WO2022072260A1 - Systems and methods relating to subcutaneous administration - Google Patents

Systems and methods relating to subcutaneous administration Download PDF

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Publication number
WO2022072260A1
WO2022072260A1 PCT/US2021/052134 US2021052134W WO2022072260A1 WO 2022072260 A1 WO2022072260 A1 WO 2022072260A1 US 2021052134 W US2021052134 W US 2021052134W WO 2022072260 A1 WO2022072260 A1 WO 2022072260A1
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Prior art keywords
compartment
subcutaneous
fibroblasts
peripheral
preadipocytes
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PCT/US2021/052134
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French (fr)
Inventor
Samir Mitragotri
Qin QI
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President And Fellows Of Harvard College
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Publication of WO2022072260A1 publication Critical patent/WO2022072260A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/069—Vascular Endothelial cells
    • 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
    • C12M23/34—Internal compartments or partitions
    • 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
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/14—Scaffolds; Matrices
    • 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
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/08—Chemical, biochemical or biological means, e.g. plasma jet, co-culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0653—Adipocytes; Adipose tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0656—Adult fibroblasts
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M5/00—Devices for bringing media into the body in a subcutaneous, intra-vascular or intramuscular way; Accessories therefor, e.g. filling or cleaning devices, arm-rests
    • A61M5/14—Infusion devices, e.g. infusing by gravity; Blood infusion; Accessories therefor
    • A61M5/142—Pressure infusion, e.g. using pumps
    • A61M2005/14288—Infusion or injection simulation
    • A61M2005/14296—Pharmacokinetic models
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue

Definitions

  • a system comprising a first subcutaneous compartment comprising a mixture comprising fibroblasts, cell culture medium, hydrogel, and at least one of preadipocytes and adipocytes; and a first peripheral compartment comprising at least one opening providing fluid, non-cellular communication with the first subcutaneous compartment; and optionally comprising endothelial cells.
  • the system further comprises a second peripheral compartment comprising at least one opening providing fluid, non-cellular communication with the first subcutaneous compartment.
  • the first subcutaneous compartment is medial with respect to the first and second peripheral compartments; and the first and second peripheral compartments are not directly in fluid, non-cellular communication with each other.
  • each compartment comprises or is a channel, cube, rectangular prism, or cylinder.
  • the first and/or second lateral portions do not comprise the at least one openings with the first peripheral compartment and optionally, second peripheral compartment.
  • the first and second peripheral compartments comprise a medial curved channel portion where they comprise the at least one openings with the first subcutaneous compartment; a first lateral channel, cube, rectangular prism, or cylinder portion; and a second lateral channel, cube, rectangular prism or cylinder portion.
  • the system further comprises a second subcutaneous compartment comprising a mixture comprising fibroblasts, cell culture medium, hydrogel, and at least one of preadipocytes and adipocytes; and a second peripheral compartment comprising at least one opening providing fluid, non-cellular communication with the second subcutaneous compartment; and optionally comprising endothelial cells; a medial injection compartment comprising at least one opening providing fluid, non-cellular communication with the first subcutaneous compartment; and at least one opening providing fluid, non-cellular communication with the second subcutaneous compartment; wherein the first and second subcutaneous compartments are lateral to the medial injection compartment.
  • the at least one opening comprises multiple openings or slits in an interposed wall or barrier. In some embodiments of any of the aspects, the at least one opening is no greater in height or width than 500 pm. In some embodiments of any of the aspects, the at least one opening is no greater in height or width than 100 pm. In some embodiments of any of the aspects, the fluid availability aspect ratio is 5 or greater. In some embodiments of any of the aspects, the fluid availability aspect ratio is 50:3. [0011] In some embodiments of any of the aspects, the fibroblasts and at least one of preadipocytes and adipocytes form a 3D culture.
  • the first peripheral compartment comprises endothelial cells. In some embodiments of any of the aspects, the first peripheral compartment comprises endothelial cells and the second peripheral compartment does not comprise cells. In some embodiments of any of the aspects, the first and second peripheral compartments each comprise endothelial cells. In some embodiments of any of the aspects, the endothelial cells line one or more walls of the first and/or second peripheral compartments. In some embodiments of any of the aspects, the endothelial cells form a confluent single-cell monolayer on all walls of the first and/or second peripheral compartments.
  • the cells are murine or human cells. In some embodiments of any of the aspects, at least 80% of the cells are viable. In some embodiments of any of the aspects, at least 90% of the cells are viable.
  • the hydrogel comprises one or more extracellular matrix components.
  • the method further comprises the following steps prior to the step of introducing the candidate subcutaneous agent: introducing the mixture comprising fibroblasts, cell culture medium, hydrogel, and at preadipocytes into the first and/or second subcutaneous compartment and then culturing the preadipocytes and fibroblasts; inducing adipocyte differentiation and then maintaining the adipocytes and fibroblasts; twice introducing endothelial cells into the first and/or second peripheral compartment and then culturing and maintaining the endothelial cells.
  • culturing the fibroblasts and at least one of preadipocytes and adipocytes comprises one or more of: culturing for at least 5 days; culturing until the fibroblasts and at least one of preadipocytes and adipocytes reach a high density; and culturing until the fibroblasts and at least one of preadipocytes and adipocytes are confluent.
  • culturing and/or maintaining the fibroblasts and at least one of preadipocytes and adipocytes comprises exchanging the culture medium in the first and/or second subcutaneous compartment at least daily.
  • culturing and/or maintaining the fibroblasts and at least one of preadipocytes and adipocytes comprises exchanging the culture medium in the first and/or second subcutaneous compartment continuously.
  • medium exchange in the injection compartment and/or a peripheral compartment not comprising cells is performed at a perfusion rate of from 0.1 pL/min to 1000 pL/min.
  • Figs. 4A-4I depict constructing the subcutaneous tissue in an idealized geometry. (Fig. 4)
  • FIG. 4B A three-compartment commercial chip made of PDMS and glass.
  • FIG. 4B Three compartments represent various environments relevant for subcutaneous injection.
  • FIG. 4C A close-up look of the narrow slits connecting adjacent channels.
  • Fig. 4E Timeline of microfluidic cell culture.
  • FIG. 4F Rheological properties of the Matrigel: medium mixture (Mix) show temperature dependence similar to pure Matrigel (MG).
  • Figs. 6A-6F depict the measurement of transport properties and the effect of MW.
  • Fig. 6A Measurements were performed in a live cell imaging system (left), tracking the change of fluorescence intensity overtime (right).
  • Fig. 6B A good fit between theoretical estimation and experimental measurement was obtained in the subcutaneous channel for both the instantaneous spatial concentration profile (left) and the overall spatiotemporal concentration profile (right)
  • Figs. 6C-6E The effect of MW on FITC-dextran transport for R12 (Fig. 6C) D2 (Fig. 6D) and R23 (Fig. 6E).
  • Fig. 6G The comparison between D2 and existing studies as a function of MW. Scale bars: 200 pm. Error bars represent standard error of the mean.
  • Figs. 7A-7F depict the influence of molecule charge and shape on subcutaneous transport.
  • Figs. 8A-8B depict the prediction of circulation pathways and in vivo pharmacokinetics.
  • Fig. 8 A Percentage of lymphatic transport compared to vascular transport based on the ratio of
  • Fig. 8B Estimation of the time of subcutaneous absorption as a function of interstitial distance s in comparison to the chip dimension. The timescales of entering and exiting the subcutaneous space are also provided for reference. Error bars represent standard error of the mean.
  • FIG. 9 depicts an image of an exemplary embodiment of the system described herein, e.g., with connecting tubes for each inlet and outlet port.
  • Figs. 11A-11C depict estimation of the narrow-slit effects.
  • Fig. 11A Perfusion occurs in the injection compartment or peripheral compartment not comprising cells with the flow rate controlled by a pump. The other two channels are clamped on both inlet and outlet ends to prevent evaporation.
  • Fig. 1 IB In addition to the injection compartment or peripheral compartment not comprising cells, flow can occur in the adjacent subcutaneous compartment as a series of resistances via 100 narrow slits along the compartment. The disturbance flow in the subcutaneous compartment be estimated based on the aspect ratio.
  • Fig. 11C The resulting disturbance velocity in the subcutaneous compartment normalized by the inlet flow velocity.
  • Fig. 14 depicts images of the top view of the growth and expansion of mice fibroblasts and preadipocytes cultured in 3D on a chip. Scale bars: 100 [im.
  • Fig. 15 depicts images of the top view of the growth and expansion of endothelial 2D monolayer on chip. The seeding procedure was repeated once to achieve a high confluency. Scale bars: 100 im.
  • Figs. 16A-16F depict images of the top view of the cell morphology under phase contrast microscope.
  • Fig. 16A Mice fibroblasts in 2D tissue culture flask.
  • Fig. 16B Mice preadipocytes in 2D tissue culture flask.
  • Fig. 16C Mice fibroblasts in 3D on chip.
  • Fig. 16D Mice preadipocytes in 3D on chip.
  • Fig. 16E Mice endothelial cells in 2D tissue culture flask.
  • Fig. 16F Human preadipocytes and fibroblasts in 3D (top) and endothelial cells in a 2D monolayer (bottom) on chip.
  • Fig. 17 depicts a graph of the comparison of Oil Red O stained area before and after adipocyte differentiation induction for mice preadipocytes (X9) and fibroblasts (L) cells on a plate assay.
  • the medial disc or sphere portion 11, the first lateral channel, cube, rectangular prism, or cylinder portion 12, and second lateral channel, cube, rectangular prism or cylinder portion 13 are arranged along the axis of fluid flow in the first subcutaneous compartment 10 in the order of: 1) the first lateral channel, cube, rectangular prism, or cylinder portion 12, 2) the medial disc or sphere portion 11, and 3) second lateral channel, cube, rectangular prism or cylinder portion 13.
