WO2024254436A2 - Glande de prostate humaine bio-modifiée et modèle de maladie - Google Patents

Glande de prostate humaine bio-modifiée et modèle de maladie Download PDF

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Publication number
WO2024254436A2
WO2024254436A2 PCT/US2024/032983 US2024032983W WO2024254436A2 WO 2024254436 A2 WO2024254436 A2 WO 2024254436A2 US 2024032983 W US2024032983 W US 2024032983W WO 2024254436 A2 WO2024254436 A2 WO 2024254436A2
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channel
layer
microfluidic device
microns
cells
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WO2024254436A3 (fr
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Linan Jiang
Cynthia MIRANTI
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University of Arizona
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University of Arizona
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions

Definitions

  • the disclosed invention is generally in the field of microfluidic devices.
  • MCC monolayered cell culture
  • Microfluidic systems have already enabled various biological studies.
  • Multicompartmental 3D microfluidic cell culture devices often termed as organs-on-chips (OoC)
  • organs-on-chips have been introduced to address limitations of in vitro modeling. So far, numerous OoC devices have been proposed to reproduce various functions of organs and tissues, such as lung, liver, kidney, intestine, gut, and bone.
  • OoC devices have been proposed to reproduce various functions of organs and tissues, such as lung, liver, kidney, intestine, gut, and bone.
  • an organ-on-a-chip system for in vitro modeling of a human prostate gland is yet to be developed. Therefore, a model system that recreates the architectural features to recapitulate the biology and physiology of the human prostate gland in vivo is needed.
  • Microfluidic devices and organ-on-a-chip platforms based on microfluidic devices are described herein.
  • an in vitro human prostate disease model based on the disclosed microfluidic devices is described in the Examples.
  • the microfluidic devices include multiple individual compartments separated by porous membranes. Co-cultures of tissue types involved in prostate diseases are implemented while providing physiological environment and communications among the tissue types.
  • the microfluidic device includes multiple individual compartments separated by porous membranes (see, e.g., Figure 2b).
  • the microfluidic device includes at least two layers and at least one porous membrane that is adjacent to each of and between two neighboring layers.
  • the two adjacent layers of the microfluidic device can be arranged in any suitable positions, such as in parallel with each other.
  • the device may contain more than two layers where each layer is separated from its adjacent layer by a porous membrane.
  • the two layers (when the microfluidic device includes two layers) or at least two layers (when the microfluidic device includes more than two layers) of the microfluidic device each include one or more channels, such as one channel or two or more channels.
  • the microfluidic device includes a feed area (see, e.g., Figure 3a, 1110), an exit area (see, e.g., Figure 3a, 1120), and a material exchange area (see, e.g., Figure 3a, 1130).
  • the channel or each channel (when two or more channels are present) in a layer is aligned with the channel or each channel (when two or more channels are present) in its neighboring layer or each neighboring layers, such that a region of the channel or each channel in the layer overlaps with a region of the channel or each channel in its neighboring layer or each neighboring layer, and a region of the channel or each channel in the layer does not overlap with any region of the channel or each channel in its neighboring layer or each neighboring layer.
  • a first channel in a first layer is aligned with a second channel in a second, adjacent layer, such that a first region (see, e.g., Figure lb, 1040a) of the first channel in the first layer overlaps with a second region (see, e.g., 1050a not visible in Figure lb) of the second channel in the second layer, and a third region (see, e.g., Figure lb, 1060a) of the first channel does not overlap with any region of the second channel in the material exchange area.
  • a first region see, e.g., Figure lb, 1040a
  • a second region see, e.g., 1050a not visible in Figure lb
  • a third region see, e.g., Figure lb, 1060a
  • the channel or each channel in a layer may include more than one region that overlaps with the channel or each channel in its neighboring layer or each neighboring layer, such as two or more overlapping regions, for example, two, three, four, or five, or more than five overlapping regions (see, e.g., Figure la, 1040a, 1040b, 1040c, 1040d, 1040e, 1040f, and 1040g); and/or more than one region that does not overlap with any region in its neighboring layer or each neighboring layer, such as two or more non-overlapping regions, for example, two, three, four, or five, or more than five non-overlapping regions (see, e.g., Figure la, 1060a, 1060b, 1060c, 1060d, 1060e, 1060f, and 1060g).
  • the microfluidic device can include one or more reservoirs in fluid connection with one or more channels in at least one layer of the microfluidic device, optionally positioned
  • each channel in the microfluidic device can have any suitable shape, as long as it can be aligned with a channel in the neighboring layer such that one or more overlapping region(s) and one or more non-overlapping regions are achieved with the channel in the neighboring layer, within the material exchange area of the microfluidic device.
  • Suitable shapes for each channel in the microfluidic device include, but are not limited to, straight and meander, such as a bent, curved, or serpentine shape.
  • each channel in the microfluidic device is a straight or meandering channel, such as a channel having a bent, curved, or serpentine configuration.
  • the shape of the channel or each channel in a layer may be the same as or different from the channel or each channel in its neighboring layer.
  • the channel or each channel in a first layer of a microfluidic device may be different from the channel or each channel in a second layer.
  • a first channel in the first layer of a microfluidic device can be substantially straight, while a second channel in the second layer is a meandering channel, such as a channel having a bent, curved, or serpentine configuration; or vice versa.
  • a first channel in the first layer is a meandering channel, such as a channel having a bent, curved, or serpentine configuration, while each of a second and third channel in the second layer can be substantially straight.
  • a first channel in the first layer can be substantially straight, which each of a second and third channels is a meandering channel, such as a channel having a bent, curved, or serpentine configuration.
  • a first channel in the first layer of a microfluidic device is meandering channel, such as a channel having a bent, curved, or serpentine configuration;
  • a second channel in the second layer can be substantially straight, and
  • a third channel in the third layer is a meandering channel, such as a channel having a bent, curved, or serpentine configuration.
  • the pore sizes and/or porosities of the membranes between different layers are selected based on the types of cells in the channel(s) to permit diffusion of substances secreted by the cells between the adjacent channels, or to allow cell invasion through the pores to the adjacent channels, or a combination thereof.
  • the substances secreted by the cells are soluble in water or an aqueous solution, such as an aqueous buffer.
  • secreted substances by cells include, but are not limited to PSA, TGF , PDGF, TNFa, exosomes, Wnts, cytokines, FGF, KGF, and HGF.
  • the pore sizes and porosities of each porous membrane can be the same or different, optionally they are different.
  • a disease model can be constructed using the microfluidic devices described herein by (i) placing a first type of cells in a first channel of the first layer, and (ii) flowing a first cell culture medium through the first channel.
  • the first type of cells may be placed at any location within the first channel.
  • the first type of cells is placed on a bottom surface of the porous membrane that functions as the ceiling of the channel in the first layer (see, e.g., Figure 3c, normal endothelial cells placed on bottom surface 313 of the porous membrane 310).
  • a second type of cells is placed in the first channel and/or a second channel in the second layer, and in step (ii) a second cell culture medium flows through the first and/or second channel.
  • no cells are placed in the second channel in a second layer of the microfluidic device.
  • the microfluidic device includes a third channel in the first or second, or a third layer
  • a third type of cells is placed in the third channel
  • step (ii) flowing a third cell culture medium through the third channel is placed in step (ii)
  • the microfluidic device includes a third channel in the first, second, or third layer
  • no cells are placed in the third channel.
  • more than one type of cells and/or tissues can be placed in a single channel.
  • at least two types of cells are placed in the first channel of the first layer.
  • the cells being placed in the channel(s) of the microfluidic device can be in any suitable form.
  • the cells can be placed in the chan nek s ) of the microfluidic device in either 2D form (for example, in the form of a monolayer) or a 3D form (for example, in the form of organoids, spheroids, one or more types of cells embedded in hydrogel or ex vivo tissue, etc.).
  • Each channel in the microfluidic device may contain a different type of cells or the same type of cells, or a combination thereof.
  • microfluidic devices described herein can be used in drug screening.
  • steps (i) and (ii) as described above are followed along with (iii) flowing a drug solution or suspension, or flowing a medium containing one or more carriers containing one or more drugs (also referred to herein as “drug carrier medium”), through the first channel and/or the second channel.
  • the drug carrier medium contains a suitable medium and one or more drug carriers that contain a suitable concentration of drug(s), such as a high concentration of drugs, where the drugs can be enclosed/embedded in the carriers, such as small-volume capsules and/or beads.
  • the drug carrier can be formed by any suitable materials, such as gelatin, collagen, Matrigel, agarose, alginate, etc.
  • the method may further include after step (iii), flowing a cell culture medium (without any drugs) through the first channel and/or the second channel.
  • a different or the same drug solution or suspension, or drug carrier medium can be flown through the channel(s) of each layer.
  • a different drug solution or suspension refers to a solution or suspension that contains a different drug or different combination of drugs, a different drug concentration, etc.
  • the same drug solution or suspension refers to a solution or suspension that contains the same drug(s), each drug having the same concentration.
  • a different drug carrier medium refers to a medium that contains a different carrier, a different carrier concentration, a different drug or combination of drugs, a different drug loading, etc.
  • the same drug carrier medium refers to a medium that contains the same carrier, same drug(s), same carrier concentration, and same drug loading.
  • a first drug solution/suspension or a first drug carrier medium flows through the channels of the first layer, and a second, different drug solution/suspension or drug carrier medium flows through the channels of the second layer.
  • a drug solution/suspension or drug carrier medium flows through the channels of the first layer, and the same drug solution/suspension or drug carrier medium flows through the channels of the second layer.
  • the dosing regime, time, frequency, etc. for flowing the drug solution/suspension or drug carrier medium in each layer may be the same or different.
  • the drug solution/suspension or carrier medium flows through the channels of the first layer at a different dosing regime, time, and/or frequency compared to the drug solution/suspension or drug carrier medium flowing through the channels of the second layer.
  • the devices described herein can be used to form an organ-on-a chip platform, such as one which mimics the prostate gland and/or portions of the tissues therein.
  • the prostate gland on a chip can be in fluid communication, such as via flow convection or diffusion, with one or more additional and different organs on a chip to study the interactions between different organs, optionally during the development of and/or possible treatments of diseases therein.
  • Figures la-lc show an exemplary microfluidic device containing two flow channels with selectively overlapping regimes separated by a porous membrane.
