of the features/characteristics discussed herein, one or more of these features/characteristics may also be disclaimed in other alternative embodiments and the present disclosure provides support for such disclaimers and these associated alternative embodiments.
Exemplary, non-limiting embodiments of a device and a method for studying interactions of a first cell type with a second cell type are disclosed hereinafter.
There is provided a device (e.g., a microfluidic device) comprising a first region, the first region configured to hold at least a first cell type (e.g., target cells such as tumor cells); a second region disposed, the second region configured to hold at least a second cell type (e.g., effector cells such as immune cells); and an array of barriers (for example, an array of structures or an array of microstructures) disposed in a position that is between the first and second regions, wherein the first region is in fluid communication with or fluidly coupled to the second region (e.g., via at least one flow passage/path that is disrupted by the array of barriers), and wherein the array of barriers is configured to selectively allow movement of cells (i.e., the second cell type or immune cells) to an interaction zone (for example, an entrapment area) to allow interaction of the first and second cell types in the interaction zone. The first and second regions may be disposed on a support layer. Advantageously, in various embodiments, the device may provide the capability of compartmentalizing and controlling the interaction between tumor and immune cell populations that the device is intended to examine. Further advantageously, various embodiments of the device, which may be a compartmentalized microfluidic device, may control and direct tumor-immune cell interactions as well as create a complex three-dimensional tumor microenvironment to more closely mimic physiological conditions in-vivo.
Thus, various embodiments of the device may, for example, be used for early discovery or screening of drug/immune therapy combinations, and/or tumor-infiltrating lymphocyte migration studies (e.g., for the study of tumour-immune cell interactions in the presence of chemotherapeutic drugs.). That is, in various embodiments, the device may, for example, be used to yield results of screening for novel combination drug therapies, which may yield potentially effective therapies for cancer treatment (in the early drug discovery stage).
In various embodiments, the microfluidic device comprises a first region configured to hold tumor cells; a second region configured to hold immune cells; and an array of microstructures disposed between the first and second regions, wherein the first region is in fluid communication with the second region, and wherein the array of microstructures is configured to selectively allow movement of immune cells, from the second region to an interaction zone that is at least partially disposed within the first region, for interaction with tumor cells in the interaction zone. In some embodiments, the interaction zone is also at least partially disposed within the second region.
In various embodiments, the selective movement of cells (i.e., the second cell type or immune cells) to the interaction zone comprises selective movement of cells from the second region to the interaction zone. In various embodiments, the interaction zone is an interaction zone of the first region. For example, the interaction zone may in some embodiments be defined as a zone that is completely disposed in the first region. In some other embodiments, the interaction zone may be a zone that spans across the first region and the second region and optionally other regions. Depending on the exact configuration of the array of microstructures and the cell types, the location where interactions between the first and second cell types occur may vary. The selective movement of second cell type may partially arise from the array of barriers (e.g., an array of microstructures) between the first and second regions which present a migratory barrier to the second cell type during the movement from the second region to the interaction zone of the first region. In various embodiments, the array of barriers (e.g., an array of microstructures) are additionally configured to substantially prevent movement of the tumor cells from the first region to the second region e.g., in the presence of an external force such as a rotational or centrifugal force.
In various embodiments, the device is a two layered structure. The device may comprise a seeding layer and a support layer. For example, the device may further comprise a seeding layer disposed over the support layer, the latter having the various regions disposed thereon. The device may comprise ports for providing access to the regions, for example, via ports corresponding to each of said regions for providing access to each of the regions. In various embodiments, the seeding layer comprises a first seeding port that allows access to (e.g., is in fluid communication with, coupled to, or fluidically connected to) the first region for seeding/removal the first cell type in the first region; and a second seeding port that allows access to the second region for seeding/removing the second cell type and/or removal of air bubbles in the second region.
In various embodiments, the device further comprises one or more third regions for one or more of the following functions: seeding/removal of the second cell type, removal of air bubbles and to hold/provide/remove tumor microenvironment (TME) materials. Tumor microenvironment materials may, for example, include artificial extra cellular matrices (e.g., Matrigel), cellular materials (e.g., cancer associated fibroblasts), or both. Tumor microenvironment materials may also include one or more molecular components (e.g., Polysaccharides, Collagen, Glycoproteins, Cytokines), natural materials (e.g., Alginate, Chitosan), and/or cellular/tissue-based components (e.g., lymph vessels, blood vessels, endothelial cells, myeloid derived suppressor cells, macrophages, T-cells and NK-cells). Advantageously, in various embodiments, including TME materials in the device may enhance the physiological relevance of experiments performed on the device (e.g., immune-tumor interaction experiments) by allowing for the creation of a complex three-dimensional TME surrounding the central tumor spheroid being studied.