  • the first and second peripheral compartments 20, 21 each comprise a medial curved channel portion 22 where they comprise the at least one openings 30, 31 with the first subcutaneous compartment 10.
  • the first and second peripheral compartments 20, 21 each comprise a first lateral channel, cube, rectangular prism, or cylinder portion 23 and a second lateral channel, cube, rectangular prism or cylinder portion 24.
  • Various embodiments of the systems described herein comprise at least one opening that provides fluid, non-cellular communication between two compartments.
  • the openings can be provided in any shape (e.g., circular, ovoid, rectangular, etc), profde, or when multiple openings are present, any combination thereof.
  • the at least one opening comprises an opening or slit in an interposed wall or barrier.
  • the at least one opening comprises multiple openings or slits in an interposed wall or barrier.
  • the at least one opening has a high fluid availability aspect ratio. That is, along the length of the compartment wall, there is more wall than openings so that at any moment in time less than half of the fluid along the wall of the compartment is available for fluid exchange through the openings.
  • the fluid aspect ratio can be expressed as the ratio of solid wall to opening along the length of the compartment’s (or portion of the compartment’s) wall. For example, if every 50 pm along a wall, there is a 3 pm slit connecting a first compartment and a second compartment, only sees 3/50 of the first compartment’s fluid is available for exchange to the second compartment.
  • An advantage of a high fluid availability aspect ration is that flow in a second compartment is not disturbed when perfusion occurs in a first channel. This can protect, e.g., the soft hydrogel-based subcutaneous tissue in a subcutaneous compartment from excess fluid shear.
  • the structures of the systems described herein can be formed, such as by etching, 3-D printing, machining, or micro-machining. In some embodiments, the system described herein is etching-free.
  • the system described herein can be made of a biocompatible material(s) according to the design and application requirements. It should be noted that the designs depicted in the Figures are exemplary and the system described herein is not limited to the configurations shown in the Figures.
  • the system and/or portions thereof can be made of a flexible material, including but not limited to, a biocompatible material such as polydimethyl siloxane (PDMS), polyurethane or polyimide.
  • PDMS polydimethyl siloxane
  • the system and/or portions thereof can also be made of non-flexible materials like glass, silicon, polysulfone, hard plastic, and the like, as well as combinations of these materials.
  • a biocompatible polymer refers to materials which do not have toxic or injurious effects on biological functions.
  • Biocompatible polymers include natural or synthetic polymers. Examples of biocompatible polymers include, but are not limited to, collagen, poly(alpha esters) such as poly(lactate acid), poly(glycolic acid), polyorthoesters and polyanhydrides and their copolymers, polyglycolic acid and polyglactin, cellulose ether, cellulose, cellulosic ester, fluorinated polyethylene, phenolic, poly-4-methylpentene, polyacrylonitrile, polyamide, polyamideimide, polyacrylate, polybenzoxazole, polycarbonate, polycyanoarylether, polyester, polyestercarbonate, polyether, polyetheretherketone, polyetherimide, polyetherketone, polyethersulfone, polyethylene, polyfluoroolefin, polyimide, polyolefin, polyoxadiazole, polyphenylene oxide, polyphenylene sulfide
  • a biocompatible material can also be, for example, ceramic coatings on a metallic substrate.
  • any type of coating material and the coating can be made of different types of materials: metals, ceramics, polymers, hydrogels or a combination of any of these materials.
  • Biocompatible materials include, but are not limited to an oxide, a phosphate, a carbonate, a nitride or a carbonitride.
  • the oxide the following ones are preferred: tantalum oxide, aluminum oxide, iridium oxide, zirconium oxide or titanium oxide.
  • Substrates are made of materials such as metals, ceramics, polymers or a combination of any of these.
  • Metals such as stainless steel, Nitinol, titanium, titanium alloys, or aluminum and ceramics such as zirconia, alumina, or calcium phosphate are of particular interest.
  • the mixture comprising preadipocytes (or adipocytes), fibroblasts, cell culture medium, and hydrogel comprises more preadipocytes (or adipocytes) than fibroblasts.
  • the mixture comprising preadipocytes (or adipocytes), fibroblasts, cell culture medium, and hydrogel comprises at least 2x as many preadipocytes (or adipocytes) than fibroblasts, e.g., at least 2x, 3x, 4x, 5x , 6x, 7x, 8x, 9x, lOx, 15x, 20x or more.
  • the cells described herein can be from any suitable source, e.g., they can be cell lines or primary cells.
  • the cells are mammalian cells.
  • the cells are murine cells.
  • the cells are human cells.
  • the cells are primary cells, cultured cells, passaged cells, immortalized cells, transgenic cells, genetically modified cells, diseased cells or cells from an animal with a disease, or cells differentiated from stem cells, embryonic stem cells (ESCs), or induced pluripotent stem cells (IPSCs).
  • ESCs embryonic stem cells
  • IPCs induced pluripotent stem cells
  • one or more of the compartments described herein can comprise at least 1 port, e.g., for introducing medium, agents, and/or cells.
  • one or more of the compartments described herein can each comprise 2 ports.
  • each of the compartments described herein comprises 2 ports.
  • the 2 ports are distal to each other, e.g., such that one port can serve as an inflow means and the second port can serve as an outflow means to provide fluid flow to at least 60%, at least 70%, at least 80%, at least 90%, or more of the compartment.
  • the 2 ports are lateral with respect to a medial portion of the compartment. In some embodiments of any of the aspects, the 2 ports are each located within different lateral portions of the compartment. Where 2 ports are present in a compartment, it is contemplated that they can be connected to a fluidics device, e.g., microfluidics device to provide an inflow and an outflow port in each compartment.
  • a fluidics device e.g., microfluidics device to provide an inflow and an outflow port in each compartment.
  • a fluid source can be a reservoir or other container comprising a volume of fluid such that the fluid can be caused to move from the fluid source and through the one or more compartments of the system.
  • the fluid source can be coupled to the one or more compartments of the system by any means of conducting a fluid, e.g. tubing, piping, channels, or the like. Either positive or negative fluid pressure, or both, can be used to cause the fluid to flow through the system’s compartment(s).
  • the fluidic device can further utilize gravity and/or pumps to cause the fluid to flow.
  • a fluidic device can further comprise valves to control inflow and outflow to and from the device.
  • a fluidic device can also be connected to a control system, such as a machine or computer system, to permit automated control of the valves and the fluid flow.
  • the machine may comprise a personal computer (PC), a tablet, a Personal Digital Assistant (PDA), a cellular telephone, a web appliance, a network router, switch or bridge, or any machine capable of executing a set of instructions (sequential or otherwise) that specify actions to be taken by that machine.
  • PC personal computer
  • PDA Personal Digital Assistant
  • a cellular telephone a web appliance
  • network router switch or bridge
  • the fluid flow rate can be constant. In some embodiments of any of the aspects, the fluid flow rate can be varied in a regular, cyclic pattern. In some embodiments the fluid flow rate can be varied in an irregular pattern. In some embodiments of any of the aspects, the control of the fluid flow can be automated.
  • the size, shape and configuration of the system described herein can be selected so that the system can be used as a replacement for chips provided by manufacturers or suppliers for a particular fluidics or microfluidic devices.
  • the ports can be provided in the appropriate size and shape necessary to accept the tubes and/or connectors of a particular fluidic or microfluidic device.
  • the fluid which is caused to flow through the one or more compartments of the system described herein can be any fluid appropriate for maintaining or culturing the cells present in the compartments of the device.
  • different fluids can be caused to flow through different compartments.
  • Fluids can comprise cell culture medium, solutions, buffers, nutrients, tracer compounds, dyes, antimicrobials, or other compounds not toxic to the cells being cultured in the cell culture system described herein.
  • suitable fluids for culturing or maintaining the cells described herein can include DMEM (ATCC) supplemented with 10% FBS.
  • Fluids suitable for maintaining or culturing preadipocytes and/or fibroblasts can include DMEM/F12 (ATCC) supplemented with 15% FBS or fibroblast basal medium with low-serum growth kit (ATCC). Fluids suitable for maintaining or culturing any of the cells described herein can include full subcutaneous culture medium (e.g., either DMEM or DMEM/F12 with 20% FBS for mice) at 10 d/min. Fluids suitable for maintaining or culturing adipocytes and/or fibroblasts can include full subcutaneous cell culture medium (e.g., either DMEM or DMEM/F12 with 20% FBS for mice) with 10 /ig/ml insulin.
  • DMEM/F12 ATCC
  • ATCC fetal growth kit
  • one or more walls of a compartment described herein can be treated or coated with one or more cell adhesive materials to promote attachment of cells. Such attachment materials and treatments are known in the art.
  • Non-limiting examples of types of attachment molecules include collagen; collagen Type I, collagen Type II; collagen Type III; collagen Type IV; collagen Type V; collagen Type VI; collagen Type VII; collagen Type VIII; collagen Type IX, collagen Type X; collagen Type XI; collagen Type XII; collagen Type XIII; collagen Type XIV; extracellular matrix, MATRIGELTM; laminin; proteoglycan; vitronectin; fibronectin; poly-D-lysine; elastin; hyaluronic acid; glycoasaminoglycans; integrin; polypeptides, oligonucleotides, DNA, and/or polysaccharide.
  • the systems described herein can be utilized in methods of culturing, studying, analyzing and/or maintaining the described cells, e.g, the behavior and charateristics of the cells under conditions provided by the described systems.
  • the systems permit the in vitro study, analysis, and characterization of subcutaneous administration characteristics of a candidate subcutaneous agent.