  • Figure la shows a top view of the microfluidic device, illustrating the alignment of the two channels.
  • Figure lb shows a zoom-in view of the device shown in Figure la.
  • Figure 1c shows a cross-sectional view at location (A- A) of the device shown in Figure la.
  • Figure 2a shows the fabrication of a PDMS microchannel replicated from mater molds for an exemplary microfluidic device.
  • Figure 2b shows an exploded view of the exemplary microfluidic device.
  • Figure 3a shows an exemplary microfluidic device for a human prostate disease model.
  • Figure 3b shows an A-A cross-sectional view of the microfluidic device of Figure 3a, where a vasculature system including normal endothelial cells (such as in the form of a monolayer) and immune cells are placed in the first channel; stroma including fibroblast and smooth muscle cells (such as in the form of stroma embedded in 3D ECM hydrogel) are placed in the second channel; and normal prostate epithelial cells or cancer cells (such as in the form of a monolayer or organoids or in 3D Matrigel, or a combination thereof).
  • a vasculature system including normal endothelial cells (such as in the form of a monolayer) and immune cells are placed in the first channel; stroma including fibroblast and smooth muscle cells (such as in the form of stroma embedded in 3D ECM hydrogel) are placed in the second channel; and normal prostate epithelial cells or cancer cells (such as in
  • Figure 3c shows that in this exemplary human prostate disease model, stroma were converted to CAFs by cancer cells; cancer cells invaded into stroma and further invaded into bloodstream; and immune cells responded to the invasion by cancer cells.
  • These models can be used in drug treatment tests, where a drug solution or suspension can flow through either one of the channels or a combination thereof.
  • Figure 4a shows an exemplary microfluidic device for cancer in situ model.
  • Figure 4b shows an A-A cross-sectional view of the microfluidic device of Figure 4a, where stroma including fibroblast and smooth muscle cells are placed in the first channel; epithelium and locally induced tumor are placed in the second channel; and the third channel is for administering treatment, such as Dox, IPTG, or DHT, or a combination thereof.
  • Figure 4c shows that the treatment induced local tumor in epithelium; tumor cells converted normal stroma to CAFs, and tumor invasion, following the cell placement and drug treatment as shown in Figure 4b.
  • Figure 5 a shows an exemplary microfluidic device for modeling the effect of tumor on healthy/normal epithelium.
  • Figure 5b shows an A-A cross-sectional view of the microfluidic device of Figure 5 a, where stroma including fibroblast and smooth muscle cells are placed in the first channel; cancer cells are placed in the second channel; and normal epithelium are placed in the third channel.
  • Figure 5c shows the co-culture of cancer cells with normal epithelium and stroma, operate under conditions (e.g. flow rate) and selected membrane pore size to allow diffusion of cancer cell secretion (0.8um or 8um pore membrane) and invasion of cancer cells (8um pore membrane), following the cell placement as shown in Figure 5b.
  • This exemplary model can be used to study the interaction between tumor cells and microenvironment, stroma conversion to CAF by cancer cells with or without physical contact, the effect of tumor cells and surrounding normal epithelium cells, and/or changes in normal epithelium.
  • Figure 6a shows an exemplary system including two connected exemplary microfluidic devices for modeling prostate tumor metastasis to a secondary organ (for example a bone chip).
  • Figure 6b shows an A-A cross-sectional view of the prostate chip and a B-B cross- sectional view of the bone chip in the system of Figure 6a.
  • Figure 7a shows an exemplary microfluidic device containing channel arrays for a high-throughput drug screening disease model, where two sets of channel arrays are separated by a porous membrane, with overlapping alignment regimes between the upper channel array and the lower channel array, and a uniform depth for the upper and lower channels.
  • Figure 7b shows an A-A cross-sectional view of the microfluidic device of Figure 7a.
  • Figure 7c shows a B-B cross-sectional view of the microfluidic devices in the system of Figure 7a.
  • This exemplary device can be used for high-throughput drug screening disease model targeting tumor/stroma/stroma-tumor interaction, such as by injecting culture medium with or without drug into the lower and/or upper channel arrays; delivering the drug into upper or lower channel array to specifically target tumor, or stroma, or stroma-tumor interaction or delivering a combination of drugs into one of or both channel arrays; and scheduling drug concentration, dose, time, frequency, etc. for each channel array or for individual channels in each array.
  • Figure 8a shows another exemplary microfluidic device containing channel arrays for a high-throughput drug-screening disease model, where two sets of channel arrays are separated by a porous membrane, with overlapping alignment regimes between the upper channel array and the lower channel array.
  • the upper channels have uniform depth, and the lower channels contain wells at the overlapping regimes.
  • Figure 8b shows an A-A cross-sectional view of the microfluidic device of Figure 8a.
  • Figure 8c shows a B-B cross-sectional view of the microfluidic devices in the system of Figure 8a.
  • This exemplary device can be used for high- throughput drug screening disease model targeting tumor/stroma/stroma-tumor interaction, such as by placing tumor cells (or stroma) on the membrane surface in the lower channels; placing stroma (or tumor cells) on the membrane surface in the upper channels; injecting suspension of drug-containing capsules/particles in culture medium in the lower channels, such that the drug capsules/particles are collected in the wells, where the drug-containing capsules/particles dissolve in culture medium in a time-dependent manner to release drug locally in the wells; and varying medium flows in the top and/or bottom channels to result in various local drug concentration in the overlapping regimes.
  • Figures 9a- 9d show an exemplary disease model by placing stroma and tumor cells in separate channels of an exemplary microfluidic device with no initial direct physical contact.
  • Figure 9a shows the exemplary microfluidic device where tumor cells and normal stroma are placed in separate channels on the porous membrane.
  • Figure 9b shows a zoom-in view of the channels of the microfluidic device of Figure 9a.
  • Figure 9c shows invasion of tumor cells into stroma, and stroma conversion into CAFs in the tumor microenvironment, following the cell placement as shown in Figure 9b.
  • Figure 9d shows a zoom-in view of the channels of the microfluidic device of Figure 9c.
  • Figures 10a- lOd show another exemplary disease model by placing stroma and tumor cells in the channel of an exemplary microfluidic device with direct physical contact.
  • Figure 10a shows the exemplary microfluidic device where tumor cells are placed on a monolayer of stroma, with direct physical contact.
  • Figure 10b shows a zoom-in view of the channels of the microfluidic device of Figure 10a.
  • Figure 10c shows enhanced invasion of tumor cells into neighboring space facilitated by invading CAFs converted by tumor cells, following the cell placement as shown in Figure 10b.
  • Figure lOd shows a zoom-in view of the channels of the microfluidic device of Figure 10c.
  • Figures 1 la-11c show simulations of the convection-diffusion flow in an exemplary microfluidic device.
  • Figure I la is a schematic illustrating a 2D physical model used for simulations depicting transport of signaling molecules through the porous membrane due to diffusion from the secreting layer to the receiving layer channel. The gradient map in the two channels illustrates concentration changes from high (black) to low (white).
  • Figure 1 lb is a graph showing the 2D simulation result of streamwise normalized concentration profiles of secreted molecules along both channels under various flowrates, at a distance 40pm away from membrane, with the corresponding rainbow maps of planar concentration distributions.
  • Figure 11c is a graph showing the simulation result of cross-stream normalized concentration profiles of secreted molecules, at mid-distance between channel inlet and outlet, under various flowrates. All results were obtained from a device with 8p m-pore membrane, except curves in dotted lines in Figure l ib and Figure 11c, which were obtained from a device with 0.8um-pore membrane at a flow rate of 3O l/hr.
  • Figure 12 is a graph showing migration rate of invading cancer cells for various initial cell plating conditions based on measured time-dependent distance between the edge of original plating channel of cancer cells and the farthest cancer cell migration front in the neighboring channel.
  • the filled symbols 22Rvl/BHP or C4-2/BHP
  • Figures 13 a- 13c are graphs showing cytokine production in a prostate cancer-on-chip model as shown in the exemplary disease model of Figures lOa-lOd.
  • Figure 13a shows the levels of TGFP in the straight channel.
  • Figure 13b shows the levels of TGF in the serpentine channel.
  • Figure 13c shows the levels of PDGF in the serpentine channel.
  • the data were measured in conditioned medium by ELISA. PDGF was below detectable levels (not shown) in the medium collected from straight channels of both experiments.
  • the conditioned medium was collected every two days starting at Day 7, from straight and serpentine channels of 8pm- pore devices.
  • microfluidic devices and organ-on-a-chip platforms based on these microfluidic devices are described herein.
  • an in vitro human prostate disease model based on the disclosed microfluidic devices is described in the Examples.
  • the microfluidic device includes multiple individual compartments separated by porous membranes (see, e.g., Figures la-lc, 2b, 3a-3c, 4a-4c, 5a-5c, 6a, 6b, 7a-7c, and 8a-8c).
  • the disclosed microfluidic devices are preferably optical transparent, accommodating standard microscopy imaging and in-situ monitoring. Using color-tagged cells, activities and responses of cells in co-cultures can be tracked.
  • the models disclosed herein advances disease study (such as prostate disease study) using a complete organ involving complex tissue types and appropriate microenvironment.
  • disease study such as prostate disease study
  • the microfluidic devices and models built therefrom offer high spatial resolution, high sensitivity, and controllability, and can be used for high-throughput screening in drug disco very.
  • co-cultures of various types of tissue involved in a disease of interest can be implemented while providing physiological environment and communications among the different tissue types.
  • prostate disease related models such as prostate tumor progression and invasion into heathy tissues within the organ, as well as into a different organ, can be investigated using various configurations of the disclosed microfluidic devices.
  • model based on the disclosed microfluidic devices promotes studies to dissect the interplay mechanisms in the disease progress, such as effects of microenvironments, stroma's role in tumor progression, and immune system responses. Accordingly, the model can be used to study and validate biological pathways in disease progression.
  • the model can be used for drug discovery. Desired local drug concentration and concentration distribution profile can be realized by balancing convectiondiffusion among the compartments through fine-tuning culture medium flow rates and drug administering scheme.
  • the microfluidic device disclosed herein includes at least two layers and a porous membrane, each layer contains a channel.
  • the first layer of the microfluidic device can have any suitable orientation relative to the second layer of the microfluidic device.