In various embodiments, the device comprises one or more third regions, for example, at least one, at least two, at least three, at least four or at least five third regions. The device may comprise one or more third region(s) for seeding/removal of the second cell type and/or removal of air bubbles. In one example, the device comprises a single third region for seeding/removal of the second cell type and/or removal of air bubbles. The device may comprise one or more third region(s) configured to hold tumor microenvironment materials. In one example, the device comprises a plurality of third regions (e.g., three third regions), each configured to hold tumor microenvironment materials. In various embodiments, at least one third region (or each of the third region) is in fluid communication with or fluidly coupled to the second region (e.g., via at least one channel/passage/path). Further, in various embodiments, the array of barriers (e.g., the array of microstructures) comprises microstructures disposed between at least one of the third regions and the second region. In some embodiments, the microstructures may be disposed between each of the third regions and the second region. Even further, in various embodiments, at least one of the third region (or each of the third region) is in fluid communication with the first and second regions, wherein the array of barriers (e.g., the array of microstructures) comprises microstructures disposed between at least one third region and the first region and/or between at least one third region and the second region and/or between two different third regions. In some embodiments, the microstructures may be disposed between each of the third regions and the first region, and/or between each of the third regions and the second region. In various embodiments, at least a portion/part of the third region(s) is sandwiched between the first region and the second region. In various embodiments, at least one of the third regions or each of the third regions substantially surrounds the first region. In one embodiment, at least one of the third regions (or a plurality of or all of the third regions) completely surrounds the first region. In other embodiments, where there are two or more separate third regions for example, one of the third regions may completely surround the first region and the other third region (s) may flank or substantially surround the first region. At least part of the third region (or at least part of each of the third region) may be in the interaction zone. In other words, in some embodiments, at least part of the interaction zone may be disposed in one or more third regions. Accordingly, in some embodiments, the interaction zone may span across the first region, the second region and one or more third regions. In various embodiments, the third region(s) are disposed on the support layer.
In various embodiments, the device further comprises a third port (e.g., disposed on said seeding layer) that allows access to (e.g., is in fluid communication with or fluidly coupled to) the third region for one or more of: seeding/removal of the second cell type, removal of air bubbles or to hold/provide/remove tumor microenvironment materials. In various embodiments, the device disclosed herein may allow for retrieval of cells via the various ports described (e.g., the first, second and third seeding ports). The extracted cells may be analysed to yield useful results (e.g., to identify potentially effective therapies for cancer treatment in the early drug discovery stage).
In various embodiments, one or more of the regions or each region (i.e., the first region, second region and third region) may comprise a shape defined by tapering of a bigger area to a smaller area. In one example, the shape may resemble a bulbous shape or a teardrop shape. Further, in various embodiments, the diameter of the curvature of the bigger area of each region may be same, e.g., to allow/accommodate the use of an automatic handler such as a bioprinter to seed/extract cells from the bigger areas.
In various embodiments, one or more regions may each have depth(s) that are deeper than other region(s). In various embodiments, the regions may have different depths from one another in order to accommodate the sizes of the different cells or tumor microenvironment materials being seeded for the particular biological model/application being simulated on the device. In some embodiments, the first region may have a depth that is deeper than the second and/or third region(s). For example, the first region has a depth of no less than about 50 μm to no more than about 500 μm, no less than about 50 μm to no more than about 400 μm, no less than about 50 μm to no more than about 300 μm, no less than about 50 μm to no more than about 290 μm, no less than about 50 μm to no more than about 280 μm, no less than about 50 μm to no more than about 270 μm, no less than about 50 μm to no more than about 260 μm or no less than about 50 μm to no more than about 250 μm. In such examples, the second and/or third region(s) may have a depth of no less than about 15 μm to no more than about 100 μm, no less than about 20 μm to no more than about 90 μm, no less than about 25 μm to no more than about 80 μm, no less than about 25 μm to no more than about 70 μm, no less than about 25 μm to no more than about 60 μm, or no less than about 25 μm to no more than about 50 μm, relative to the top surface of the support layer.
In various embodiments, the second cell type (e.g., immune cells) is capable of moving (or migrating) to the interaction zone only through the spaces between the array of barriers (or array of microstructures).
In various embodiments, the first and second cell types are different cell types. In various embodiments, the different regions/compartments may each contain a single cell population or a coculture of cell types (e.g., fibroblasts and macrophages; cancer-associated fibroblasts (CAFs) and tumour-associated macrophage (TAMs)).
In various embodiments, the first and second cell types may be of different sizes.
In various embodiments, the first cell type is a tumour cell. The first cell type may be derived from a human (e.g., a human sample and/or a patient-derived cell line) or from a non-human mammal/an animal model (e.g., derived from a syngeneic mouse model).