  • an in vitro method of determining the subcutaneous administration characteristics of a candidate subcutaneous agent comprising i) introducing a candidate subcutaneous agent into the injection compartment and/or a peripheral compartment not comprising cells, of a system described herien; and ii) measuring at least one of: the amount and/or change in the amount of subcutaneous agent in the outflow of fluid from one or more of the compartments; the amount and/or change in the amount of subcutaneous agent present in one or more of the compartments; and the amount and/or change in the amount of subcutaneous agent in the outflow of fluid from a peripheral compartment comprising endothelial cells.
  • a peripheral compartment not comprising endothelial cells can indicate and/or model subcutaneous administration characteristics via lymphatic pathways.
  • a peripheral compartment not comprising vascular endothelial cells can indicate and/or model subcutaneous administration characteristics via lymphatic pathways.
  • a peripheral compartment comprising lymphatic endothelial cells and not comprising vascular endothelial cells can indicate and/or model subcutaneous administration characteristics via lymphatic pathways.
  • a peripheral compartment comprising endothelial cells can indicate and/or model subcutaneous administration characteristics via vascular pathways.
  • the candidate agent can be introduced into an injection compartment and/or a peripheral compartment not comprising cells by adding it directly via an injection port, or by adding it to media provided by the inflow port of the compartment.
  • the compartment to which the candidate agent is introduced is perfused with media during the introduction of the candidate agent, regardless of the means by which the candidate agent itself is introduced to the compartment.
  • the compartment to which the candidate agent is introduced is perfused with media from at least the the introduction of the candidate agent until the measuring steps are complete, regardless of the means by which the candidate agent itself is introduced to the compartment.
  • the compartment to which the candidate agent is introduced is perfused at a rate of from about 0.01 pL/min to about 10,000 pL/min. In some embodiments of any of the aspects, the compartment to which the candidate agent is introduced is perfused at a rate of from 0.01 pL/min to 10,000 pL/min. In some embodiments of any of the aspects, the compartment to which the candidate agent is introduced is perfused at a rate of from 0.1 pL/min to 1,000 pL/min. In some embodiments of any of the aspects, the compartment to which the candidate agent is introduced is perfused at a rate of from 1 pL/min to 100 pL/min.
  • the compartment to which the candidate agent is introduced is perfused at a rate of from 5 pL/min to 50 pL/min. In some embodiments of any of the aspects, the compartment to which the candidate agent is introduced is perfused at a rate of from 1 pL/min to 50 pL/min. In some embodiments of any of the aspects, the compartment to which the candidate agent is introduced is perfused at a rate of from 10 pL/min to 100 pL/min. In some embodiments of any of the aspects, the compartment to which the candidate agent is introduced is perfused at a rate of from 50 pL/min to 100 pL/min.
  • the subcutaneous compartment of the system comprises adipocytes.
  • the adipocyte can be added to the subcutaneous compartment, or preadipocytes can be added to the subcutaneous compartment and differentiated in the compartment.
  • the methods described herein can comprise introducing a mixture comprising preadipocytes, fibroblasts, cell culture medium, and extracellular matrix material into the first and/or second subcutaneous compartment and then culturing the preadipocytes and fibroblasts; inducing adipocyte differentiation; and then maintaining the adipocytes and fibroblasts.
  • the mixture and/or cell is introduced into the system at a low temperature, e.g., to minimize cell metabolism or modulate viscosity.
  • the mixture comprising preadipocytes, fibroblasts, cell culture medium, and extracellular matrix material is at less a temperature of less than 20 C when it is introduced into the first and/or second subcutaneous compartment.
  • the mixture comprising preadipocytes, fibroblasts, cell culture medium, and extracellular matrix material is at less a temperature of less than 10 C when it is introduced into the first and/or second subcutaneous compartment.
  • the mixture comprising preadipocytes, fibroblasts, cell culture medium, and extracellular matrix material is at a temperature of 4 C or less when it is introduced into the first and/or second subcutaneous compartment.
  • maintaining or “culturing” refers to continuing the viability of a population of cells.
  • a maintained tissue will have a population of metabolically active cells. The number of these cells can be roughly stable over a period of at least 1 day or can grow.
  • differentiated In the context of cell ontogeny, the term “differentiated”, or “differentiating” is a relative term.
  • a “differentiated cell” is a cell that has progressed further down the developmental pathway than the cell it is being compared with.
  • stem cells can differentiate to lineage-restricted precursor cells, which in turn can differentiate into other types of precursor cells further down the pathway, and then to an end-stage differentiated cell, which play a characteristic role in a certain tissue type, and may or may not retain the capacity to proliferate further.
  • differentiation refers to the process whereby a cell moves further down the developmental pathway and begins expressing markers and phenotypic characteristics known to be associated with a cell that are more specialized and closer to becoming terminally differentiated cells. Differentiation is a developmental process whereby cells assume a more specialized phenotype, e.g., acquire one or more characteristics or functions distinct from other cell types.
  • the differentiated phenotype refers to a cell phenotype that is at the mature endpoint in some developmental pathway (a so called terminally differentiated cell). In many, but not all tissues, the process of differentiation is coupled with exit from the cell cycle.
  • preadipocyte differentiation to adipocytes can be induced by perfusing differentiation medium daily for three days.
  • the differentiation medium contains full subcutaneous cell culture medium (e.g., either DMEM or DMEM/F12 with 20% FBS for mice) supplemented with 5.0 rM dexamethasone, 5 mM methylisobutylxanthine and 100 . g/ml insulin.
  • the differentiated cells can be maintained using full subcutaneous cell culture medium (e.g., either DMEM or DMEM/F12 with 20% FBS for mice) with 10 jUg/ml insulin for daily media exchange.
  • the cells described herein can be cultured for any amount of time prior to the introduction of a candidate agent.
  • the preadipocytes/adipocytes and/or fibroblasts are cultured for at least one of the following periods prior to introduction of a candidate agent: at least 5 days; until the preadipocytes/adipocytes and/or fibroblasts reach a high density; and until the preadipocytes/adipocytes and/or fibroblasts are confluent.
  • high density means that the number of cells no longer increases.
  • the endothelial cells are cultured for at least one of the following periods prior to introduction of a candidate agent: at least 3 days; until the endothelial cells reach a high density; and until the endothelial cells are confluent.
  • high density means at least 90% confluency.
  • media can be perfused continuously or exchanged at intervals.
  • Media can be perfused or exchanged by causing direct fluid flow via inflow/outflow ports in the relevant compartment, or media can be perfused or exchanged in a first compartment by causing fluid flow in a second compartment that is in fluid, non-cellular communication with the first compartment.
  • the second compartment does not comprise cells.
  • culturing the preadipocytes/adipocytes and/or fibroblast and/or maintaining the adipocytes and fibroblasts comprises exchanging the culture medium in the first and/or second subcutaneous compartment at least daily.
  • culturing the preadipocytes/adipocytes and/or fibroblast and/or maintaining the adipocytes and fibroblasts comprises exchanging the culture medium in the first and/or second subcutaneous compartment continuously.
  • media exchange in the injection compartment and/or a peripheral compartment not comprising cells is performed at a perfusion rate of from about 0.01 pL/min to about 10,000 pL/min. In some embodiments of any of the aspects, media exchange in the injection compartment and/or a peripheral compartment not comprising cells is performed at a perfusion rate of from 0.01 pL/min to 10,000 pL/min. In some embodiments of any of the aspects, media exchange in the injection compartment and/or a peripheral compartment not comprising cells is performed at a perfusion rate of from 0.1 pL/min to 1,000 pL/min.
  • media exchange in the injection compartment and/or a peripheral compartment not comprising cells is performed at a perfusion rate of from 10 pL/min to 100 pL/min. In some embodiments of any of the aspects, media exchange in the injection compartment and/or a peripheral compartment not comprising cells is performed at a perfusion rate of from 50 pL/min to 100 pL/min.
  • culturing or maintaining endothelial cells comprises exchanging the culture medium in the first and/or second peripheral compartment at least daily. In some embodiments of any of the aspects, culturing or maintaining endothelial cells comprises exchanging the culture medium in the first and/or second peripheral compartment continuously. In some embodiments of any of the aspects, medium exchange in the first and/or second peripheral compartment is performed at a perfusion rate of from about 0.01 pL/min to about 500 pL/min. In some embodiments of any of the aspects, medium exchange in the first and/or second peripheral compartment is performed at a perfusion rate of from 0.01 pL/min to 500 pL/min.
  • medium exchange in the first and/or second peripheral compartment is performed at a perfusion rate of from 0.02 pL/min to 200 pL/min. In some embodiments of any of the aspects, medium exchange in the first and/or second peripheral compartment is performed at a perfusion rate of from 2 pL/min to 20 pL/min. In some embodiments of any of the aspects, medium exchange in the first and/or second peripheral compartment is performed at a perfusion rate of from 1 pL/min to 100 pL/min. In some embodiments of any of the aspects, medium exchange in the first and/or second peripheral compartment is performed at a perfusion rate of from 1 pL/min to 50 pL/min.
  • the terms “candidate compound” or “candidate agent” refer to a compound or agent and/or compositions thereof that are to be screened and/or analyzed for their subcutaneous administration behavior/characteristics.
  • the terms “compound” or “agent” are used interchangeably and refer to molecules and/or compositions.
  • the amount of an agent in one or more compartments and/or outflows is determined at a particular point in time. In some embodiments of any of the aspects, the amount of an agent in a compartment and/or outflow is determined at at least two particular points in time, thereby permitting the change in amount and/or rate of change of the amount to be determined.
  • the amount of an agent present in a compartment and/or the outflow of a compartment can be determined by any method known in the art.
  • the agent can be detectably labelled and the amount/concentration of the agent can be determined with an appropriate detector for the label type.
  • the label can be FITC and the detection can be done by fluorescence microscopy.
  • Suitable types of labels can include a light-absorbing dye, a fluorescent dye, or a radioactive label. Detectable labels, methods of detecting them, and methods of incorporating them into reagents (e.g. antibodies and nucleic acid probes) are well known in the art.