  • the first layer of the microfluidic device is in parallel with the second layer of the microfluidic device.
  • the porous membrane is typically adjacent to each of and between the first and second layers of the microfluidic device.
  • the microfluidic device 1000 includes two layers (a first layer 100 and a second layer 200) in parallel with each other, and a first porous membrane 300 adjacent to each of and between the first layer 100 and second layer 200.
  • the porous membrane sandwiched between the first and second layers of the microfluidic device separates the flows in each channel, while allows communication and interaction between cells in one or more of the channels of the microfluidic device.
  • the porous membrane also allows diffusion of solutes from one or more channels into neighboring channels.
  • the microfluidic device has more than two layers, each layer containing a channel.
  • the microfluidic device has three layers, four layers, five layers, six layers, seven layers, eight layers, nine layers, ten layers, etc.
  • each pair of adjacent layers are separated by a porous membrane.
  • the microfluidic device includes three layers and two porous membranes (see, e.g., Figures 3a, 3b, 4a, and 4b).
  • the first layer and second layers are in parallel with each other, and the first porous membrane is sandwiched between the first and second layers.
  • the third layer is positioned on top of the second layer and in parallel with the first and second layers, and the second porous membrane is sandwiched between the third and second layers.
  • the third channel of the third layer can have any suitable orientation relative to the channels of the first and second layers.
  • a region of the third channel of the third layer overlaps with a region of the second channel of the second layer.
  • the microfluidic device 1300 includes three layers (a first layer 130, a second layer 230, and a third layer 430), and two porous layers (a first porous membrane 330a and a second porous membrane 330b).
  • the first porous membrane 330a (such as having an average pore diameter of 0.4-10 microns) is adjacent to each of and between the first layer 130 and second layer 230, and the second porous membrane 330b (such as having an average pore diameter of 0.4-8 microns) is adjacent to each and between the second layer 230 and third layer 430.
  • one or more layers of the microfluidic device contains more than one channel.
  • the channels in the same layer can have any suitable relative orientations.
  • two channels are contained in one layer and the two channels are in parallel with each other (see, e.g., Figures 5a and 5b).
  • the channel in the layer adjacent to such layer which is typically separated by a porous membrane, can have any suitable orientation relative to each of the channels of such layer.
  • one layer of the microfluidic device contains two channels (i.e., first and second channels), and the layer adjacent to this layer, separated by a porous membrane, contains one channel (i.e., third channel); the third channel may have one or more regions overlap with one of the first and second channels or both the first and second channels.
  • each of the layers and porous membrane of the microfluidic device is formed of an inert, transparent material.
  • transparent material suitable for forming the layers and porous membranes include, but are not limited to, polydimethylsiloxane (“PDMS”), polyester, acrylic, nylon, polycarbonate, polylactic acid, polyethylene terephthalate glycol, polyether ether ketone, polyether ketone ketone, polypropylene, and glass, and combinations thereof.
  • the layers and porous membranes can be formed of the same material or at least two different materials.
  • the microfluidic device includes two or three layers and one or two porous membranes, where each of the layers is formed of PDMS or glass and each of the porous membrane is formed of polyester or glass. Glass can cut down on oxygen to create a reduced oxygen environment, based on the hypothesis that both the prostate gland, prostate tumors, and the bone have relatively low oxygenation relative to other organs.
  • each of one or more channels of the microfluidic device has a fluid inlet and a fluid outlet, and optionally one or more channels of the microfluidic device does not have a fluid inlet and/or a fluid outlet.
  • each channel of the microfluidic device includes a fluid inlet and a fluid outlet. The inlet and outlet are configured such that fluid flows in a certain direction through the channel. The flow direction of fluid in each channel can be the same or different.
  • the micro fluidic device includes two channels, each including a fluid inlet and a fluid outlet.
  • the inlet and outlet are configured such that fluid flows in a direction through the first channel and fluid flows in a second direction through the second channel.
  • the first direction and the second direction can be the same or different.
  • the first direction is the same as the second direction.
  • two or more of the microfluidic devices can be fluidly connected with each other, such as via at least one channel of each of the connecting microfluidic devices, for studying interactions between two organs and/or cancer metathesis at a secondary organ.
  • a first microfluidic device 2100 is fluidly connected to a second microfluidic device 2200, where the first channel 2110 of the first microfluidic device is connected to the first channel 2210 of the second microfluidic device via a conduit 1910.
  • the microfluidic devices in a platform can be connected in any other desired way.
  • the porous membrane (when one porous membrane is included in the microfluidic device) or each porous membrane (when two or more porous membranes are included in the microfluidic device) has a top surface and a bottom surface.
  • the top and bottom surfaces of the porous membrane can function as the floor and the ceiling of the channels in the two adjacent layers of the microfluidic device, respectively.
  • the top surface 301 of the porous membrane 300 functions as the floor of the second channel 201 in the second layer 200
  • the bottom surface 303 of the porous membrane 300 functions as the ceiling of the first channel 101 in the first layer 100.
  • a first porous membrane 310a is sandwiched between a first layer 110 and a second layer 210, while a second porous membrane 310b is sandwiched between the second layer 210 and a third layer 410.
  • the top surface 311a of the porous membrane 310a functions as the floor of the second channel 211 in the second layer 210
  • the bottom surface 313a of the porous membrane 310a functions as the ceiling of the first channel 111 in the first layer 110.
  • the top surface 31 lb of the porous membrane 310b functions as the floor of the third channel 411 in the third layer 410
  • the bottom surface 313b of the porous membrane 310 functions as the ceiling of the second channel 211 in the first layer 210.
  • cells and/or tissues when placed inside the channel(s), they can be placed on the top and/or bottom surfaces of each pours membrane sandwiched between two adjacent layers to optionally facilitate transport of the cells and/or secreted solutes by the cells between neighboring channel(s) (see, e.g., Figure 3c and 5c).
  • each layer of the microfluidic device includes one channel (see, e.g., Figure 2b, 101 and 201), two channels (see, e.g., Figure 5b, 131, 231a, and 231b), or more than two channels, such as an array of channels (see, e.g., Figure 7b, 141a, 141b, 141c, etc. and Figure 7c, 241a, 241b, 241c, etc.).
  • the microfluidic device 1000 includes a first layer 100 containing a first channel 101 and a second layer 200 containing a second channel 201.
  • the microfluidic device 1300 includes a first layer 130 containing a first channel 131, and the second layer 230 includes a second channel 231a and a third channel 231b.
  • the first layer 140 and second layer 240 each include an array of channels (141a, 141b, 141c, etc. in the first layer 140 and 241a, 241b, 241c, etc. in the second layer 240).
  • the porous membrane or each porous membrane (when two or more porous membranes are included) of the microfluidic device contains a plurality of pores.
  • the pores in the plurality of pores of the porous membrane or each porous membrane can have the same diameter or two or more different diameters.
  • the pores in the plurality of pores of the porous membrane or each porous membrane can have an average diameter ranging from about 0.4 micron to about 15 microns, from about 0.4 micron to about 10 microns, from about 0.4 micron to about 8 microns, from about 0.4 micron to about 5 microns, from about 0.4 micron to about 1 microns, from about 0.8 micron to about 15 microns, from about 1 micron to about 15 microns, from about 0.8 micron to about 10 microns, from about 1 micron to about 10 microns, from about 0.8 micron to about 8 microns, from about 0.8 micron to about 5 microns, from about 2 micron to about 15 microns, or from about 5 micron to about 15 microns, such as from about 0.4 micron to about 15 microns, from about 0.4 micron to about 10 microns, from about 0.4 micron to about 8 microns.
  • the pores in the plurality of pores of the porous membrane have different diameters.
  • the pores of different diameters can be in different groups/area, or with any desired arrangement on the porous membrane.
  • a first group of pores in the plurality of pores has a first average diameter ranging from about 0.4 micron to 10 micron, from about 0.4 micron to about 8 microns, from about 0.4 micron to about 5 microns, or from about 0.4 micron to about 1 microns
  • a second group of pores in the plurality of pores has a second average diameter ranging from about 0.8 micron to about 15 microns, from 0.8 micron to about 10 microns, from about 1 micron to about 15 microns, or from about 1 micron to about 10 microns, and wherein the first diameter is different from the second diameter.
  • the pores in the plurality of pores of the first porous membrane can have the same or different average diameter compared to the pores in the plurality of pores of the second porous membrane.
  • the pores in the plurality of pores of the first porous membrane has the same average diameter of the pores in the plurality of pores of the second porous membrane, where the average diameter ranges from about 0.4 micron to about 15 microns, from about 0.4 micron to about 10 microns, from about 0.4 micron to about 5 microns, from about 0.4 micron to about 1 microns, from about 0.8 micron to about 15 microns, from about 1 micron to about 15 microns, from about 0.8 micron to about 10 microns, from about 1 micron to about 10 microns, from about 0.8 micron to about 8 microns, from about 0.8 micron to about 5 microns, from about 2 micron to about 15 microns, or from about 5 micron to about 15 microns.
  • the pores in the plurality of pores of the first porous membrane has a first average diameter ranging from about 0.4 micron to 15 micron, from about 0.4 micron to 10 micron, from about 0.4 micron to about 8 microns, from about 0.4 micron to about 5 microns, or from about 0.4 micron to about 1 microns
  • the pores in the plurality of pores of the second porous membrane has a second average diameter ranging from about 0.8 micron to about 15 microns, from 0.8 micron to about 10 microns, from about 1 micron to about 15 microns, or from about 1 micron to about 10 microns, and wherein the first diameter is different from the second diameter.
  • the microfluidic device includes a feed area (see, e.g., Figure 3a, 1110), an exit area (see, e.g., Figure 3a, 1120), and a material exchange area (see, e.g., Figure 3a, 1130).
  • a region of the channel of the first layer overlaps with a region of the channel of the second layer.
  • the channel of the first layer and the channel of the second layer can have one overlapping region or two or more discrete overlapping regions.
  • the overlapping region or each of the two or more overlapping regions can have any suitable shape and dimensions.
  • each channel can be the same or different, where each overlapping region can be in planar (squared, circular, etc.), or in 3D (dome, cone, truncated cone, cylinder, etc.).
  • the channel of the first layer also has one region or two or more regions that do not overlap with any region of the channel of the second layer.