In various embodiments, the first cell type may be co-cultured with one or more other cell types different from the first cell type. The one or more other cell types may be a cell in a tumour microenvironment (TME). For example, the one or more other cell type may be a fibroblast (e.g., a cancer-associated fibroblast (CAF)). In one instance, a tumour microenvironment cell type (e.g., fibroblasts) may be seeded in the first region via the first seeding port (prior to seeding the first cell type e.g., in the form of tumour spheroids) such that the tumour microenvironment cell type may adhere to the spaces in between the array of barriers (e.g., artificial pillar barriers) and form a more physiologically accurate environment for the first cell type e.g., in the form of tumour spheroids.
In various embodiments, the second cell type is an immune cell or a combination of different classes/types of immune cells. For example, the second cell type may be a lymphocyte including T cells such as a CD8+ T cell/a cytotoxic T cell, and a Natural Killer (NK) cell. The second cell type may be derived from a human (e.g., a human sample and/or a patient-derived cell line) or from a non-human mammal/an animal model (e.g., derived from syngeneic mouse model).
In various embodiments, the second cell type may be co-cultured with one or more other cell types different from the second cell type. The one or more other cell types may be a cell in a tumour microenvironment (TME). For example, the one or more other cell type may be one or more of a macrophage (e.g., a tumour-associated macrophage (TAM)), an effector T cell (e.g., a helper T cell/a CD4 T cell, a memory T cell), or a regulatory T cell or a combination thereof.
In various embodiments, the first cell type may be in singularized or spheroid forms. The first cell type may also be in the form of cell aggregates. Accordingly, in various embodiments, the device disclosed herein may be configured to allow loading and compacting of (single) cells into a cellular mass, and/or to trap cellular masses larger than a single cell (e.g., via the array of barriers or microstructures).
In various embodiments, the array of barriers/structures (or array of microstructures) may have an arrangement that resembles a curved shape, a non-polygonal shape and/or a shape that is devoid of sharp corners and/or edges. For example, the arrangement may resemble an overall ellipsoidal shape, circular shape, oval shape or part thereof.
In various embodiments, the array of barriers (or array of microstructures) may be organised in a radial manner, for example in a radiating fashion from a common point of radiation e.g., a common center. That is, in various embodiments, when the array of barriers is organised in a radial manner, the barriers may share the common point of radiation from which the pillars appear to emanate from and the barriers may each be disposed at a distance with respect to the common point of radiation. When organised in a radial manner, the barriers/microstructures may be further arranged in radiating rows, each row being perceptibly distinguished from the other rows. For example, adjacent barriers/microstructures within a row may be separated by a substantially fixed/constant distance from each other and the value of this fixed distance may vary from one row to another such that it is possible to perceptibly make out one row from another. Additionally or alternatively, each row may be sufficiently spaced out/apart from each other such that it is possible to perceptibly identify different groupings of the barriers/microstructures as different rows. The radiating rows may each have a shape that resembles an arc of a full or partial circle such that the microstructures/barriers in each row appears to line the circumference of the arc. Accordingly, in various embodiments, the spaces between adjacent rows also resemble an arc of a full or partial circle.
In various embodiments, the array of barriers may be organised in a manner where the distances between the barriers are uniform, or non-uniform e.g., the array of barriers may be organised in a manner where there is a gradient of distances between the barriers from the outermost row of barriers (furthest from the interaction zone) to the innermost row of the barriers (closest to the interaction zone). For example, when the array of barriers or microstructures are organised in a radial manner, the barriers in the outermost row may be spaced apart at a distance of no less than about 25 μm, no less than about 20 μm, no less than about 15 μm, no less than about 10 μm, no less than about 9 μm, no less than about 8 μm, no less than about 7 μm, no less than about 6 μm or no less than about 5 μm; and the barriers in the innermost row may be spaced apart at a distance of no more than about 15 μm, no more than about 10 μm, no more than about 9 μm, no more than about 8 μm, no more than about 7 μm, no more than about 6 μm or no more than about 5 μm. Consequently, the barriers within the innermost row may be spaced apart at a distance much lesser than the distance in the outermost row; and the barriers within the intermediate rows (between outermost row and innermost row) may be spaced apart at distances falling between those of the outermost row and innermost row e.g., based on graduated increments or decrements. Likewise, the array of barriers may also be organised in a manner where there is a gradient of distances between adjacent rows of barriers (row-to-row distance). For example, the row-to-row distances may change from the outermost rows of barriers (furthest from the interaction zone) to the innermost rows of the barriers (closest to the interaction zone). It will be appreciated that other combinations of non-uniform distances may also be applied.