  • detectable labels can include labels that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as fluorescence, chemifluoresence, or chemiluminescence, or any other appropriate means.
  • the detectable labels used in the methods described herein can be primary labels (where the label comprises a moiety that is directly detectable or that produces a directly detectable moiety) or secondary labels (where the detectable label binds to another moiety to produce a detectable signal, e.g., as is common in immunological labeling using secondary and tertiary antibodies).
  • the detectable label can be linked by covalent or non-covalent means to the reagent.
  • a detectable label can be linked such as by directly labeling a molecule that achieves binding to the agent via a ligand-receptor binding pair arrangement or other such specific recognition molecules.
  • Detectable labels can include, but are not limited to radioisotopes, bioluminescent compounds, chromophores, antibodies, chemiluminescent compounds, fluorescent compounds, metal chelates, and enzymes.
  • the agent is labelled with a fluorescent compound.
  • a detectable label can be a fluorescent dye molecule, or fluorophore including, but not limited to fluorescein, phycoerythrin, phycocyanin, o-phthaldehyde, fluorescamine, Cy3TM, Cy5TM, allophycocyanine, Texas Red, peridenin chlorophyll, cyanine, tandem conjugates such as phycoerythrin-Cy5TM, green fluorescent protein, rhodamine, fluorescein isothiocyanate (FITC) and Oregon GreenTM, rhodamine and derivatives (e.g., Texas red and tetrarhodimine isothiocynate (TRITC)), biotin, phycoerythrin, AMCA,
  • a detectable label can be a radiolabel including, but not limited to 3 H, 125 1, 35 S, 14 C, 32 P, and 33 P.
  • Enzymes contemplated for use to detectably label an antibody reagent include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alphaglycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • an agent can also be labeled with a detectable tag, such as c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS, or biotin.
  • a detectable tag such as c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS, or biotin.
  • Other detection systems can also be used, for example, a biotin-streptavidin system.
  • the antibodies immunoreactive (i. e. specific for) with the biomarker of interest is biotinylated. Quantity of biotinylated antibody bound to the biomarker is determined using a streptavidin-peroxidase conjugate and a chromagenic substrate.
  • streptavidin peroxidase detection kits are commercially available, e. g. from DAKO;
  • a reagent can also be detectably labeled using fluorescence emitting metals such as 152 EU, or others of the lanthanide series. These metals can be attached to the reagent using such metal chelating groups as diethylenetriaminepentaacetic acid (DTP A) or ethylenediaminetetraacetic acid (EDTA).
  • DTP A diethylenetriaminepentaacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • FIA fluorescence-linked immunoassay
  • CLIA chemiluminescence immunoassays
  • ELIA electrochemiluminescence immunoassay
  • CIA counting immunoassay
  • LFIA lateral flow tests or immunoassay
  • MIA magnetic immunoassay
  • the terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount.
  • the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.
  • a “increase” is a statistically significant increase in such
  • a "subject” means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters.
  • Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.
  • the subject is a mammal, e.g., a primate, e.g., a human.
  • the terms, “individual,” “patient” and “subject” are used interchangeably herein.
  • the subject is a mammal.
  • the mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of a disease.
  • a subject can be male or female.
  • protein and “polypeptide” are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.
  • protein and “polypeptide” refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function.
  • modified amino acids e.g., phosphorylated, glycated, glycosylated, etc.
  • amino acid analogs regardless of its size or function.
  • Protein and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps.
  • polypeptide proteins and “polypeptide” are used interchangeably herein when referring to a gene product and fragments thereof.
  • exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.
  • the terms also refer to fragments or variants of the polypeptide that maintain at least 50% of the activity or effect.
  • nucleic acid or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof.
  • the nucleic acid can be either single -stranded or double-stranded.
  • a single -stranded nucleic acid can be one nucleic acid strand of a denatured double- stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double -stranded DNA.
  • the nucleic acid can be DNA.
  • nucleic acid can be RNA.
  • Suitable DNA can include, e.g., genomic DNA or cDNA.
  • Suitable RNA can include, e.g., mRNA.
  • the methods described herein relate to measuring, detecting, or determining the level of at least one agent.
  • detecting or “measuring” refers to observing a signal from, e.g. a probe, label, or target molecule to indicate the presence of an analyte in a sample. Any method known in the art for detecting a particular label moiety can be used for detection. Exemplary detection methods include, but are not limited to, spectroscopic, fluorescent, photochemical, biochemical, immunochemical, electrical, optical or chemical methods. In some embodiments of any of the aspects, measuring can be a quantitative observation.
  • contacting refers to any suitable means for delivering, or exposing, an agent to at least one cell.
  • exemplary delivery methods include, but are not limited to, direct delivery to cell culture medium, perfusion, injection, or other delivery method well known to one skilled in the art.
  • contacting comprises physical human activity, e.g., an injection; an act of dispensing, mixing, and/or decanting; and/or manipulation of a delivery device or machine.
  • compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
  • the term "consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
  • a system comprising: a first subcutaneous compartment comprising: i. a mixture comprising fibroblasts, cell culture medium, hydrogel, and at least one of preadipocytes and adipocytes; and a first peripheral compartment comprising: i. at least one opening providing fluid, non-cellular communication with the first subcutaneous compartment; and ii. optionally comprising endothelial cells.
  • first and/or second lateral portions do not comprise the at least one openings with the first peripheral compartment and optionally, second peripheral compartment.
  • first and second peripheral compartments comprise a medial curved channel portion where they comprise the at least one openings with the first subcutaneous compartment; a first lateral channel, cube, rectangular prism, or cylinder portion; and a second lateral channel, cube, rectangular prism or cylinder portion.
  • a second subcutaneous compartment comprising: i. a mixture comprising fibroblasts, cell culture medium, hydrogel, and at least one of preadipocytes and adipocytes; and a second peripheral compartment comprising: i. at least one opening providing fluid, non-cellular communication with the second subcutaneous compartment; and ii. optionally comprising endothelial cells;
  • a medial injection compartment comprising: i. at least one opening providing fluid, non-cellular communication with the first subcutaneous compartment; and ii. at least one opening providing fluid, non-cellular communication with the second subcutaneous compartment; wherein the first and second subcutaneous compartments are lateral to the medial injection compartment.
  • the medial injection compartment comprises: a medial disc or sphere portion that comprises at least one of the openings with each of the first and second subcutaneous compartments; a first lateral channel, cube, rectangular prism, or cylinder portion; and a second lateral channel, cube, rectangular prism or cylinder portion.
  • the first and/or second lateral portions do not comprise the at least one openings with the first and second subcutaneous compartments.
  • the injection port is in the medial disc or sphere portion.
  • the at least one opening comprises multiple openings or slits in an interposed wall or barrier.
  • the system of any of the preceding paragraphs, wherein the at least one opening is no greater in height or width than 500 pm.
  • the system of any of the preceding paragraphs, wherein the at least one opening is no greater in height or width than 100 pm.
  • the system of any of the preceding paragraphs, wherein the fluid availability aspect ratio is 5 or greater.
  • the system of any of the preceding paragraphs, wherein the fluid availability aspect ratio is 50:3.
  • fibroblasts and at least one of preadipocytes and adipocytes form a 3D culture.
  • the mixture comprising fibroblasts, cell culture medium, hydrogel, and at least one of preadipocytes and adipocytes comprises more preadipocytes and/or adipocytes than fibroblasts.
  • the mixture comprising fibroblasts, cell culture medium, hydrogel, and at least one of preadipocytes and adipocytes comprises at least 2x as many preadipocytes and/or adipocytes as fibroblasts.
  • the mixture comprising fibroblasts, cell culture medium, hydrogel, and at least one of preadipocytes and adipocytes comprises at least 3x as many preadipocytes and/or adipocytes as fibroblasts.
  • the first peripheral compartment comprises endothelial cells.
  • the first peripheral compartment comprises endothelial cells and the second peripheral compartment does not comprise cells.
  • the first and second peripheral compartments each comprise endothelial cells.
  • the endothelial cells line one or more walls of the first and/or second peripheral compartments.
  • the system of any of the preceding paragraphs, wherein the endothelial cells form a confluent single-cell monolayer on all walls of the first and/or second peripheral compartments.
  • the system of any of the preceding paragraphs, wherein the cells are murine or human cells.
  • the system of any of the preceding paragraphs, wherein at least 90% of the cells are viable.
  • each compartment further comprises at least 2 ports.
  • An in vitro method of determining the subcutaneous administration characteristics of a candidate subcutaneous agent comprising: introducing a candidate subcutaneous agent into the injection compartment and/or a peripheral compartment not comprising cells, of the system of any of paragraphs 1- 34; and measuring at least one of: the amount and/or change in the amount of subcutaneous agent in the outflow of fluid from one or more of the compartments; the amount and/or change in the amount of subcutaneous agent present in one or more of the compartments; and the amount and/or change in the amount of subcutaneous agent in the outflow of fluid from a peripheral compartment comprising endothelial cells.
  • the method of paragraph 35 wherein the following are measured: the amount and/or change in the amount of subcutaneous agent present in each of the compartments; and the amount and/or change in the amount of subcutaneous agent in the outflow of fluid from a peripheral compartment comprising endothelial cells.
  • a peripheral compartment not comprising vascular endothelial cells indicates subcutaneous administration characteristics via lymphatic pathways.
  • a peripheral compartment comprising lymphatic endothelial cells but not comprising vascular endothelial cells indicates subcutaneous administration characteristics via lymphatic pathways.
  • a peripheral compartment comprising endothelial cells indicates subcutaneous administration characteristics via vascular pathways.
  • a peripheral compartment comprising vascular endothelial cells indicates subcutaneous administration characteristics via vascular pathways.
  • the candidate subcutaneous agent is introduced into the injection compartment and/or a peripheral compartment not comprising cells at a perfusion rate of from 0. 1 pL/min to 1,000 pL/min.