  • the channel or each channel (when two or more channels are present) in a layer is aligned with the channel or each channel (when two or more channels are present) in its neighboring layer or each neighboring layers, such that a region of the channel or each channel in the layer overlaps with a region of the channel or each channel in its neighboring layer or each neighboring layer, and a region of the channel or each channel in the layer does not overlap with any region of the channel or each channel in its neighboring layer or each neighboring layer.
  • a first channel in a first layer is aligned with a second channel in a second, adjacent layer, such that a first region (see, e.g., Figure lb, 1040a) of the first channel in the first layer overlaps with a second region (see, e.g., 1050a, not visible in Figure lb) of the second channel in the second layer, and a third region (see, e.g., Figure lb, 1060a) of the first channel does not overlap with any region of the second channel in the material exchange area.
  • a first region see, e.g., Figure lb, 1040a
  • a second region see, e.g., 1050a, not visible in Figure lb
  • a third region see, e.g., Figure lb, 1060a
  • a first channel in a first layer is aligned with a second channel and a third channel in a second, adjacent layer, such that a first region (see, e.g., Figure 5a, 1340a) of the first channel in the first layer overlaps with a second region (see, e.g., 1350a not visible in Figure 5a) of the second channel in the second layer, a third region (see, e.g., Figure 5a, 1360a) of the first channel overlaps with a fourth region (see, e.g., 1370a not visible in Figure 5a) of the third channel in the second layer, and a fifth region (see, e.g., Figure 5a, 1380a) of the first channel does not overlap with any region of the second and third channel in the material exchange area.
  • a first region see, e.g., Figure 5a, 1340a
  • a second region see, e.g., 1350a not visible in Figure 5a
  • a third region see,
  • a second channel in a second layer (which is between a first and third layer) is aligned with a first channel in the first layer and with a third channel in the third layer, such that a first region (see, e.g., Figure 3a, 1140a) of the second channel overlaps with a second region (see, e.g., 1150a not visible in Figure 3a) of the first channel, the first region (see, e.g., Figure 3a, 1140a) of the second channel also overlaps with a fourth region (see, e.g., 1170a not visible in Figure 3a) of the third channel, and a fifth region (see, e.g., Figure 3a, 1180a) of the second channel does not overlap with any region of the first and third channels in the material exchange area.
  • the region of such channel that overlaps with the channels in the two adjacent layers can be the same or different.
  • the regions of the second channel that overlap with the first and third channels are the same.
  • the regions of the second channel that overlap with the first and third channels may be different.
  • each channel (see, e.g., Figure 7b, 141a) in a first array of channels in a first layer is aligned with each channel in a second array of channels in a second layer, such that the channel in the first array has a region (see, e.g., Figure 7a, 1440a, 1440b, 1440c, 1440d, 1440e, etc.) that overlaps with a region (see, e.g., 1450a, 1450b, 1450c, 1450d, 1450e, etc. in Figure 7a, which overlaps with 1440a, 1440b, 1440c, 1440d, 1440e, etc.
  • the channel or each channel in a layer may include more than one region that overlaps with the channel or each channel in its neighboring layer or each neighboring layer, such as two or more overlapping regions, for example, two, three, four, or five, or more than five overlapping regions (see, e.g., Figure la, 1040a, 1040b, 1040c, 1040d, 1040e, 1040f, and 1040g); and/or more than one region that does not overlap with any region in its neighboring layer or each neighboring layer, such as two or more non-overlapping regions, for example, two, three, four, or five, or more than five non-overlapping regions (see, e.g., Figure la, 1060a, 1060b, 1060c, 1060d, 1060
  • one or more layers of the microfluidics device contains a multiplechannel array (see, e.g., Figures 7a and 8a).
  • the array of channels in each layer can have any suitable relative orientations.
  • the channels are in parallel with each other in the array.
  • the channels in the layer adjacent to such layer which is typically separated by a porous membrane, can have any suitable orientation relative to the channel array of such layer.
  • one layer of the microfluidic device contains a 16-channel array, and the layer adjacent to this layer, separated by a porous membrane, contains a 16-channel array; the two channel arrays may have 16x16 overlapping regions.
  • the layer containing multiple channels can be arranged in any suitable position or orientation in the microfluidic device.
  • the number of the channels in the channel array in the first layer can be the same as or different from the number of channels in the channel array in the second layer.
  • the geometry and dimensions of the channels in the channel array in the first layer can be the same as or different from the channels in the channel array in the second layer.
  • the channels in a channel array in a first, lower layer (see, e.g., Figures 8b and 8c, 150) of a microfluidic device are fluidly connected to reservoirs at the overlapping regions of the channels (see, e.g., Figure 8c, 1540a, 1540b, 1540c, 1540d, and 1540e).
  • the reservoirs are in fluid communication with the channel and can be positioned in any suitable position relative to the channel, such as fluidly connected to the floor of the channel optionally at each overlapping region of the channel (see, e.g., Figure 8c, 1540a, 1540b, 1540c, 1540d, and 1540e).
  • reservoirs can be configured to retain 3D tissues, such as tumor spheroids or organoids, in the co-culture of the disease model, and/or to retain drug capsules, releasing drug at an extended period of time with local concentration gradient.
  • the reservoirs can have any suitable geometry and dimensions, and can be selected based on the specific function. For example, the reservoirs has a volume larger than the volume of the carrier(s) in the drug carrier medium and a height that is larger than the height of the channel to which it is connected, to facilitate deployment of the carriers when the drug carrier medium flown over/through.
  • each of the layers and porous membranes of the microfluidic device can have any suitable height, depending on the specific applications of the microfluidic device.
  • each of the layers and porous membranes of the microfluidic device has a height ranging from about 50 microns to about 4 mm, from about 100 microns to about 4 mm, from about 50 microns to about 2 mm, from about 100 microns to about 2 mm, from about 50 microns to about 1 mm, from about 100 microns to about 1 mm, from about 10 microns to 1 mm, from about 10 microns to about 4 mm, from about 10 microns to about 2 mm, from about 50 microns to about 500 microns, from about 100 microns to about 200 microns, from about 10 microns to 100 microns, or from about 15 microns to about 50 microns.
  • the layers and porous membranes can have the same height or at least two different heights.
  • the microfluidic device includes two or three layers and one or two porous membranes, where each of the layers has a height ranging from about 50 microns to about 500 microns, and each of the porous membranes has a height ranging from about 15 microns to about 50 microns.
  • the layers can have the same height or at least two different heights and the porous membranes can have the same height or at least two different heights.
  • a layer of larger height such as a height of 2 mm or 4 mm, is particular useful for applications using hydrogels with organoids, tumor spheroids, embedded cells, etc. b. Channel Dimensions and Shapes
  • the channels of the microfluidic device can have any suitable dimensions and shapes, and can be arranged in any suitable relative positions, as long as desired overlapping region(s) and non-overlapping region(s) of two channels in neighboring layers can be achieved in the material exchange area.
  • Suitable shapes for each channel in the microfluidic device include, but are not limited to, straight and meander, such as a bent, curved, or serpentine shape.
  • each channel in the microfluidic device is independently a straight or meandering channel, such as a channel having a bent, curved, or serpentine configuration.
  • one channel is substantially straight (along the length) and the other channel can have a wavy shape, such that one or more regions of the straight channel overlap with one or more regions of the wavy channel; and one or more regions of the straight channel do not overlap with any region of the wavy channel, in the material exchange area.
  • a first channel in the first layer can be wavy (see, e.g., Figure 5a, 131), and a second and third channels in the second layer can each be substantially straight (along the length) (see, e.g., Figure 5a, 231a and 231b), such that one or more regions of the first channel overlap with one or more regions of each of the second and third channels; and one or more regions of the first channel do not overlap with any region of the second and third channels, in the material exchange area.
  • the second channel (see, e.g., Figure 3a, 211) in the second layer can each be substantially straight (along the length) and the first (see, e.g., Figure 3a, 111) and third (see, e.g., Figure 3a, 411) channels in the first and third layers, respectively, can be wavy, such that two or more regions of the second channel overlap with two or more regions of each of the second and third channels, and two or more regions of the second channel do not overlap with any region of each of the first and third channels, in the material exchange area.
  • wavy shapes that can be used to form the wavy channel include, but are not limited to, serpentine, sinewave, and triangle-wave, and combinations thereof.
  • the width of the channel can be uniform or varied.
  • each channel in the microfluidic device can independently has a width ranging from about 50 microns to about 4 mm, from about 100 microns to about 4 mm, from about 50 microns to about 2 mm, from about 100 microns to about 2 mm, from about 50 microns to about 1 mm, from about 100 microns to about 1 mm, from about 10 microns to 1 mm, from about 10 microns to about 4 mm, from about 10 microns to about 2 mm, from about 50 microns to about 500 microns, from about 100 microns to about 200 microns, from about 10 microns to 100 microns, or from about 15 microns to about 50 microns; and/or a length ranging from 5 mm to 100 mm, from 5 mm to 90 mm, from 5 mm to 80 mm, from 5 mm to 70 mm, from 5 mm to 60 mm, from 5 mm to 50 mm, from 5 mm to 40 mm, from 5 mm
  • each channel can be uniform along the length of the channel or varied along the length of the channel.
  • the height of the channel is uniform along the length of the channel.
  • the height of the channel can vary.
  • the width of the channel can the uniform or be varied along the length of the channel.
  • the width of one or more channels in the microfluidic device is larger at the overlapping region(s) than the rest of the channel.
  • Organ-on-a-chip platforms based on the microfluidic devices are also described.
  • the platforms can be formed using any of the microfluidic devices described herein.
  • the platform may be formed using any one, or two or more microfluidic devices described herein.
  • at least one channel of each microfluidic device can be in fluid connection with another microfluidic device in the platform.
  • the platform contains at least one type of tissue in at least one channel of the microfluidic device.
  • the organ-on-a-chip platform contains two or more types of tissue in a single channel.
  • one channel contains fibroblasts and smooth muscle cells in suitable ratio to mimic what is seen in vivo, such as a ratio of approximately 80% fibroblasts to 20% smooth muscle cells.