In various embodiments, the distances between the barriers within a row and/or between different/adjacent rows may be determined based on a size distribution of the second cell type. The distances between the barriers within a row and/or between different/adjacent rows may also be determined based on an attempt to artificially mimic barriers naturally found between tissue types within a body (e.g., an organism) or based on the extent to which a user desires to challenge the ability of the second cell type (e.g., an immune cell) to migrate towards a target (or a first cell type, e.g., a tumour cell). The distances between the barriers within a row and/or between different/adjacent rows may further also be determined based on the intrinsic properties of the second cell type, e.g., size, migration characteristics, deformability. The various embodiments described above may advantageously provide selective permeability at a radial micropillar structure.
In various embodiments, the array of barriers (or array of microstructures) may be organised as substantially concentric rows of microstructures. In various embodiments, the concentric rows are substantially circular in shape. When the array of barriers is organised as substantially concentric rows of microstructures, the microstructures may share a common center and the microstructures may each be disposed at a distance with respect to the common center. In various embodiments, the microstructures/barriers within each row may have substantially the same distance or are substantially equidistant from the common center. In various embodiments, organising the array of barriers as substantially concentric rows of microstructures may be a form or a subset of organising the array of barriers in a radial manner. In various embodiments, when the microstructures are organised in substantially concentric rows (e.g. the rows are substantially circular in shape or part of a circle), the respective/adjacent rows are separated from one another, for example, by a distance of no less than about 10 μm, a distance of no less than about 20 μm, a distance of no less than about 30 μm, a distance of no less than about 40 μm, a distance of no less than about 50 μm, no less than about 60 μm, no less than about 70 μm, no less than about 80 μm, no less than about 90 μm or no less than about 100 μm. The distances between concentric rows may be uniform or non-uniform. In various embodiments, the space created between the concentric rows of microstructures may usefully provide space for the device to hold tumor microenvironment materials in the vicinity of the interaction zone of the first region. Therefore, concentric rows of microstructures may at least partially line the boundaries of the third region (or each of the plurality of third regions), for example to separate a third region from another third region, to separate a third region from the first region, and/or to separate a third region from the second region such that the second cell type has to migrate/move across the respective rows of microstructures when transiting from one region to another. In various embodiments, the concentric rows of microstructures may be further arranged such that the distance between the microstructures in the row closest to the first region is smaller than the distance between the microstructures in the row furthest from the first region, i.e., the distances between the microstructures are non-uniform across different rows (although the distances between the microstructures within the same row may be uniform). Further, in various embodiments, the size or dimensions of the microstructures in the row closest to the first region may be smaller than the size or the dimension of the microstructures in the row furthest from the first region. In various embodiments, size may be defined as the amount of estate space occupied on the support layer and may directly be correlated to the volume, cross-sectional area etc. of the microstructures.
In various embodiments, the array of barriers (or array of microstructures) may be organised in a grid-like manner. For example, the spaces existing between the barriers/microstructures appear to look like a network of substantially perpendicular lines intersecting one another. The array of barriers may be further organised in a manner where the distances between the barriers are uniform. For example, the barriers may be spaced apart at a distance of no more than about 5 μm, no more than about 10 μm, no more than about 15 μm, no more than about 20 μm or no more than about 25 μm. It will be appreciated that other distances may also be applied. In various embodiments, the distances between the barriers may be determined based on a size distribution of the second cell type. The distances between the barriers may also be determined based on an attempt to artificially mimic barriers naturally found between tissue types within a body (e.g., an organism) or based on the extent to which a user desires to challenge the ability of the second cell type (e.g., an immune cell) to migrate towards a target (or a first cell type, e.g., a tumour cell). The distances between the barriers may further also be determined based on the intrinsic properties of the second cell type, e.g., size, migration characteristics, deformability.
The organisation of the array of microstructures may be a combination of two or more of the different organisational patterns described above.
In various embodiments, the array of barriers (or array of microstructures) comprises pillars. In other words, the device may comprise micropillars. For example, the pillars have a non-polygonal cross-section (e.g., circular, oval, elliptic, oblong). Alternatively, the pillars may also have a polygonal cross-section, such as a parallelogram like a square, rectangle or rhombus. In various embodiments, the height of the pillars is the same as the height of the support layer of the device. Thus, depending on the depth of region at which the pillars are disposed (e.g., when they line the boundaries of the regions), the heights of the pillars may vary accordingly. The pillars may have a height of no less than about 50 μm to no more than about 500 μm, no less than about 50μm to no more than about 400 μm, no less than about 50 μm to no more than about 300 μm, no less than about 50 μm to no more than about 290 μm, no less than about 50 μm to no more than about 280 μm, no less than about 50 μm to no more than about 270 μm, no less than about 50 μm to no more than about 260 μm, no less than about 50 μm to no more than about 250 μm; no less than about 15 μm to no more than about 100 μm, no less than 20 μm to no more than about 90 μm, no less than 25 μm to no more than about 80 μm, no less than 25 μm to no more than about 70 μm, no less than 25 μm to no more than about 60 μm, no less than 25 μm to no more than about 50 μm, or no less than about 15 μm and no more than about 100 μm. In various embodiments, the pillars lining the boundaries of the first region have heights that are greater than the pillars lining the boundaries of a third region and/or second region.