  • any of paragraphs 35-42 further comprising the following steps prior to the step of introducing the candidate subcutaneous agent: introducing the mixture comprising fibroblasts, cell culture medium, hydrogel, and at preadipocytes into the first and/or second subcutaneous compartment and then culturing the preadipocytes and fibroblasts; inducing adipocyte differentiation and then maintaining the adipocytes and fibroblasts; twice introducing endothelial cells into the first and/or second peripheral compartment and then culturing and maintaining the endothelial cells.
  • culturing and/or maintaining the fibroblasts and at least one of preadipocytes and adipocytes comprises exchanging the culture medium in the first and/or second subcutaneous compartment at least daily.
  • culturing and/or maintaining the endothelial cells comprises one or more of: culturing for at least 3 days; culturing until the endothelial cells reach a high density; and culturing until the endothelial cells are confluent.
  • Example 1 A Subcutaneous Tissue Chip for Assessing Biologies Transport
  • An ideal in vitro model for subcutaneous transport should possess at least the following components: (i) adipocytes as the major cell type and fibroblasts forming the connective tissue (II) the interstitial space containing the extracellular matrix (ECM) within which the drug molecules traverse, and (iii) capillaries and lymphatics embedded in the subcutaneous space to enter systemic circulation 1- 3 .
  • adipocytes as the major cell type and fibroblasts forming the connective tissue
  • ECM extracellular matrix
  • capillaries and lymphatics embedded in the subcutaneous space to enter systemic circulation 1- 3 With the development of organs-on-chips, these key components can be constructed on a microfluidic device using a combination of 2D and 3D cellular cultures 9-10 . The majority of similar systems utilizes custommade devices which provide the most flexibility in chip design and setup.
  • a commercial chip with a three-compartment design for this study which is made of poly dimethylsiloxane (PDMS) on a glass slide (Fig. 4A, Fig. 9). Each channel is 100 rm in depth. The top and bottom compartments are 200 rm wide and the central compartment is 500 rm wide (Fig. 4B).
  • This chip geometry can mimic the subcutaneous transport pathway involving drug injection, transport through the ECM and uptake by blood and lymphatic capillariesl (Fig. 10): the top channel serves as the injection channel containing the liquid solution of interest (Fig.
  • the central channel serves as the subcutaneous channel containing a mixture of adipose cells and fibroblasts organized in a 3D structure; the bottom channel represents vessels which are either lymphatics or capillaries with endothelial cells grown in a 2D monolayer lining the channel walls.
  • the three compartments are connected by narrow slits (3 pm wide X 100 pm deep X 50 pm long with 50 pm spacings, Fig. 4C) which blocks cell migration across channels and enables sequential diffusion of macromolecules from the injection channel.
  • these slits play an important role of controlling the rate of mass transport across the channels without using microvalves. Due to the high aspect ratio design, flow in the neighboring channel is not disturbed when perfusion occurs in one channel (Figs. 11A-11C), thus protecting the soft hydrogel-based subcutaneous tissue from excess fluid shear 12 .
  • These narrow slits also enable media exchange via slow diffusion.
  • the slit size is three to four orders of magnitude larger than the macromolecules and steric hindrance is negligible.
  • the inventors developed a protocol to seed three types of cells from either mice or human into the chip in sequence and maintained the cell culture for at least 10 days (Fig. 4E).
  • the inventors focused on mice cells due to the availability of in vivo data for validation. Fibroblasts and preadipocytes were mixed with cold MATRIGELTM liquid and quickly perfused into the subcutaneous channel. The device was then incubated at 37°C for the mixture to polymerize. Based on our rheological measurements (Fig. 4F, Figs.
  • the inventors seeded endothelial cells into the vessel channel at a high density twice to ensure sufficient attachment of endothelial cells onto the channel walls (Fig. 41, Fig. 15).
  • the inventors left the vessel channel empty such that molecules can diffuse freely.
  • preadipocytes and fibroblasts are difficult to differentiate especially due to their similar morphologies. Based on their 2D morphologies and 3D morphologies when cultured independently (Figs. 16C-16D), preadipocytes are slightly smaller, less elongated and have fewer protrusions. The inventors found that fibroblasts grew and expanded faster than preadipocytes both on the chip as well as in flasks. Therefore, the inventors seeded more preadipocytes to prevent the confluent subcutaneous co-culture from being disproportionate.
  • the inventors examined the tissue structure in the subcutaneous and vascular channels using actin and nucleus staining (Fig. 5G). The inventors confirmed the 3D character of the subcutaneous tissue spanning the entire channel depth (z). In the vessel channel, endothelial cells formed a complete lumen with a confluent monolayer lining the inside of the channel walls, mimicking blood capillaries. This 2D monolayer structure allows perfusion through the channel. The 2D and 3D cellular structures (Fig. 5H) pose distinct types of barriers for the transport mechanism. [00158] Measurement of transport properties and the effect of MW. The inventors measured subcutaneous transport by perfusing fluorescently-labeled macromolecules into the injection channel.
  • the inventors acquired a time series of images under live conditions (Fig. 6A) for at least three hours.
  • the inventors derived analytical expressions describing the concentration changes in each channel using three rate constants: the permeability from the injection channel to the subcutaneous channel If. the diffusivity in the subcutaneous channel D2 and the permeability from the subcutaneous channel into the vessel channel R23 (lymphatic or vascular).
  • Obtaining these expressions is not trivial considering the chip geometry but is much simpler than numerical simulations for data analysis 12 13 .
  • the inventors fitted three rate constants and a good fit was obtained between expected values and experimental values for all three constants (Fig. 6B, Figs. 19A-19B).
  • MATRIGELTM control similar to other in vitro 3D cultures 25,34 . Therefore, it is necessary to include cellular components in in vitro models to provide a more physiological transport environment for several reasons. First, they reduce the interstitial space available for diffusion, which may contribute to interspecies (Figs. 16E-16F) and intertissue differences. Secondly, fibroblasts can produce a network of glycosaminoglycans, collagen and elastin 1 , which reorganizes the existing structure of ECM and its mechanical properties 25,34 .
  • the inventors also investigated the effect of molecular shape using fluorescently labeled IgG which have MW comparable to 150k dextran.
  • the inventors observed a significant reduction in rate constants for IgG (Fig. 7D-7F), consistent with previous findings 6 .
  • IgG usually have spherical shapes in comparison to the linear dextran.
  • the hydrodynamic radius of IgG (5 nm) is thus much bigger than those for dextran (0.8 nm).
  • the inventors observed hindered diffusion in both the subcutaneous tissue as well in the endothelial barrier.
  • the increase in hydrodynamic radius can significantly reduce its diffusivity.
  • serum IgG carry a slightly negative charge under physiological conditions 38 which should facilitate their transport as mentioned earlier, overall the shape effect dominates.
  • lymphatic absorption In vivo measurement of lymphatic absorption is rare due to technical difficulties associated with the surgical preparation in animal models 3 . Based on the limited amount of data in sheep, over 80% transport occurs through the lymphatics after subcutaneous administration for proteins with MW greater than 30kDa, agreeing with our predictions.
  • In vitro measurement comparing a monolayer of vascular endothelial cells and lymphatic endothelial cells showed no significant difference in permeation under static conditions 15 . The difference can be reconciled by a less confluent vascular monolayer in in vitro experiments and the mechanism of lymphatic junctions opening under interstitial pressure 3 . Narrow slits in our model therefore resembles the open structure of lymphatic pores.
  • This length scale depends on the species and the injection site.
  • the Df measured on our device with a 500 pm diffusive distance can therefore be adjusted for predicting the overall rate of absorption instead of selecting different channel geometries.
  • the inventors estimated a rate of absorption close to 100 hours, comparable to the estimate from pharmacokinetic modeling in rats 39 .
  • Microfluidic device and processing Microfluidic chips with three-compartment designs were purchased (catalog # 108011, Synvivo, Inc.). The basic device setup and manipulation follows manufacturer instructions (downloadable at synvivobio.com). In brief, each of the three inlet and three outlet ports was connected to a Tygon ND-100-80 non-DEHP medical tubing (Saint Gobain). During incubation, these tubings were closed using clamps (catalog # 202003, Synvivo, Inc.). During liquid exchange, clamps were removed.
  • Ax is the travel distance of the slits which equals 50 rm.
  • the hindrance factor H is calculated based on the ratio A of dextran molecule’s hydrodynamic radius (4.5 A) relative to the slit size using the
  • Measurements were taken immediately after loading the cold sample and repeated for 1 hour until the sample was fully polymerized 41 .
  • the rheometer was cooled to 2C and equilibrated for 2 minutes before samples were loaded. Measurements were repeated at increasing temperatures until 37 C with 1 -minute equilibration between each temperature.
  • mice subcutaneous fibroblasts L, ATCC
  • mice subcutaneous preadipocytes X9, ATCC
  • mice aortic endothelial cells MAOEC, iXCells
  • human subcutaneous preadipocytes PCS-210-010, ATCC
  • human dermal fibroblasts NHDF, C-12302, PromoCell
  • human endothelial cells EA.hy926, ATCC
  • the injection and vessel channels were perfused with full subcutaneous culture medium (e.g., either DMEM or DMEM/F12 with 20% FBS for mice) at 10 d/min. This media exchange was repeated daily for at least 5 days.
  • full subcutaneous culture medium e.g., either DMEM or DMEM/F12 with 20% FBS for mice
  • adipocyte differentiation was induced by perfusing differentiation medium into the injection channel daily for three days.
  • the differentiation medium contains full subcutaneous cell culture medium (e.g., either DMEM or DMEM/F12 with 20% FBS for mice) supplemented with 5.0 pM dexamethasone, 5 mM methylisobutylxanthine and 100 p g/ml insulin. These supplements were used at higher concentrations than established protocols 42 to account for their low partitioning into the subcutaneous channel.