  • the platform includes a microfluidic device described herein and at least two different types of tissues, where the two different types of tissues can be in the same channel or different channels. The amount of tissue in each channel can be assessed based on the number of the cells that are initially placed within the channel, and by measuring markers known to be associated with each cell type at the end.
  • two different types of tissues are in the channels of the microfluidic device, wherein at least 70%, at least 80%, or at least 90% of the first type of tissue is in a first channel, and at least 70%, at least 80%, or at least 90% of the second type of tissue is in a second channel.
  • two different types of tissues are in the channels of the microfluidic device, wherein at least 90% of the first type of tissue and at least 90% of the second type of tissue are in the same channel.
  • three different types of tissues are in the channels of the microfluidic device, wherein at least 70% of the first type of tissue is in a first channel, at least 20% of the second type of tissue is in the first channel, and at least 90% of the third type of tissue is in a second channel.
  • three different types of tissues are in the channels of the microfluidic device, wherein at least 90% of the first type of tissue is in a first channel, at least 90% of the second type of tissue is in a second channel, and at least 90% of the third type of tissue is in a third channel.
  • four different types of tissues are in the channels of the microfluidic device, wherein at least 90% of the first type of tissue is in a first channel, at least 90% of the second type of tissue is in a second channel, at least 90% of the third type of tissue is in the second channel, and at least 90% of the fourth type of tissue is in a third channel.
  • microfluidic devices and organ-on-a-chip platforms formed therefrom are illustrated in Figures la-lc, 2b, 3a-3c, 4a-4c, 5a-5c, 6a, 6b, 7a-7c, and 8a-8c.
  • each of these exemplary microfluidic devices can be modified to incorporate other configurations (e.g., overall device configuration, such as arrangement of layers, shape, size, etc.; channel configuration, such as channel arrangement, shape, size, etc.; inlet and outlet arrangement, etc.), other dimensions, other orientations (e.g., channel orientation, inlet and outlet orientation, etc.), and other tissue types, depending on the specific application.
  • other configurations e.g., overall device configuration, such as arrangement of layers, shape, size, etc.; channel configuration, such as channel arrangement, shape, size, etc.; inlet and outlet arrangement, etc.
  • other orientations e.g., channel orientation, inlet and outlet orientation, etc.
  • any one of the channels in the devices exemplified in Figures la-lc, 2b, 3a-3c, 4a-4c, 5a-5c, 6a, 6b, 7a-7c, and 8a-8c can be modified to be in a straight/wavy shape or in a different wavy shape.
  • any one of the channels in the devices exemplified in Figures la-lc, 2b, 3a-3c, 4a-4c, 5a-5c, 6a, 6b, 7a-7c, and 8a-8c can be modified to include at least two wavy channels and one straight channel. This can facilitate visualization of migration in the different channels.
  • the first channel may be loaded with epithelial/cancer cells
  • the second channel (which is in the middle layer) can be loaded with fibroblasts/smooth muscle cells
  • the third channel can be loaded with endothelial cells (see, e.g., Figure 3c).
  • the third channel can have a shorter height compared to the first and second channels to create increased sheer stress/flow velocity to mimic vasculature physiology.
  • Such an arrangement mimics prostate organ/tumor architecture.
  • any one of the devices exemplified in Figures la-lc, 2b, 3a-3c, 4a-4c, 5a- 5c, 6a, 6b, 7a-7c, and 8a-8c can be modified to include three or more channels in three layers, having any other shapes and/or in any other arrangements.
  • organ-on-chip platforms exemplified in Figures 6a and 6b can be modified to use any other and/or additional microfluidic devices described herein for study of interaction between any other organs or disease models.
  • microfluidic devices and organ-on-a-chip platforms disclosed herein can be produced using methods known in the art.
  • the microfluidic devices can be prepared using photolithography, a specific example of which is described in the Example section below.
  • the organ-on-a-chip platforms can be prepared by placing cells in the respective channels of the microfluidic devices and flowing culture mediums through the channels containing the cells.
  • the specific types of cells and their placement in the channels of the microfluidic devices depend on the specific disease or organ being studied.
  • An exemplary procedure for preparing prostate cancer models based on the disclosed microfluidic device is described in the Example section below.
  • microfluidic devices have a wide variety of uses in biomedical applications, such as for constructing a disease model (e.g., bio-Engineered human prostate disease model), studying disease mechanisms, and studying how disease influences other organs, etc.
  • a disease model e.g., bio-Engineered human prostate disease model
  • studying disease mechanisms e.g., studying how disease influences other organs, etc.
  • Prostate disease related models such as prostate tumor progression and invasion into heathy tissues within the organ, as well as into different organs, can be investigated using various configurations of the devices. Role of infectious agents in prostatitis or mechanisms of benign prostate hyperplasia development can be interrogated. Additionally, the disclosed microfluidic devices can be used to dissect the interplay mechanisms in various prostate diseases, including the effects of microenvironmental factors such as stroma, vascular, sensory neurons, and immune system responses. Physical components, such as variations in matrix, pH, and oxygen tension can be modeled in prostate disease states. The model can be used to study and validate genetic and biological pathways in disease progression.
  • microfluidic devices can be used for drug discovery, drug screening, and drug dose regimen test, Desired local drug concentration and concentration distribution profiles can be realized by balancing convection-diffusion among the compartments through fine-tuning cell culture medium flow rates and drug administering schemes.
  • the devices described herein can be used for high throughput screening in drug discovery.
  • the microfluidic device disclosed herein can be used for constructing a disease model.
  • the method generally includes (i) placing cells in one or more channels of the microfluidic device and (ii) flowing suitable culture medium through each channel containing the cells to form tissue.
  • the cells may be placed in each of the channels of the microfluidic device, or selectively placed in one or more channels of the microfluidic device but not all.
  • the cells being placed in one channel can be a single type or a mixture of different types, in 2D form or 3D form.
  • the cells can be placed in the channel(s) of the microfluidic device in a 2D form (for example, in the form of a monolayer) or a 3D form (for example, in the form of organoids, spheroids, one or more types of cells embedded in hydrogel or ex vivo tissue, etc.).
  • a 2D form for example, in the form of a monolayer
  • a 3D form for example, in the form of organoids, spheroids, one or more types of cells embedded in hydrogel or ex vivo tissue, etc.
  • Each channel in the microfluidic device may contain a different type of cells or the same type of cells, or a combination thereof.
  • each channel in the same layer may contain the same or different type of cells; or at least one channel contains a type of cells that is different from the other channels in the same layer.
  • at least one channel in one or more layers of the microfluidic device does not contain any cells.
  • a different type of cells is placed in step (i) in the channel(s) of each layer of a microfluidic device.
  • a first type of cells is placed, and in the channel(s) of one or more layers, no cells are placed.
  • the placement of cells in the microfluidic device can be in any suitable pattern, and can be selected based on the specific cells, tissues, disease models, dimensions of the channels, configuration of the channels, etc.
  • the microfluidic device contains two layers, and each layer contains one or more channels.
  • a different type of cells is placed in each channel of each layer (see, e.g., Figure 5b, a first type of cells is placed in the first channel 131 of the first layer 130 of the microfluidic device 1300, a second type of cells is placed in the second channel 231a of the second layer, and a third type of cells is placed in the third channel 231b of the second layer; and see also, e.g., Figure 9a, a first type of cells is placed in the first channel 161 of the first layer 160 of the microfluidic device 1600, and a second type of cells is placed in the second channel 261 of the second layer 260).
  • more than one type of cells can be placed in a single channel.
  • one or more types of cells are placed (see, e.g., Figure 10a, tumor cells placed on a layer of stroma in the first channel 171), and in the channel(s) of the second layer, no cells are placed (see, e.g., Figure 10a, no cells in the second channel 271).
  • the microfluidic device contains three layers, and each layer contains one or more channels.
  • a different type of cells is placed (see, e.g., Figure 3b, a first type of cells is placed in the first channel 111 of the first layer 110 of microfluidic device 1100, a second type of cells is placed in the second channel 211 of the second layer 210, and a third type of cells is placed in the third channel 411 of the third layer 410).
  • step (i) in the channel(s) of at least one layer one or more types of cells are placed, and the channel(s) of at least one layer does not contain any cells.
  • more than one type of cells can be placed in a single channel.
  • At least one type of tissue is/are formed in the channels of the microfluidic device.
  • a different type of tissue is formed in each channel following step (ii).
  • two or more different types of cells are placed in a single channel, two or more different types of tissues are formed in the single channel following step (ii).
  • the relative amounts of the type of tissue formed in each channel can be determined at desired time points following step (ii), such as 1 day, 2 days, 3, days, 4 days or 5 days following step (ii), to determine the initially formed tissue composition.
  • the initial composition of the tissue formed in the channel is typically predominantly one type of tissue, i.e. initially at least 90% of at least one type of tissue is in the channel.
  • one or more cells placed in one channel infiltrate into the adjacent channel (which does not have the same cells placed therein in step (i)) via the pores of the porous membrane.
  • the method further includes, prior to step (i), coating the channels with a suitable matrix.
  • the matrix used for coating each channel is known and can be selected based on the specific cell types cultured in the channel. Specific examples of the matrix suitable for coating the channel prior to cell placement are described in the Examples section below.
  • the microfluidic device used for constructing a disease model includes at least two layers, each layer containing a channel, and a porous membrane sandwiched between the two layers.
  • the method includes: (i) placing one or more types of cells in the first channel and optionally one or more types of cells in the second channel; and (ii) flowing a first culture medium through the first channel and optionally a second culture medium through the second channel.
  • one channel of the microfluidic device initially contains epithelial cells and the other channel initially contains fibroblasts or smooth muscle cells, or a combination of both therein.
  • one channel of the microfluidic device initially contains normal fibroblasts and the other channel initially contains tumor cells (see, e.g., Figure 9a).
  • one channel of the microfluidic device initially has tumor cells placed on a monolayer of fibroblasts therein and another channel does not have any cells placed therein (see, e.g., Figure 10a).
  • the cancer cells and fibroblasts/smooth muscle cells can be placed in channels of any suitable position and shape.
  • step (ii) cells are cultured to form tissues under any suitable conditions.
  • step (ii) is performed at about 37 C for a time period ranging from 3 to 21 days or from 7 to 18 days.