In various embodiments, the device may further comprise one or more obstacles configured to or arranged to direct fluid flow in a passive manner. In one example, an obstacle may be arranged between the second region and the array of barriers (or array of microstructures). The obstacle may be arranged to follow the shape of the array of barriers at its circumference (e.g., a curved shape, a non-polygonal shape and/or a shape that is devoid of sharp corners and/or edges). In various embodiments, barriers which direct fluid flow may also be present at other places within the device.
In various embodiments, the device is symmetrical about a plane (or a central axis) that cuts across the centre of the support layer and/or seeding layer.
In various embodiments, the first and second regions are symmetrical about a same/common line of symmetry. In various embodiments, the first region, second region and third region (or each of the third regions) are symmetrical about a same/common line of symmetry. In various embodiments, the first region, second region, and/or third region (or each of the third regions) has/have only a single line of symmetry.
In various embodiments, the device may be manufactured using soft lithography. To improve the resolution of the microstructures within the device as well as the depth of each region, in various embodiments, the manufacturing method may alternatively be converted to an injection molding microfabrication method.
An exemplary device is described below for illustrative purposes. In one example, the device may be a microfluidic device that allows for the observation of cellular interactions, which has the components of: (a) a fluidic chamber partitioned by a radial micropillar barrier into multiple compartments, with said barrier exhibiting a gradient of distances between micropillars and (b) a loading and cell retrieval mechanism which utilizes centrifugal force to seed and recover cells of interest. In the above exemplary embodiments, the microfluidic device may comprise micropillars that act as a trap for the tumor cell mass, and additionally present a barrier to the free movement of lymphocytes. Also, the device may be configured to allow cells to be loaded into the micropillar trap and retrieved from the device via centrifugal forces.
There is also provided a chip (or plate) device comprising a plurality of the devices (i.e., microfluidic devices) provided herein. For example, the chip may be a multiplexed chip comprising a plurality of the devices arranged in an A×B array, where A and B are integers that are independently selected from 1 to 50. Advantageously, in various embodiments, the device design (i.e., the microfluidic device sign) may be scaled to conform to standard industrial dimensions (e.g., the size of a standard microscope size or a well plate). That is, the various embodiments of the device disclosed herein may provide a design with built-in amenability to scale-up using at least two types of multiplexed devices in the form of a chip or plate. Therefore, in various embodiments, the chip (or plate) device may enable massively parallel in vitro screens of immune-tumour interactions. These screens can be conducted with a library of immunotherapy drug candidates to determine which patients respond well to those drugs.
In various embodiments, the chip is be designed and dimensioned to fit conventional centrifugation machines such that a longitudinal axis or a line of symmetry of each of the device (or the regions within the device) contained within the chip approximately passes through the axis of rotation of the centrifugation machines (to allow the centrifugal force to direct the cells contained therein to one end of the region it is contained in) when the chip is securely positioned in the machines for centrifugation. In various embodiments, when the chip is rotated 180 degrees and securely repositioned in the centrifugation machines for centrifugation, the longitudinal axis or the line of symmetry of each of the device (or the regions within the device) still approximately passes through the axis of rotation (to allow the centrifugal force to direct the cells contained therein to the other end of the region it is contained in) of the centrifugation machines.
In various embodiments, usefully, a scaled-up (chip or plate) version of the device (i.e., microfluidic device) may be mass produced and made accessible in the form of a commercial consumable, along with a protocol/instructions on how to successfully use the consumable. Such consumables may also be further tagged for traceability purposes.
Various embodiments of the device disclosed herein may provide a design with built-in amenability to scale-up using at least two types of multiplexed devices in the form of a chip or plate.
There is also provided a method of interacting a first cell type with a second cell type (or studying interactions between a first cell type with a second cell type, or screening a sample comprising a first cell type). The method may include providing the first cell type in a first region of the device provided herein; applying an external force (e.g. rotational force or centrifugal force) to the first cell type to direct the first cell type to an interaction zone of the first region; providing the second cell type in the second region of the device provided herein; and allowing the first cell type to migrate from the first region to the interaction zone of the second region for interaction with the second cell type in the interaction zone, with or without application of an external force. Advantageously, in various embodiments, the method provides an in-vitro method for e.g., screening individual tumor sample responses to new and existing combinatorial cancer therapies involving human immune cells.