  • the differentiated cells were maintained using full subcutaneous cell culture medium (e.g., either DMEM or DMEM/F12 with 20% FBS for mice) with 10 rg/ml insulin for daily media exchange.
  • the seeding procedure was repeated with endothelial cells trypsinized from a second culture flask followed by incubation with a normal chip placement for at least 4 hours. Excess cells were washed by endothelial culture medium. Daily media exchange was performed using endothelial culture medium (20% FBS for mice) at 2 d/min.
  • the device was incubated for 1 hour to permeabilize fixed cells. After washing with DPBS for 30 min, ActinRed 555 ReadyProbes (2 drops per 200 ul of DPBS, ThermoFisher) and Hoechst 33342 (2 drops per 200 ul of DPBS, ThermoFisher) were perfused to the injection channel. ActinGreen 488 ReadyProbe (2 drops per 1 ml of DPBS, ThermoFisher) and Hoechst 33342 (2 drops per 1 ml of DPBS) were perfused to the endothelial channel. The device was incubated for at least 3 hours followed by a wash with DPBS, a 30-minute incubation and a second wash with DPBS.
  • Adipocyte differentiation on a plate assay To compare the effect of adipocyte differentiation between fibroblasts and preadipocytes at matching cell densities, the inventors seeded them separately on 6-well plates. Mice cells were maintained to confluency following the same procedure as in tissue culture flasks. Adipocyte differentiation was induced using cell culture medium supplemented with 0.5 p M Dexamethasone, 0.5 mM methylisobutylxanthine and 10 rg/ml insulin for three days. Differentiated cells were maintained using cell culture medium supplemented with 10 jUg/ml insulin for two days. Cells were then fixed using 4% PFA and permeabilized with 0.2% Triton X-100 in DPBS. Cells were incubated with Oil Red O solution for 1 hour and washed before imaging. The stock Oil Red O solution (300 mg/L in isopropanol) was diluted to 180 mg/L with distilled water and filtered before use.
  • Image processing and three-dimensional z-stack reconstruction were done using ImageJTM software (Figs. 20A-20D). Images were first rotated using bilinear interpolation to align with the channel directions. They were then cropped to separate files for subcutaneous and vessel channels for quantitative analysis. For rendering purposes, channels were combined with individually adjusted brightness to account for lower fluorescence intensity in the subcutaneous channel. For Oil Red O and live/dead staining, the inventors combined z-stack images using ZProjection with maximum intensity. For quantification analysis, fluorescence intensities in each channel were converted to binary values (black and white). A threshold area was used in the analyze particle function to detect individual cells or lipid droplets. Lipid content was determined as the total stained area.
  • Live and dead cell counts were determined from the particle counts. Cell viability may be underestimated due to overlapping live cells unable to be detected.
  • dead stains were processed using a median filter with a radius of 2.0.
  • the confocal 3D rendering of actin and nucleus staining was performed with interpolation using ImageJ 3D Project. The fluorescence intensity across channel depth in each channel was plotted using plot Z-profile.
  • FITC-dextran (MW 10k, 40k and 150k), FITC-DEAE-dextran (40k), FITCCM-dextran (40k) were obtained from Sigma Aldrich.
  • Mouse FITC-IgG was obtained from Southern Biotechnology Associates. Acquisition of images fortransport measurement were performed using an inverted microscope (Axio Observer Zl, Zeiss) with an incubator chamber (Zeiss) to maintain 37°C, 5% CO2 and controlled humidity for live cell imaging. Image acquisition was configured by establishing a calibration curve of fluorescence intensity vs. concentration on the chip using a 5X objective. At the beginning of each experiment, 1 mg/ml solution in DPBS was flown into the injection channel. For cellular devices, images were acquired at 1-minute intervals for 3 to 6 hours. For acellular devices used for control experiments, images were acquired at 30-second intervals for 2 hours.
  • the inventors therefore approximated the injection and vessel channels as well-stirred compartments with only temporal variations in concentration represented by Ci(t) and (t) respectively.
  • the concentration profde in the subcutaneous channel is denoted Cz(x, t).
  • the inventors assumed Ci » C2 » C3 throughout the experiment, which is validated by comparing their fluorescence intensities at the end of each experiment (Fig. 22E). This assumption allowed us to calculate R12, D and R23 individually to obtain analytical solutions and bypass simulation approaches which are more time-consuming and less user-friendly. After converting all fluorescence intensities to concentrations, the inventors performed data fitting in MATLAB using Isqcurvefit.
  • D 2 is the diffusivity in the subcutaneous space.
  • e is the distance to the fixed concentration source and T is a reflective distance such that at
  • Example 3 The effect of flow conditions on microfluidic processing
  • a finite element simulation of the flow profile on a multi -compartment microfluidic chip with interconnecting narrow slits reveal that when pressure driven flow occurs in one channel to drive perfusion, the neighboring channel experiences a disturbance flow no greater than 5% the inlet velocity due to the high aspect ratio between the slit and channel dimensions 12 .
  • the inventors approximated the flow through slits as multiple resistances connected in sequence and parallel 43 (Fig.
  • the inventors considered the influence of flow conditions on nutrient transfer during both perfusion and static incubation.
  • the Peclet number describing ratio of advection rate to diffusion rate is written as: .
  • the inventors estimated the Peclet number to be at least 200000 for the slowest perfusion rate used (2 d/min). Therefore, the perfusion process is convection dominated and the entire liquid content in the perfusion channel was replaced.
  • this ratio is typically
  • n The number density of cells n is assumed to be only a function of z.
  • the inventors only consider binary hydrodynamic interactions which result in shear-induced diffusion 45,47 jdiffu ion - Due to the cell deformability, they migrate away from the wall under stokes flow with a velocity viift(z), generating a lift flux jm 48 .
  • the lift flux can be written as:
  • the shear-induced diffusional flux /diffusion is proportional to the product of the number densities of two particles multiplied by the z velocity difference between them and is an integral over all possible spatial configurations 47 . According to previous work 46 , this flux can be simplified to the following expression:
  • the inhomogeneity in the concentration profile increases with Ca (Fig. 13). Therefore, it is important that the inventors maintain a low flow rate during cell seeding.

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Abstract

Described herein are systems with multiple compartments for culturing more or more cell types and, e.g., modeling or reproducing subcutaneous tissue or environments. The systems described herein are suitable for, e.g., in vitro methods of characterizing the subcutaneous administration charactertistics of agents.

Description

SYSTEMS AND METHODS RELATING TO SUBCUTANEOUS ADMINISTRATION
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63/085,216 filed September 30, 2020, the contents of which are incorporated herein by reference in their entirety.
TECHNICAL FIELD
[0002] The technology described herein relates to in vitro systems for modeling or studying subcutaneous administration.
BACKGROUND
[0003] Subcutaneous drug administration is a preferred route of administration due to ease and relative low invasiveness. However, for this route of administration to be effective the drug or formulation must be able to transit the subcutaneous environment and enter a vessel (blood or lymphatic) for systemic administration to operate. The most advanced in vitro model so far is an acellular system made of a buffer solution and a membrane with a single measurable quantity. This simple setup only offers limited information regarding the complex transport process involving the interaction between subcutaneous microstructure and drug properties such as molecular weight, shape, charge and formulation viscosity. Currently there is an urgent need of reliable preclinical models to study transport processes in the subcutaneous environment.
SUMMARY
[0004] Described herein is a fluidic system utilizing 3D subcutaneous cultures and optionally, 2D vascular or lymphatic barriers. The system is responsive to the effects of molecular weight, charge and size influencing transport rates. It is demonstrated herein that lymphatic transport played a dominant role in the clearance of macromolecules, and the overall rate of absorption can be predicted from the subcutaneous diffusion.
[0005] In one aspect of any of the embodiments, described herein is a system comprising a first subcutaneous compartment comprising a mixture comprising fibroblasts, cell culture medium, hydrogel, and at least one of preadipocytes and adipocytes; and a first peripheral compartment comprising at least one opening providing fluid, non-cellular communication with the first subcutaneous compartment; and optionally comprising endothelial cells.
[0006] In some embodiments of any of the aspects, the system further comprises a second peripheral compartment comprising at least one opening providing fluid, non-cellular communication with the first subcutaneous compartment. In some embodiments of any of the aspects, the first subcutaneous compartment is medial with respect to the first and second peripheral compartments; and the first and second peripheral compartments are not directly in fluid, non-cellular communication with each other. In some embodiments of any of the aspects, each compartment comprises or is a channel, cube, rectangular prism, or cylinder.
[0007] In some embodiments of any of the aspects, the first subcutaneous compartment comprises a medial disc or sphere portion that comprises at least one of the openings with the first peripheral compartment and optionally, second peripheral compartment; a first lateral channel, cube, rectangular prism, or cylinder portion; and a second lateral channel, cube, rectangular prism or cylinder portion. In some embodiments of any of the aspects, the first subcutaneous compartment comprises a disc or sphere portion that comprises at least one of the openings with the first peripheral compartment and optionally, second peripheral compartment; a first channel, cube, rectangular prism, or cylinder portion; and a second channel, cube, rectangular prism or cylinder portion. In some embodiments of any of the aspects, the first and/or second lateral portions do not comprise the at least one openings with the first peripheral compartment and optionally, second peripheral compartment. In some embodiments of any of the aspects, the first and second peripheral compartments comprise a medial curved channel portion where they comprise the at least one openings with the first subcutaneous compartment; a first lateral channel, cube, rectangular prism, or cylinder portion; and a second lateral channel, cube, rectangular prism or cylinder portion. In some embodiments of any of the aspects, the first and second peripheral compartments comprise a curved channel portion where they comprise the at least one openings with the first subcutaneous compartment; a first channel, cube, rectangular prism, or cylinder portion; and a second channel, cube, rectangular prism or cylinder portion.