  • each of the first and second culture media has an adequate input flow rate such that normalized concentration 10% (vol) or less of the medium, including secreted factors by cells (such as PSA, TGFP, PDGF, TNFa, exosomes, Wnts, cytokines, FGF, KGF, HGF, etc.) in the first channel diffuses through the first porous membrane into the second channel, and optionally 10% (vol) or less of the medium, including secreted factors by cells, in the second channel, diffuses through the porous membrane into the first channel.
  • other conditions such as shear stress, diffusion level, and coating/matrix, etc. are also selected to establish the appropriate physiological microenvironment for proper cell growth, cross-talks among the tissues, and other cell activities.
  • At least one type of tissue preferably two different types of tissues, is/are formed in the channels of the microfluidic device.
  • one or more cells of the tissue or each tissue are in the adjacent channel (which does not have the same cells placed therein in step (i)) (see, e.g., Figure 9b and 10b).
  • two different types of tissues are formed in the channels of the microfluidic device, wherein at least 90% of the first type of tissue is in the first channel, and at least 90% of the second type of tissue is in the second channel.
  • one or more cells of the first type of tissue is in the second channel and/or one or more cells of the second type of tissue is in the first channel.
  • the amount of tissue in each channel can be assessed using the number of cells that are initially placed within the channel, and by measuring markers known to be associated with each cell type at the end.
  • two different types of tissues are formed in the channels of the microfluidic device, wherein at least 90% of the first type of tissue and at least 90% of the second type of tissue are in the first channel, and the second channel does not have more than 10% of the first or second type of tissue.
  • one or more cells of the first type of tissue and/or one or more cells of the second type of tissue is in the second channel.
  • the two cells can have an 80/20% ratio to mimic what is seen in vivo.
  • three different types of tissues are formed in the channels of the microfluidic device, wherein at least 90% of the first type of tissue is in the first channel, at least 90% of the second type of tissue is in the first channel, and at least 90% of the third type of tissue is in the second channel.
  • one or more cells of the first type of tissue and/or one or more cells of the second type of tissue is in the second channel, and/or one or more cells of the third type of tissue is in the first channel.
  • the method further includes, prior to step (i), coating the channels with a suitable matrix, first channel with a first matrix and coating the second channel with a second matrix.
  • one layer of the microfluidic device may include two or more channels having any suitable shape and orientation relative to the other channels, or the microfluidic device includes a third layer and a second porous membrane each having any suitable shape and orientation relative to the other layers and channels, such as those described above and exemplified in the Figures.
  • any cells of a suitable type such as cells of a single type or cells of two or more different types, for example, endothelial cells, immune cells, pericytes, etc.
  • a suitable culture medium can flow therethrough in step (ii) to form a tissue and optionally one or more cells placed therein can diffuse into the adjacent channel in step (ii).
  • the culture media has an adequate flow rate such that normalized concentration of 10% (vol) or less of the medium diffuses through the porous membrane into the adjacent channel.
  • other conditions such as shear stress, diffusion level, and coating/matrix, etc. are also selected to establish the appropriate physiological microenvironment for proper cell growth, cross-talks among the tissues, and other cell activities.
  • two or more different tissues are formed in the channels of the microfluidic device, such as three different tissue types, four different tissue types, five different tissue types, etc.
  • one or more of such additional channels can be left empty (i.e., not having cells placed therein in step (i)), and one or more cells placed in an adjacent channel can infiltrate into the empty channel in step (ii).
  • the microfluidic device disclosed herein can be used for drug screening, such as to determine dosing regimen. Any drugs can be tested using the microfluidic device, such as anti-cancer agents, anti-inflammatories, growth factors, steroids, antibodies, etc.
  • the method includes step (i) placing cells in one or more channels of the microfluidic device and step (ii) flowing culture medium through one or more channels, as described above to first construct a disease model, and further include (iii) flowing a drug solution or suspension, or flowing a medium containing one or more carriers containing one or more drugs (also referred to herein as “drug carrier medium”), through one or more channels.
  • the drug carrier medium contains a suitable medium and one or more drug carriers that contain a suitable concentration of drug(s), such as a high concentration of drugs, where the drugs can be enclosed/embedded in the carriers, such as small-volume capsules and/or beads.
  • the drug carrier can be formed by any suitable materials, such as gelatin, collagen, Matrigel, agarose, alginate, etc.
  • the method may further include after step (iii), flowing a cell culture medium (without any drugs) through the first channel and/or the second channel.
  • the drug solution or suspension can be administered in channels that initially contain at least 70%, at least 80%, or at least 90% of one type of tissues (or of a combination of two or more tissues, such as a combination of smooth muscle cells and fibroblasts), or in channels that do not have cells placed therein in step (i) but may have one or more cells infiltrated therein from adjacent channels following step (ii).
  • the drug solution or suspension has a sufficient flow rate and initial concentration such that a concentration gradient is produced along the channel it is administered into due to the intake of the drugs by cells in the device.
  • the method may further include during or after step (iii), measuring cell responses to the drug.
  • cell responses that can be measured include, but are not limited to, cell death as indicated by release of solutes (such as LDH), or measured using live/dead assay or cell counting; molecular events (such as TUNEL, LC3/autophagy) by staining; loss of specific pathway signaling targeted by drug either through the use of cells reporters within the cells or by immunostaining; loss of cell adhesion (such as loss of adhesion to ECM as measured by ratio of area of attached cell layer to area of cell original surface before drug treatment (fully confluent layer); loss of cell adhesion to neighboring cells (such as measured by numbers of individual intact cell groups, where before drug treatment, the cells formed a full layer); change in cell migration rate or direction- measured by time-dependent imaging of cells; increased/decreased expressions in certain proteins as measured by ELISA, PCR, flow cytometry, immunostaining, etc.
  • the flow of the drug solution or suspension in each channel can have any suitable direction and can be the same or different. Further, drug concentration, treatment period, dosage, input channel(s) can be controlled by designing medium control flow scheme.
  • the microfluidic device can include one or more reservoirs connected to one or more channels in at least one layer of the microfluidic device.
  • the microfluidic device includes an array of reservoirs connected to each channel in the first layer, located at each overlapping region of each channel (see, e.g., Figure 8c, 1540a, 1540b, 1540c, 1540d, and 1540e) in the first layer.
  • the reservoirs are in fluid communication with the channel and can be positioned in any suitable position relative to the channel, such as fluidly connected to the floor of the channel, such as at the overlapping regions of the channel (see, e.g., Figure 8c, 1540a, 1540b, 1540c, 1540d, and 1540e).
  • the reservoirs can have any suitable geometry and dimensions, as long as it has a volume larger than the volume of the carrier(s) in the drug carrier medium and a height that is larger than the height of the channel to which it is connected, to facilitate deployment of the carriers when the drug carrier medium flown over/through.
  • the carriers in the drug carrier medium can be deployed by flowing the drug carrier medium through the channel connecting to the reservoirs at overlapping regions.
  • the carriers containing the drugs can be then retained in the reservoirs (see, e.g., Figure 8c, 550a, 550b, 550c, 550d, and 550e).
  • the drug carriers retained in the reservoirs can release drug locally at the overlapping regions in flowing culture medium that does not contain any drugs.
  • a microfluidic device comprising: at least two layers, each layer comprising a channel; and a first porous membrane, wherein the first layer is in parallel with the second layer, wherein the first porous membrane is adjacent to each of and between the first and second layers, wherein the microfluidic device comprises a material exchange area, wherein within the material exchange area, a first region of a first channel overlaps with a second region of a second channel, and wherein a third region of the first channel does not overlap with any region of the second channel in the material exchange area.
  • microfluidic device comprises a feed area and an exit area, wherein within the feed area, each channel comprises a fluid inlet, and within the exit area, each channel comprises a fluid outlet, wherein each of the fluid inlets and fluid outlets are configured such that fluid flows in a first direction through the first channel and fluid flows in a second direction through the second channel, and wherein the first direction and the second direction are the same or different, optionally wherein the first direction is the same as the second direction.
  • the first porous membrane comprises a plurality of pores, and optionally wherein the pores in the plurality of pores have the same average diameter or comprise two or more groups, where each group of pores has a different average diameter. 4.
  • the plurality of pores have an average diameter ranging from about 0.4 micron to about 15 microns, from about 0.4 micron to about 10 microns, from about 0.4 micron to about 5 microns, from about 0.4 micron to about 1 microns, from about 0.8 micron to about 15 microns, from about 1 micron to about 15 microns, from about 0.8 micron to about 10 microns, from about 1 micron to about 10 microns, from about 0.8 micron to about 8 microns, from about 0.8 micron to about 5 microns, from about 2 micron to about 15 microns, or from about 5 micron to about 15 microns; or wherein the plurality of pores comprises two or more groups, wherein a first group of pores has a first average diameter ranging from about 0.4 micron to 5 micron, and a second group of pores has a second average diameter ranging from 0.8 micron to about 15 microns, and wherein the first average diameter is different from the second
  • each of the first layer, the second layer, and the first porous membrane independently has a height ranging from about 50 microns to about 4 mm, from about 100 microns to about 4 mm, from about 50 microns to about 2 mm, from about 100 microns to about 2 mm, from about 50 microns to about 1 mm, from about 100 microns to about 1 mm, from about 10 microns to 1 mm, from about 10 microns to about 4 mm, from about 10 microns to about 2 mm, from about 50 microns to about 500 microns, from about 100 microns to about 200 microns, from about 10 microns to 100 microns, or from about 15 microns to about 50 microns, and optionally wherein the first layer, the second layer, and the first porous membrane have the same height or have at least two different heights.
  • each of the first layer, the second layer, and the first porous membrane is independently formed of an inert, transparent material, optionally wherein the transparent material is polydimethylsiloxane, polyester, acrylic, nylon, polycarbonate, polylactic acid, polyethylene terephthalate glycol, polyether ether ketone, polyether ketone ketone, polypropylene, or glass, or a combination thereof, optionally wherein the first layer, the second layer, and the first porous membrane are formed of the same material or are formed of at least two different materials.
  • the first or second layer further comprises a third channel, and wherein within the material exchange area, the third channel comprises a fourth region that overlaps with the first region of the first channel or the second region of the second channel, and/or overlaps with a fifth region of the second or first channel, and wherein the third channel comprises a sixth region that does not overlap with any region of the second or first channel in the material exchange area.