In various embodiments, the method of studying interactions of a first cell type with a second cell type, includes providing a device disclosed herein that is structurally configured to facilitate the desired interactions between the cell types. Such device, for example, may comprise at least a first region configured to hold a first cell type, a second region configured to a second cell type, and an array of microstructures disposed between the first and second regions, wherein the first region is in fluid communication with the second region, and wherein the array of microstructures is configured to selectively allow movement of the second cell type from the second region to an interaction zone that is at least partially disposed within the first region, to allow interaction of the first cell type and the second cell type in the interaction zone. Thereafter, the method may then involve seeding the first cell type in the first region of the device. The method may also comprise applying a first external force to direct the first cell type to the interaction zone. A second cell type may be seeded in the second region of the device. The seeding of the second cell type may be carried out after the first cell type has been directed to the interaction zone by the external force. After the second cell type has been seeded, the second cell type may be allowed to migrate from the second region (e.g., across the microstructures) to the interaction zone for interaction with the first cell type in the interaction zone. Such migration of the second cell type and interaction of the second cell type with the first cell type may then be monitored.
In various embodiments, the first cell type is provided in the first region of the device and/or the second cell type is provided in the second region of the device manually/by hand (e.g., by applying an external force or by passive flow) or by using an automatic handler (e.g., a bioprinter).
In various embodiments, applying a first external force to the device to, for example, load the first cell type (e.g., tumor cells, in the form of tumor fragments for example) may be performed through a first centrifugation. In various embodiments, after the first cell type is seeded into the device, by applying the external forces (e.g., rotational or centrifugal forces), the first cell type may be directed away from the first seeding port of the device and towards the first interaction zone that is at least partially located in the first region.
In various embodiments, monitoring migration of the second cell type and/or interaction of the second cell type with the first cell type comprises monitoring with an imaging device/apparatus, for example, observing the device via microscopy (e.g., confocal microscopy).
In various embodiments, the method further comprises capturing one or more images (e.g., microscopic images) comprising the array of barriers and/or the first region and/or the interaction zone or parts thereof with an image capturing device, for example a microscope enabled with video/image capturing capabilities (e.g., linked to a computer).
In various embodiments, the method further comprises tracking/monitoring cell properties/characteristics/behaviour/interaction such as cell viability, cell count, distances travelled (e.g., distances travelled by the second cell type), cytokine/chemokine activity/measurement, clustering/recruitment of cells (e.g., recruitment of the second cell type), cell morphology, cell motility and/or speed of migration or the like. For example, the cell viability of the first cell type and/or the second cell type may be tracked/monitored. As another example, cell count of the second cell type located within the array of barriers and/or located at the interaction zone may be tracked/monitored. The tracking/monitoring may be performed via computer vision, fluorescence measurements and/or immunoassays (e.g., ELISPOT). As one example, the tracking/monitoring may be performed via an automated computer vision algorithm using MATLAB that identifies key cellular interactions. In various embodiments of the method disclosed herein, if fluorescence microscopy is relied on for the generation of quantitative results, fluorescent signals can vary slightly between testing runs and can be prone to bleaching. This may have an effect on the accuracy of the automated analysis results. Therefore, in various embodiments, improved immunostaining fluorophores may be used and the exposure of the tagged cells to incident light from the microscope used may be limited.
In various embodiments, the method further comprises applying a second external force to direct cells present within the interaction zone away from the interaction zone for retrieval and analysis. This may include applying an external force of the same nature of the first external force to the device for a second time to direct cells located in the first region (e.g., the first cell type and/or recruited second cell type) away from the interaction zone and/or towards a first seeding port of the device, and/or to direct non-recruited (e.g., non-migratory) second cell type away from the second region and/or towards a third region/a third port of the device. In this step, the device may be oriented such that the device is rotated by no less than about 90 degrees and no more than about 270 degrees, no less than about 100 degrees and no more than about 260 degrees, no less than about 110 degrees and no more than about 250 degrees, no less than about 120 degrees and no more than about 240 degrees, no less than about 130 degrees and no more than about 230 degrees, no less than about 140 degrees and no more than about 220 degrees, no less than about 150 degrees and no more than about 210 degrees, no less than about 160 degrees and no more than about 200 degrees, no less than about 170 degrees and no more than about 190 degrees, or about 180 degrees from its original position/orientation, for example the device may be rotated about an axis that is substantially perpendicular to the line of symmetry of the device/first region/second region/third region or substantially parallel to (or lying in) a plane that cuts across the centre/midline of the device (e.g., including the support layer and/or seeding layer).
Accordingly, in various embodiments, the method comprises a method of centrifuging that is able to direct cells toward and away from the first interaction zone (or the tumor trap area) for the purposes of loading and retrieval respectively. That is, in various embodiments, the method disclosed herein may use centrifugal forces to position tumour masses in microfluidic devices for the purposes of trapping such cellular fractions and for the subsequent purpose of cell retrieval. The method provided herein may further involve a second centrifugation for the purposes of removing the first and second cell types (e.g., tumor cells and immune cells respectively) e.g., after a pre-determined period, for example. In various embodiments, the external forces (e.g., centrifugal forces) used in the second centrifugation may be in opposite direction to the external forces in the first centrifugation, relative to interaction zone.