[0008] In some embodiments of any of the aspects, the system further comprises a second subcutaneous compartment comprising a mixture comprising fibroblasts, cell culture medium, hydrogel, and at least one of preadipocytes and adipocytes; and a second peripheral compartment comprising at least one opening providing fluid, non-cellular communication with the second subcutaneous compartment; and optionally comprising endothelial cells; a medial injection compartment comprising at least one opening providing fluid, non-cellular communication with the first subcutaneous compartment; and at least one opening providing fluid, non-cellular communication with the second subcutaneous compartment; wherein the first and second subcutaneous compartments are lateral to the medial injection compartment. In some embodiments of any of the aspects, the system further comprises a second subcutaneous compartment comprising a mixture comprising fibroblasts, cell culture medium, hydrogel, and at least one of preadipocytes and adipocytes; and a second peripheral compartment comprising at least one opening providing fluid, non-cellular communication with the second subcutaneous compartment; and optionally comprising endothelial cells; a injection compartment comprising at least one opening providing fluid, non- cellular communication with the first subcutaneous compartment; and at least one opening providing fluid, non-cellular communication with the second subcutaneous compartment. In some embodiments of any of the aspects the medial injection compartment comprises: a medial disc or sphere portion that comprises at least one of the openings with each of the first and second subcutaneous compartments; a first lateral channel, cube, rectangular prism, or cylinder portion; and a second lateral channel, cube, rectangular prism or cylinder portion. In some embodiments of any of the aspects the injection compartment comprises: a disc or sphere portion that comprises at least one of the openings with each of the first and second subcutaneous compartments; a first channel, cube, rectangular prism, or cylinder portion; and a second channel, cube, rectangular prism or cylinder portion. In some embodiments of any of the aspects, the first and/or second lateral portions do not comprise the at least one openings with the first and second subcutaneous compartments. In some embodiments of any of the aspects, the first and second subcutaneous compartments each comprise a medial curved channel portion where they comprise at least one of the openings with the medial injection compartment; a first lateral channel, cube, rectangular prism, or cylinder portion; and a second lateral channel, cube, rectangular prism or cylinder portion. In some embodiments of any of the aspects, the first and second subcutaneous compartments each comprise a curved channel portion where they comprise at least one of the openings with the medial injection compartment; a first channel, cube, rectangular prism, or cylinder portion; and a second channel, cube, rectangular prism or cylinder portion. In some embodiments of any of the aspects, the medial curved channel portion of the first subcutaneous compartment comprises at least one of the openings with the first peripheral compartment; and the medial curved channel portion of the second subcutaneous compartment comprises at least one of the openings with the second peripheral compartment. In some embodiments of any of the aspects, the curved channel portion of the first subcutaneous compartment comprises at least one of the openings with the first peripheral compartment; and the curved channel portion of the second subcutaneous compartment comprises at least one of the openings with the second peripheral compartment.
[0009] In some embodiments of any of the aspects, the medial injection channel, first subcutaneous compartment, and/or second subcutaneous compartment further comprises an injection port. In some embodiments of any of the aspects, the injection channel, first subcutaneous compartment, and/or second subcutaneous compartment further comprises an injection port. In some embodiments of any of the aspects, the injection port is in the disc or sphere portion, e.g., the medial disc or sphere portion.
[0010] In some embodiments of any of the aspects, the at least one opening comprises multiple openings or slits in an interposed wall or barrier. In some embodiments of any of the aspects, the at least one opening is no greater in height or width than 500 pm. In some embodiments of any of the aspects, the at least one opening is no greater in height or width than 100 pm. In some embodiments of any of the aspects, the fluid availability aspect ratio is 5 or greater. In some embodiments of any of the aspects, the fluid availability aspect ratio is 50:3. [0011] In some embodiments of any of the aspects, the fibroblasts and at least one of preadipocytes and adipocytes form a 3D culture. In some embodiments of any of the aspects, the mixture comprising fibroblasts, cell culture medium, hydrogel, and at least one of preadipocytes and adipocytes comprises more preadipocytes and/or adipocytes than fibroblasts. In some embodiments of any of the aspects, the mixture comprising fibroblasts, cell culture medium, hydrogel, and at least one of preadipocytes and adipocytes comprises at least 2x as many preadipocytes and/or adipocytes as fibroblasts. In some embodiments of any of the aspects, the mixture comprising fibroblasts, cell culture medium, hydrogel, and at least one of preadipocytes and adipocytes comprises at least 3x as many preadipocytes and/or adipocytes as fibroblasts.
[0012] In some embodiments of any of the aspects, the first peripheral compartment comprises endothelial cells. In some embodiments of any of the aspects, the first peripheral compartment comprises endothelial cells and the second peripheral compartment does not comprise cells. In some embodiments of any of the aspects, the first and second peripheral compartments each comprise endothelial cells. In some embodiments of any of the aspects, the endothelial cells line one or more walls of the first and/or second peripheral compartments. In some embodiments of any of the aspects, the endothelial cells form a confluent single-cell monolayer on all walls of the first and/or second peripheral compartments.
[0013] In some embodiments of any of the aspects, the cells are murine or human cells. In some embodiments of any of the aspects, at least 80% of the cells are viable. In some embodiments of any of the aspects, at least 90% of the cells are viable.
[0014] In some embodiments of any of the aspects, each compartment further comprises at least 2 ports. In some embodiments of any of the aspects, the at least 2 ports are connected to a microfluidics system to provide an inflow and an outflow port in each compartment.
[0015] In some embodiments of any of the aspects, the hydrogel comprises one or more extracellular matrix components.
[0016] In one aspect of any of the embodiments, described herein is an in vitro method of determining the subcutaneous administration characteristics of a candidate subcutaneous agent, the method comprising: introducing a candidate subcutaneous agent into the injection compartment and/or a peripheral compartment not comprising cells, of the system described herein; and measuring at least one of: the amount and/or change in the amount of subcutaneous agent in the outflow of fluid from one or more of the compartments; the amount and/or change in the amount of subcutaneous agent present in one or more of the compartments; and the amount and/or change in the amount of subcutaneous agent in the outflow of fluid from a peripheral compartment comprising endothelial cells.
[0017] In some embodiments of any of the aspects, the following are measured:the amount and/or change in the amount of subcutaneous agent present in each of the compartments; and the amount and/or change in the amount of subcutaneous agent in the outflow of fluid from a peripheral compartment comprising endothelial cells. In some embodiments of any of the aspects, a peripheral compartment not comprising vascular endothelial cells indicates subcutaneous administration characteristics via lymphatic pathways. In some embodiments of any of the aspects, a peripheral compartment comprising lymphatic endothelial cells but not comprising vascular endothelial cells indicates subcutaneous administration characteristics via lymphatic pathways. In some embodiments of any of the aspects, a peripheral compartment comprising endothelial cells indicates subcutaneous administration characteristics via vascular pathways. In some embodiments of any of the aspects, a peripheral compartment comprising vascular endothelial cells indicates subcutaneous administration characteristics via vascular pathways.
[0018] In some embodiments of any of the aspects, the candidate subcutaneous agent is introduced into the injection compartment and/or a peripheral compartment not comprising cells at a perfusion rate of from 0.1 pL/min to 1,000 pL/min.
[0019] In some embodiments of any of the aspects, the method further comprises the following steps prior to the step of introducing the candidate subcutaneous agent: introducing the mixture comprising fibroblasts, cell culture medium, hydrogel, and preadipocytes into the first and/or second subcutaneous compartment and then culturing the preadipocytes and fibroblasts; inducing adipocyte differentiation and then maintaining the adipocytes and fibroblasts. In some embodiments of any of the aspects, the method further comprises the following steps prior to the step of introducing the candidate subcutaneous agent: introducing the mixture comprising fibroblasts, cell culture medium, hydrogel, and at preadipocytes into the first and/or second subcutaneous compartment and then culturing the preadipocytes and fibroblasts; inducing adipocyte differentiation and then maintaining the adipocytes and fibroblasts; twice introducing endothelial cells into the first and/or second peripheral compartment and then culturing and maintaining the endothelial cells.
[0020] In some embodiments of any of the aspects, the mixture comprising fibroblasts, cell culture medium, hydrogel, and at least one of preadipocytes and adipocytes is at less a temperature of less than 10 C when it is introduced into the first and/or second subcutaneous compartment. In some embodiments of any of the aspects, the mixture comprising fibroblasts, cell culture medium, hydrogel, and at least one of preadipocytes and adipocytes is at less a temperature of 4 C or less when it is introduced into the first and/or second subcutaneous compartment.
[0021] In some embodiments of any of the aspects, culturing the fibroblasts and at least one of preadipocytes and adipocytes comprises one or more of: culturing for at least 5 days; culturing until the fibroblasts and at least one of preadipocytes and adipocytes reach a high density; and culturing until the fibroblasts and at least one of preadipocytes and adipocytes are confluent. In some embodiments of any of the aspects, culturing and/or maintaining the fibroblasts and at least one of preadipocytes and adipocytes comprises exchanging the culture medium in the first and/or second subcutaneous compartment at least daily. In some embodiments of any of the aspects, culturing and/or maintaining the fibroblasts and at least one of preadipocytes and adipocytes comprises exchanging the culture medium in the first and/or second subcutaneous compartment continuously. In some embodiments of any of the aspects, medium exchange in the injection compartment and/or a peripheral compartment not comprising cells is performed at a perfusion rate of from 0.1 pL/min to 1000 pL/min.