  • the third channel comprises a third fluid inlet within the feed area, and a third fluid outlet within the exit area, wherein the third fluid inlet and outlet are configured such that fluid flows in a third direction through the third channel, and wherein the third direction is the same as the first and second directions, or is different from the first and/or second direction(s).
  • microfluidic device of any one of paragraphs 1-12 further comprising a third layer and a second porous membrane, wherein the third l yer comprises a fourth channel, wherein the third layer is positioned on top of the second layer and in parallel with the first and second layers, wherein the second porous membrane is adjacent to each of and between the third and second layers, and wherein within the material exchange area, the fourth channel comprises a seventh region that overlaps with the second region, the fifth region, and/or an eighth region of the second channel, and wherein the fourth channel comprises a ninth region that does not overlap with any region of the second channel in the material exchange area.
  • the second porous membrane comprises a plurality of pores, and optionally wherein the pores in the plurality of pores have the same average diameter or comprise two or more groups, wherein each group of pores has a different average diameter.
  • each of the third layer and second porous membrane has a height ranging from about 50 microns to about 500 microns, from about 100 microns to about 200 microns, from about 10 microns to 100 microns, or from about 15 microns to about 50 microns, and optionally wherein the third layer and second porous membrane has the same height or two different heights.
  • each layer of the microfluidic device comprises two or more channels.
  • microfluidic device of any one of paragraphs 1-17 further comprising one or more reservoirs in fluid connection with at least one channel, optionally wherein the one or more reservoirs are fluidly connected to the floor of the at least one channel.
  • a microfluidic device comprising: at least two layers, each layer comprising two or more channels; and a first porous membrane, wherein the first layer is in parallel with the second layer, wherein the first porous membrane is adjacent to each of and between the first and second layers, wherein the microfluidic device comprises a material exchange area, wherein within the material exchange area, a first region of each channel in the first layer overlaps with a second region of at least one channel in the second layer, and wherein a third region of each channel in the first layer does not overlap with any region of any channel in the second layer in the material exchange area.
  • a method for constructing a disease model using the microfluidic device of any one of paragraphs 1-19 comprising:
  • step (ii) is performed at about 37 C for a time period ranging from 3 to 21 days or from 7 to 18 days.
  • step (ii) the first cell culture medium, and optionally the second cell culture medium, has an adequate flow rate such that normalized diffusion of 10% or less of the first cell culture medium diffuses through the first porous membrane into the second channel, optionally normalized concentration of 10% or less of the second cell culture medium diffuses through the first porous membrane into the first channel.
  • step (ii) at least two different types of tissues are formed in the channels of the microfluidic device, wherein at least 90% of at least one type of tissue is in the first channel, optionally at least 90% of at least one type of tissue is in the second channel.
  • step (i) placing a third type of cells in the third channel and in step (ii) flowing a third cell culture medium through the third channel.
  • step (ii) the third cell culture medium has an adequate flow rate such that normalized concentration of 10% or less of the third cell culture medium diffuses through the first porous membrane into the first or second channel.
  • step (i) placing a fourth type of cells in the fourth channel and in step (ii) flowing a fourth cell culture medium through the fourth channel.
  • step (ii) the fourth cell culture medium has an adequate flow rate such that normalized concentration of 10% or less of the fourth cell culture medium diffuses through the second porous membrane into the second channel.
  • step (ii) at least three or at least four different types of tissues are formed in the microfluidic device.
  • step (i) the cells is placed in the form of a monolayer, an organoid, or a spheroid, or the cells being placed are embedded in a hydrogel or ex vivo tissue.
  • the method comprises in step (i) the one or more type(s) of cells are placed in all channels in the first layer, and optionally the one or more type(s) of cells are placed in all channels in the second layer; in step (ii) the first cell culture medium flows through all channels in the first layer, and optionally the second cell culture medium flows through all channels in the second layer; and in step (iii) a first drug solution or suspension, or a first drug carrier medium flows through one mor more channel(s) in the first layer, and/or a second drug solution or suspension, or a second drug carrier medium flows through one or more channel(s) in the second layer, wherein the first and second drug solution or suspension are the same or different.
  • the method further comprises optionally in step (i) placing a fourth type of cells in the third channel, optionally in step (ii) flowing a fourth cell culture medium through the third channel, and in step (iii) flowing a drug solution or suspension through the third channel.
  • step (iii) the drug solution or suspension has an adequate flow rate and/or concentration such that a concentration gradient is produced along the first and/or second channels, and optionally along the fourth channel.
  • An organ-on-a-chip platform comprising: one or more the microfluidic device of any one of paragraphs 1-19, and one or more types of tissue predominantly in the first channel and optionally one or more types of tissue predominantly in the second channel, wherein when two or more microfluidic devices are included, at least one channel of each microfluidic device is in fluid connection with another microfluidic device in the platform.
  • a first type of tissue is predominantly in the first channel and optionally a second type of tissue is predominantly in the second channel, and optionally wherein one or more cells of the first type of tissue are in the second channel and/or one or more cells of the second type of tissue are in the first channel.
  • the platform further comprises a third tissue predominantly in the third channel, optionally wherein one or more cells of the third tissue are in the second or first channel.
  • the platform further comprises a fourth tissue predominantly in the fourth channel, optionally wherein one or more cells of the fourth tissue are in the second channel.
  • An exemplary device includes two channels separated by a porous membrane, and inlet and outlet holes for connection with external flow operating system.
  • One of the two channels is a straight channel having the dimension as described in Jiang, et al., Biomicrofluidics, 13, 064116 2019, and the other one is a 59 mm-long serpentine channel, 1mm wide and 500 m high.
  • the serpentine channel consists of 16 orthogonal channel segments, perpendicular to the main channel axis, extending in both directions from the centerline axis of the straight channel to distal locations at a distance of 2.5mm.
  • the membrane sandwiched between the two channels has a pore size of 0.8 pm or 8pm, selected to conduct a designed experiment.
  • Figures la-lc and 2a-2b illustrate the device design CAD drawings and the device assembly.
  • the exemplary device is made of optically transparent poly dimethylsiloxane (PDMS, Sylgard 184, Dow Corning Corporation) utilizing soft lithography technology.
  • PDMS optically transparent poly dimethylsiloxane
  • Two molds for the microchannels were fabricated in an aluminum block or using 3D printing technique, respectively, for the straight channel and the serpentine channel.
  • PDMS mixture prepared by mixing PDMS base and curing agent at a 10:1 w/w ratio, was dispensed on the mold. After removing air bubbles in the PDMS mixture under vacuum, the molds with the PDMS were placed on a leveled surface at room temperature until the PDMS was fully cured. The PDMS layer was peeled off from the mold to obtain microchannel replicates.
  • Porous membrane upon 1-minute treatment in an oxygen-plasma (Harrick Plasma, USA), was immediately immerged in 5% 3-aminopropyltriethoxysilane (APTES, Sigma- Aldrich) solution in water, and was incubated at 50°C for about 30min to prepare its surfaces for bonding.
  • APTES 3-aminopropyltriethoxysilane
  • the bonding surfaces of a pair of PDMS channel replicates, with inlet and outlet holes, were activated by oxygen plasma.
  • the pair of the PDMS channels, and the dried membrane were then aligned and brought into firm contact.
  • the assembled device was incubated at 50°C for 30min to establish strong bonding.
  • inlet and outlet adapter tubing were assembled to the channel inlet and outlet holes for connections with the external flow control system.
  • the design allows independent supply of the medium-constituents at desired flow rate to the two channels to maintain two cell cultures, one in each channel.
  • the communications and interaction between the two cultures are realized through diffusion of the two media through the pores.
  • drug can be administered at the inlet of one or both channels for treatment tests.
  • a channel was first washed by flowing 70% ETOH several times and incubated under UV irradiation inside the biosafety hood for sterilization. For example, after washing the channel with IxPBS multiple times, a coating solution was introduced into the channel. The device was placed inside the biosafety hood under UV for a one-hour incubation at room temperature. The coating solution was then removed from the channel leaving a thin layer coating on the surfaces of the channel. The device was then placed inside the incubator to cure the coating for one hour at 37°C. A different coating in the other channel was conducted in a similar manner subsequently. After the coated layer was cured, the channel was washed with IxPBS, and subsequently, washed with an appropriate medium, and incubated for more than 30min before placing the cells inside the channel.
  • the stroma channel was coated using 20pg/ml fibronection (Sigma F0895) or a matrix of collagen/fibronectin/PEG, whereas the tumor channel was coated with I Opg/ml laminin (original concentration 1.2mg/ml).
  • Fibronectin coating solution was diluted in IxPBS w/o Ca/Mg to a concentration of 20pg/ml
  • laminin coating solution was diluted in IxPBS w/ Ca/Mg to obtain a concentration of lOpg/ml.
  • the solution of the matrix coating for stroma is a mixture of 500pg/ml rat tail collagen (Corning 354236), 20pg/ml fibronectin (Sigma F0895), and 62.5pg/ml PEG in IxPBS w/o Ca/Mg at pH 7.0 (adjusted by adding a couple microliter of 0. IM NaOH once at a time incrementally).
  • PrECs Immortalized primary human prostate basal epithelial cells
  • PrECs were grown in keratinocyte serum-free medium (KSFM, Gibco 17005042) supplemented with bovine pituitary extract (BPE), epidermal growth factor (EGF), and 1% penicillin-streptomycin.
  • BPE bovine pituitary extract
  • EGF epidermal growth factor
  • BHPrSl s Benign human prostate stroma] cells from Simon Hayward were used as the normal stroma.
  • BHPrSl cells were grown in RPMI 1640 medium (Gibco) with 5% fetal bovine serum (FBS) and 1% penicillin-streptomycin.
  • EMP Error-associated tumor cell lines
  • C4-2 and 22 RV1 Three types of tumor cell lines, EMP, C4-2 and 22 RV1 (ATCC), were used.
  • EMP cells were generated by stably introducing Myc (M) shPten (P) and AErg (E) into PrEC cells. They were maintained in Keratinocyte Serum-Free Medium (KSFM, Gibco 17005042) supplemented with bovine pituitary extract (BPE), epidermal growth factor (EGF), and 1 % penicillin-streptomycin.
  • C4-2 and 22Rv 1 cells were obtained from ATCC and grown in RPMI 1640 medium (Gibco) with 5% FBS, 1% penicillin-streptomycin.