In various embodiments, the method further comprises extracting cells from a first port and/or a third port of the device manually/by hand or by using an automatic handler (e.g., a bioprinter). That is, the method allows for retrieval of cells. Advantageously, various embodiments of the method disclosed herein may allow automated processing and recovery (via centrifugation and automated handler processing). In various embodiments, the method further comprises analysing the extracted cells to identify genetic biomarkers. For example, in various embodiments, the method involves recovering cell populations of interest (e.g., effective or non-effective tumor infiltrating lymphocytes cells) that can be further studied for biomarker identification.
In various embodiments, the method comprises seeding the immune cell types and subsequently, after the experiment is finished, removing those immune cell types from the same port. In various embodiments, the tumor cell types are seeded and removed from the same port, and any immune cell types that have migrated into the tumor spheroid itself will be removed along with the tumor spheroid. In various embodiments, there is a possibility of seeding the immune cells from an immune cell recovery port. This cell recovery port may in fact be located on “the same side” as the tumor seeding port in terms of its proximity but does not mean that the two cell types would be seeded from the same port. That being said, in various embodiments, there is a possibility of seeding microenvironment materials such as a tumor microenvironment cell type (e.g., fibroblasts) from the tumor seeding port (prior to seeding the tumor spheroid itself) such that they adhere to the spaces in between the artificial pillar barriers and form a more physiologically accurate environment for the tumor spheroid. In this regard, in some embodiments, the device used for the method may further comprises one or more third regions which are capable of holding microenvironment materials (e.g., tumour microenvironment cell type or material) and the method comprises seeding microenvironment materials into the one or more third regions. The arrangement of the third region may be such that the third region is in fluid communication with the first and second regions, and the array of microstructures comprises microstructures disposed between the third region and the first region. In various embodiments, the array of microstructures also comprise microstructures disposed between the third region and the second region. Accordingly, after seeding of the microenvironment materials, these materials may be sandwiched between the first region (or first cell type after it has been seeded) and the second region (or second cell type after it has been seeded) so that the second cell type has to migrate through these materials to reach the first cell type for interaction. The seeding of the microenvironment materials in the third region may be carried out prior or after the seeding of the first cell type in the first region. The seeding of the microenvironment materials in the third region may also be carried out prior to or after seeding of the second cell type in the second region. In various embodiments, the seeding of the microenvironment materials in the third region is prior to applying the first external force (e.g., centrifugal force) to direct the first cell type to the interaction zone.
An exemplary method is described below for illustrative purposes. In one example, the method may be a method (or workflow) by which tumor-immune cell interactions can be screened at high-throughput, comprising the following steps: (a) automated depositing of tumour-related cellular matter into sealed devices; (b) centrifugation of multiple devices so as to aggregate and position cellular matter; (c) automated deposition of immune cells into the devices; (d) observation of tumour-immune interactions with microscopy and automated image analysis of said interactions to identify targeted interactions; (e) centrifugation of multiple devices to recover distinct cell populations; and (f) automated removal of cell populations for further analysis.
Various embodiments of the present disclosure may provide a combination of a device and a process/method which combines a unique microfluidic design with centrifugation, resulting in a unique way to isolate and combine two distinct cell types: tumor and immune cells, and separating them with a physical barrier, or a combination of physical and biological barriers by creating an artificial TME, in a single device such that their interactions—particularly tumor-infiltrating lymphocyte behavior—can be studied in an explicit manner.
Example embodiments of the disclosure will be better understood and readily apparent to one of ordinary skill in the art from the following discussions and if applicable, in conjunction with the figures. It should be appreciated that other modifications related to structural and biological changes may be made without deviating from the scope of the invention. Example embodiments are not necessarily mutually exclusive as some may be combined with one or more embodiments to form new exemplary embodiments. The example embodiments should not be construed as limiting the scope of the disclosure.
The examples describe a platform suitable for use for in-vitro chemotherapeutic and immunotherapeutic combinatorial drug testing using at least two different types of cells: tumor cells and immune cells (e.g., CD8+ T cells, Natural Killer (NK) cells, etc.) with the option of including other cell types to create a complex three-dimensional microenvironment surrounding a tumor aggregate and more accurately physiologically mimic the tumor microenvironment in vivo. The platform is comprised of a physical device, an assay protocol, and an analysis procedure designed to be amenable to process automation and scale-up.
In the following examples, a microfluidic device (i.e., a compartmentalized tumor trapping microfluidic device) for observation of immune-tumor interactions is provided. In some of the following examples, additional compartmentalized layers are provided for placement of cell-laden hydrogels or artificial extra-cellular matrix material surrounding the trapped tumor to create complex three-dimensional microenvironments.