  • Human prostate cancer spheroids isolated from a patient were used in one experiment.
  • the deidentified tissue was obtained from the tissue repository under a standard IRB protocol.
  • fluorescently labeled cells were obtained by lentiviral infection with plasmids containing either green fluorescent protein (GFP) or red fluorescence protein (RFP) cDNAs.
  • GFP green fluorescent protein
  • RFP red fluorescence protein
  • mCherry-labeled BHPrSl cells, GFP-labeled tumor cells, either C4-2 or 22Rvl, and normal PrEC cells were utilized.
  • tumor cells (C4-2 and 22RV1) with color-reporters were used for co-culture experiments with color or non-color BHPrSl cells.
  • Synthetic androgen (R1881) was obtained from Perkin Elmer.
  • R1881 was administered in the stroma medium through an in-flow adapter every two days at average concentration of 50nM.
  • HYD-1 was administered to the tumor medium supply at a concentration in a range of 14 M-50pM, on the alternate dates to the R1881 treatment.
  • Antibodies ITGa6 (GoH3) (sc- 19622), AR (sc-7305), and p63 (sc-56188) and POSTIN (sc-398631) were purchased from Santa Cruz.
  • TMPRSS2 was obtained from University of Washington.
  • HMWCK was obtained from Dako.
  • CoLlAl Gene Tex GTX112731
  • FAP Gene Tex GTX102732
  • Keratin 19 (ab76539) was obtained from Abeam.
  • the immune-staining processes were performed by flowing the solutions through the channels with the cells on each side of the membrane inside the channels.
  • the cells were first washed several times using IxPBS, they were then fixed with 4% paraformaldehyde at room temperature for about lOmin. Permeabilization was carried out using 0.2% Triton for 6 min, followed by 4 washes with lx PBS. Goat serum (5%), donkey serum (5%) or BSA (1%), was used for Ih blocking at room temperature. Cells were then incubated in designated primary antibody solutions diluted in the blocking solutions at 4 °C overnight. After 4 washes with 1 x PBS, secondary antibody solutions were flowed into the channels for 1 h-incubation at room temperature.
  • the cells were washed thoroughly using lx PBS and followed, when needed, by nucleus staining with 10 pg/ml Hoescht (Sigma- Aldrich) for 10 min. Fluorescence imaging were conducted using a Nikon Eclipse TE2000-U microscope under various objectives (4x, lOx, 20x). For taking images under higher magnifications (40x, and lOOx), the devices were opened to obtained the membrane with cells.
  • the membrane was mounted in 70%-90% glycerol solution and was sandwiched between two #1.5 glass coverslips.
  • prostate tumor cells were co-cultured with normal prostate fibroblasts in disease model described in Figures 9a-9d.
  • Co-culturing normal stroma with an oncogene-induced human tumor cell line EMP or with metastatic C4-2 or 22 RV 1 tumor cells in either 0.8um or 8um pore devices induced the expression of CAF markers aS MA and Coll Al in the stroma . This shows that secretive solute from the tumor cells can convert stroma into CAF without physical contact .
  • Tumor cells induced CollAl and aSMA in distinct stromal populations, showing heterogeneity within the CAF population.
  • co-culture of primary prostate tumor from a patient with the normal stroma also converted the stroma to CAFs where aS MA and CollAl were expressed in distinct subsets of stromal cells.
  • Coll Al and aSMA were nearly undetectable in the stroma co-cultured with normal prostate epithelial cells (PrECs).
  • Tumor cells also induced loss of Androgen Receptor (AR) expression in stroma in co-culture in the devices.
  • AR expression which was very high and localized in stroma cell nucleus when cocultured with normal PrECs, was significantly reduced in the stroma co-cultured with tumorigenic EMP cells or C4-2 cancer cells.
  • Prostate tumor cells (GFP-tagged 22RV 1 or C4-2) co-cultured with normal human stromal fibroblasts (mCherry-tagged BHPrS) in 8p m-pore devices invaded into the stroma channel through the pores of the membrane at the overlapping channel regimes.
  • BHPrS cells converted into CAFs expressing aSMA, CollAl, POSTIN and FAP, were also observed in the tumor cell channels. Collected cells from each of the BHPrS and tumor cell channels both contained mixtures of BHP and tumor cells.
  • CAF fibroblasts Upon placed into culture wells, CAF fibroblasts spread simultaneously and developed into a full layer on the well surface within a couple of days, while tumor cells suspended in the culture medium initially, and seeded on the well surface in several days. Both cells were proliferating.
  • stromal fibroblasts and tumor cells were placed in direct contact in the disease model as illustrated in Figures 10a- lOd.
  • Normal BHPrS 1 fibroblasts were plated on the membrane in the lower straight channel to obtain a confluent monolayer.
  • GFP-tagged C4-2 cancer cells were placed directly on top of the fibroblast cell layer establishing 2 cell layers in direct physical contact in the same lower channel.
  • the upper serpentine channel was left empty without cells.
  • Culture medium flow was driven through each of the upper and lower channels at a constant flow rate of 30 pL/h, and R1881 treatment was administered only in the lower channel containing both cell types.
  • Stromal fibroblasts were first observed in the upper channel on Day 2 of the co-culture, migrating from the overlapping regions of the straight seeding channel into the upper serpentine channel. In the ensuing few days, the stromal fibroblasts rapidly spread over the entire serpentine channel surface reaching all distal locations. In this configuration, the C4-2 cancer cells followed the stromal fibroblasts advancing into the serpentine channel at a very fast pace. Many C4-2 cancer cells were detected at the farthest distal locations in the serpentine channel about 2 mm away from their original straight channel by Day 12. They appeared, mostly as single cells or in small cell clusters with some noticeable individual cells, not rounded but spreading on the channel surface.
  • the C4-2 cancer cells remaining in their original channel also changed morphologically from a monolayer into a disrupted 3D layer reaching the surface facing the separation membrane of the lower channel that had been covered with stromal fibroblasts. This could be attributed to both a high proliferation rate and improved adherence to the channel surface modified by the stromal fibroblasts.
  • the direct physical contact of the cancer cells with fibroblast dramatically increased the number of invading tumor cells along with their migration range and pace.
  • cytokines secretion of cytokines, TGFp, PDGF, and PDGFR, by the tumor cells were evaluated using conditioned medium samples collected from experiments with physical contact between tumor and stroma cultures ( Figures 10a- lOd). Samples were collected separately from tumor and stroma channels at 2-day intervals from Day 7 to Day 13 of the co-culture, and analyzed using ELISA. The production of TGFP in straight channel medium (initial tumor cell/stroma plating channel) was robustly detected. Higher TGFP levels were initially produced in “22Rvl on BHP”, but slightly dropped later, while the levels in “C4-2 on BHP” slightly increased with time ( Figures 13a).
  • Drug tests were also investigated under various conditions by varying the treatment dosage, treatment frequency, treatment total times, etc. Roles of stroma on the treatment of tumor cells were also investigated by varying the co-culture arrangement, e.g., tumor cells and stroma in different channels on each side of the pore membrane, or tumor cells placed on top of a full layer of stroma in the same channel. Drug treatment was administered either in one of the channels or in both channels.
  • tumor cells were under treatment of HYD- 1 in the disease model without initial direct physical contact between tumor cells and stroma ( Figures 9a- 9d).
  • the treatment was provided at low concentrations in a range of 10-20 M HYD-1 in medium flow only to the tumor cell channel, while culture medium without drug was flowed into the stroma channel.
  • tumor cells did not stop invading into the stroma channel.
  • tumor cells became dead after 4 days.
  • a clear distribution of tumor cell color reporter intensity along the channel was observed, where cells lost green signal, indicating their death, at the drugcontaining medium flow inlet but alive in bright green color at the flow outlet .
  • tumor cells exhibiting bright green signal remained at the overlapping regimes of the two channels, where interactions between tumor cells and stromal fibroblasts were present.
  • drug HYD-1 containing culture medium was flowed into both channels of tumor and stroma. Death of tumor cells were observed along the channel including the overlapping regimes. The initially fully confluent stromal fibroblasts became deteriorating and detached from the channel surface. This showed that treatment to both tumor cells and the surrounding stroma in the tumor microenvironment was more effective.
  • Drug treatment was also tested in another disease model, where tumor cells and stroma were initially placed with direct physical contact in the same channel ( Figures lOa-lOd).
  • Drug of 50pM HYD-1 in culture medium was flowed into the tumor/stroma channel while culture medium flow was supplied to the second empty channel.
  • gradient in green signal tumor cell live/death reporter
  • An additional 3-day of treatment resulted in more dead tumor cells in the channel while only some tumor cells were alive downstream to the flow outlet.
  • treatment of 8 days most tumor cells were dead along with deteriorated stroma. This result further confirmed the role of stroma on effectiveness of drug treatment in tumor cells.

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Abstract

L'invention concerne des dispositifs microfluidiques, des plateformes d'organe sur puce (par exemple imitant la glande de la prostate humaine), et leurs utilisations. Le dispositif microfluidique comprend au moins deux couches et une membrane poreuse entre celles-ci. Chaque couche comprend un canal, et le premier canal et le second canal sont alignés de telle sorte qu'une première région d'un premier canal chevauche une région du second canal, et une seconde région du premier canal ne chevauche pas une seconde région du second canal. Un modèle de maladie utilisant les dispositifs microfluidiques comprend (i) le placement d'un premier type de cellules dans le premier canal et (ii) l'écoulement d'un premier milieu de culture cellulaire à travers le premier canal. Facultativement, à l'étape (i), un second type de cellules dans le second canal et à l'étape (ii) un second milieu de culture cellulaire s'écoule à travers le second canal. Les dispositifs microfluidiques peuvent être utilisés dans le criblage et le test de médicaments.
PCT/US2024/032983 2023-06-07 2024-06-07 Glande de prostate humaine bio-modifiée et modèle de maladie Ceased WO2024254436A2 (fr)

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ES2672201T3 (es) * 2008-07-16 2018-06-13 Children's Medical Center Corporation Dispositivo de imitación de órganos con microcanales y métodos de uso
GB2554292A (en) * 2015-04-14 2018-03-28 Harvard College Microfluidic device having offset, high-shear seeding channels

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