A three-dimensional illustration of an exemplary physical device is shown in
In
In
The fluidic layer 102 ranges between 25 and 200 μm in depth depending on the type of device and the region/zone/compartment area (i.e., depending on the application and type of cells being used, and the region/zone/compartment area) (see
Some examples of the different types of microstructures arrays and resulting barriers are shown in
Returning to
A three-dimensional illustration of another exemplary physical device 300 is shown in
The microfluidic device 300 is a physical device designed specifically with four or more different zones or compartments, each with a specific purpose: a tumor cell seeding port zone which can also function as a cell recovery port zone, an immune cell seeding port zone, a controlled tumor-immune cell interaction and observation zone, and a variable number of tumor microenvironment (TME) material (such as hydrogels, cellular material, or both) seeding port zones which can also function as cell recovery port zones.
Similar to the device 100 described with reference to
Also similar to the device 100 described with reference to
In
These concentric rows of micropillars keep the tumor spheroid in place, and allow for the organized placement (via centrifugation) of layers of tumor-microenvironment materials. There are spaces between each of the micropillar structures, allowing for effector (immune) cells migrating from the outer regions of the tumor-immune interaction zone to the innermost region (where the tumor spheroid is located) to pass through.
An example of the arrangement of the micropillar barriers in the tumor-immune interactions zone creating two distinct layers surrounding the tumor spheroid regions is shown in
In the following examples, processes (involving the devices e.g., device 100 of
In the following first example, the protocol and workflow described was developed specifically for the device 100 described with reference to
In the following second example, the protocol and workflow described was developed specifically for the device 300 described with reference to
In the examples described above e.g., with reference to
In the examples described above, microscopic images captured at a frequency of 2 to 4 images per hour are recorded throughout the duration of the experiment and analyzed using a computer vision algorithm written specifically to identify, track and flag cellular interactions of interest, namely: tumor cell viability, immune cell viability and immune cell count (within the barrier and inside the tumor trap area).
In the examples described above, the algorithm is designed to simplify and streamline the analysis process (e.g., see
In the examples described e.g., with reference to
Two different exemplary multiplexed devices (based on the device 100 described with reference to
Another exemplary multiplexed device (based on the device 300 described with reference to
In
Advantageously, various embodiments of the method/process (including the use of the device) disclosed herein provide an in vitro assay that can be scaled-up easily and used for high-throughput screening that would allow pharmaceutical companies and clinical researchers for example to study patient responses to specific therapies without posing danger to the patient, as well as provide an early-stage screening for healthcare providers for example to determine which patients may or may not respond well to a particular combination therapy.
Various embodiments of the present disclosure provide an OncoMiMIC (Onco-Multi-Metric Immuno-Combinatorial) testing platform. In various embodiments, the OncoMiMIC testing platform is advantageously adapted for forming a complex 3D Tumor Microenvironments (TME). In various embodiments, such platforms may be referred to as an OncoMiMIC-CTM (Onco-Multi-Metric Immuno-Combinatorial Complex Microenvironment) testing platform.
Various embodiments of the present disclosure provide a testing platform that comprises a class of microfluidic chips that are inexpensive. In various embodiments of the present disclosure, the comprehensive in-vitro testing platforms can screen individual tumor sample responses to new and existing combinatorial cancer therapies, thereby potentially reducing the cost of identifying and testing target drugs in the development and discovery stage.
In various embodiments of the present disclosure, the OncoMiMIC-CTM testing platform advantageously allows for an end user to place, in a precise and well-organized manner, one or several surrounding layers of tissues or artificial extra-cellular matrix (ECM) material around a tumor sample, thereby creating a more physiologically relevant, complex, three-dimensional microenvironment for the tumor. Then, in various embodiments, similar to the way in which the OncoMiMIC platform functions, the OncoMiMIC-CTM platform allows for the interaction of effector cells (immune cells) to interact with this complex tumor structure.
In various embodiments of the present disclosure, the testing platforms provide platform flexibility in that the platforms are amenable to automation and scale-up for an industry requiring high-throughput and big data.
In various embodiments of the testing platform disclosed herein, the platform provides an easily scalable design, which allows the platform to be highly amenable to automation. It has been recognized that a single screening test can take place in an individual device (e.g., see
It will be appreciated by a person skilled in the art that other variations and/or modifications may be made to the embodiments disclosed herein without departing from the spirit or scope of the disclosure as broadly described. For example, in the description herein, features of different exemplary embodiments may be mixed, combined, interchanged, incorporated, adopted, modified, included etc. or the like across different exemplary embodiments. The present embodiments are, therefore, to be considered in all respects to be illustrative and not restrictive.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10202003690W | Apr 2020 | SG | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/SG2021/050227 | 4/22/2021 | WO |