[0022] In some embodiments of any of the aspects, culturing and/or maintaining the endothelial cells comprises one or more of: culturing for at least 3 days; culturing until the endothelial cells reach a high density; and culturing until the endothelial cells are confluent. In some embodiments of any of the aspects, culturing and/or maintaining the endothelial cells comprises exchanging the culture medium in the first and/or second peripheral compartment at least daily. In some embodiments of any of the aspects, culturing and/or maintaining the endothelial cells comprises exchanging the culture medium in the first and/or second peripheral compartment continuously. In some embodiments of any of the aspects, medium exchange in the first and/or second peripheral compartment is performed at a perfusion rate of from 0.02 pL/min to 200 pL/min.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Fig. 1 depicts an exemplary emobodiment of the systems described herein.
[0024] Fig. 2 depicts an exemplary emobodiment of the systems described herein.
[0025] Fig. 3 depicts an exemplary emobodiment of the systems described herein.
[0026] Figs. 4A-4I depict constructing the subcutaneous tissue in an idealized geometry. (Fig. 4)
A three-compartment commercial chip made of PDMS and glass. (Fig. 4B) Three compartments represent various environments relevant for subcutaneous injection. (Fig. 4C) A close-up look of the narrow slits connecting adjacent channels. (Fig. 4D) Permeability across narrow slits in cell-free chips was measured using FITC-dextran (MW = 40k), which depends on slit size and agrees with the estimation based on a porous media without hindrance. (Fig. 4E) Timeline of microfluidic cell culture. (Fig. 4F) Rheological properties of the Matrigel: medium mixture (Mix) show temperature dependence similar to pure Matrigel (MG). (Fig. 4G) The uniformity of the initial cell distribution in the subcutaneous channel after seeding was examined using actin staining and confocal microscopy. (Fig. 4H) Top view (x-y) of the growth and expansion of mice fibroblasts and preadipocytes. (Fig. 41) Top view (x-y) of the growth and expansion of mice endothelial cells. Scale bars: (Fig. 4H) 50 pm, (Fig. 41) 100 pm. Error bars represent standard error of the mean.
[0027] Figs. 5A-5H depict the characterization of cellular functions and properties on a chip. (Fig. 5A) Top view of the morphology of subcutaneous cells (fibroblasts and preadipocytes) cultured in 3D. (Fig. 5B) Top view of the morphology of subcutaneous cells after adipocyte differentiation. (Fig. 5C) Presence of lipid droplets stained by Oil Red O after adipocyte induction. (Fig. 5D) Comparison of Oil Red O stained area before and after adipocyte induction. (Fig. 5E) Live/dead staining for cells (top view). (Fig. 5F) Cell viability in subcutaneous and endothelial channels. (Fig. 5G) Actin and nucleus staining visualized by confocal microscope (top and side x-z views). (Fig. 5H) Fluorescence intensity in the z direction in endothelial and subcutaneous channels indicating 2D (peaks near z = 0, 100 pm) and 3D (uniform distribution) structures. Scale bars: 200 pm. Error bars represent standard error of the mean.
[0028] Figs. 6A-6F depict the measurement of transport properties and the effect of MW. (Fig. 6A) Measurements were performed in a live cell imaging system (left), tracking the change of fluorescence intensity overtime (right). (Fig. 6B) A good fit between theoretical estimation and experimental measurement was obtained in the subcutaneous channel for both the instantaneous spatial concentration profile (left) and the overall spatiotemporal concentration profile (right) (Figs. 6C-6E) The effect of MW on FITC-dextran transport for R12 (Fig. 6C) D2 (Fig. 6D) and R23 (Fig. 6E). (Fig. 6G) The comparison between D2 and existing studies as a function of MW. Scale bars: 200 pm. Error bars represent standard error of the mean.
[0029] Figs. 7A-7F depict the influence of molecule charge and shape on subcutaneous transport. The influence of molecular charge on
Figure imgf000009_0001
(Fig. 7C). The influence of molecular shape on
Figure imgf000009_0002
(Fig. 7F). Error bars represent standard error of the mean.
[0030] Figs. 8A-8B depict the prediction of circulation pathways and in vivo pharmacokinetics. (Fig. 8 A) Percentage of lymphatic transport compared to vascular transport based on the ratio of
Figure imgf000009_0003
(Fig. 8B) Estimation of the time of subcutaneous absorption as a function of interstitial distance s in comparison to the chip dimension. The timescales of entering and exiting the subcutaneous space are also provided for reference. Error bars represent standard error of the mean.
[0031] Fig. 9 depicts an image of an exemplary embodiment of the system described herein, e.g., with connecting tubes for each inlet and outlet port.
[0032] Fig. 10 depicts a schematic of subcutaneous injection.
[0033] Figs. 11A-11C depict estimation of the narrow-slit effects. (Fig. 11A) Perfusion occurs in the injection compartment or peripheral compartment not comprising cells with the flow rate controlled by a pump. The other two channels are clamped on both inlet and outlet ends to prevent evaporation. (Fig. 1 IB) In addition to the injection compartment or peripheral compartment not comprising cells, flow can occur in the adjacent subcutaneous compartment as a series of resistances via 100 narrow slits along the compartment. The disturbance flow in the subcutaneous compartment be estimated based on the aspect ratio. (Fig. 11C) The resulting disturbance velocity in the subcutaneous compartment normalized by the inlet flow velocity.
[0034] Figs. 12A-12E depict the temperature-dependent rheological properties of the Matrigel: medium mixture. (Fig. 12A) Schematic for handling the mixture at different temperatures. (Figs. 12B- 12D) Frequency sweep measurement of the complex modulus at various temperatures indicating distinct physical state of the mixture: (Fig. 12B) liquid (Fig. 12C) transition (Fig. 12D) gel. (Fig. 12E) Flow sweep measurement of the shear-thinning property of the Matrigel at 37 C. Hysteresis was observed when the cold mixture was placed on a 37C plate and disappeared after the mixture fully polymerized and reached equilibrium.
[0035] Fig. 13 is a graph depicting the nonhomogeneous concentration distribution of cells under pressure-driven flow in channels. Two cases are plotted: strong flow of deformable cells (Ca =100), and weak flow of deformable cells (Ca=l).
[0036] Fig. 14 depicts images of the top view of the growth and expansion of mice fibroblasts and preadipocytes cultured in 3D on a chip. Scale bars: 100 [im.
[0037] Fig. 15 depicts images of the top view of the growth and expansion of endothelial 2D monolayer on chip. The seeding procedure was repeated once to achieve a high confluency. Scale bars: 100 im.
[0038] Figs. 16A-16F depict images of the top view of the cell morphology under phase contrast microscope. (Fig. 16A) Mice fibroblasts in 2D tissue culture flask. (Fig. 16B) Mice preadipocytes in 2D tissue culture flask. (Fig. 16C) Mice fibroblasts in 3D on chip. (Fig. 16D) Mice preadipocytes in 3D on chip. (Fig. 16E) Mice endothelial cells in 2D tissue culture flask. (Fig. 16F) Human preadipocytes and fibroblasts in 3D (top) and endothelial cells in a 2D monolayer (bottom) on chip. [0039] Fig. 17 depicts a graph of the comparison of Oil Red O stained area before and after adipocyte differentiation induction for mice preadipocytes (X9) and fibroblasts (L) cells on a plate assay.
[0040] Fig. 18 depicts a graph of a calibration curve on a chip for transport measurement using fluorescence imaging.
[0041] Figs. 19A-19B depict graphs of fit versus experimental measurement for the injection channel (Fig. 19A) and the vessel channel (Fig. 19B).
[0042] Figs. 20A-20D illustrate image processing in ImageJ. After rotation and cropping for individual channels, zstack images were projected to one image with fluorescence intensities converted to binary values for stained live (Fig. 20A) and dead (Fig. 20C) cells. The analyze particle function was used to detect individual live (Fig. 20B) and dead (Fig. 20D) cells. The live cell count may be underestimated due to clustering. Cell viability was calculated based on the ratio of live cells to the total number of cells.
[0043] Fig. 21 dpiect MATLAB workflow of image analysis for transport measurement.
[0044] Figs. 22A-22G depict justifications for assumptions made for transport calculations. (Fig. 22A) Instantaneous % deviation from average concentration in the injection channel. (Fig. 22B) Time evolution of the standard error of mean of the concentration in the injection channel. (Fig. 22C) Instantaneous % deviation from y-averaged concentration in the subcutaneous channel (Fig. 22D) Standard error of mean of the y-averaged concentration in the subcutaneous channel. (Fig. 22E) Instantaneous % deviation from average concentration in the vessel channel. (Fig. 22F) Time evolution of the standard error of mean of the concentration in the vessel channel. (Fig. 22G) Concentration in subcutaneous and vascular channels relative to the injection channel at the end of measuremen.
[0045] Fig. 23 depicts a graph of R23 for lymphatic transport
[0046] Fig. 24 depicts a table summarizing flow rates used for microfluidic processing and the reasoning for the flow conditions.
[0047] Figs. 25 and 26 depict two exemplary embodiments of the systems described herein, demonstrating that the rate constants in these comparments match those in linear channels after adjusting for geometic differences.
DETAILED DESCRIPTION
[0048] Described herein are systems for culture of one or more cell types, e.g., for providing an in vitro model of drug transport following subcutaneous administration. The systems comprise one or more compartments connected by at least one opening providing fluid, non-cellular communication between the compartments. As used herein, “compartment” refers to a void within a device or system shaped to permit the presence, maintenance, or growth of cells. In some embodiments of any of the aspects, a compartment can be a channel or a portion of a channel. In some embodiments of any of the aspects, a compartment can comprise one or more portions with different shapes and dimensions. Suitable shapes for compartments or portions thereof include channels, cubes, rectangular prisms, cylinders, discs, spheres, and combinations thereof.
[0049] In some embodiments, a compartment can comprise or be connected to one or more channels, ports, etc. As used herein, the term “channel” refers to any capillary, channel, tube, or groove that is deposed within or upon a substrate. A channel can be a microchannel; i.e. a channel that is sized for passing through microvolumes of liquid. As used herein, the term “port” refers to a portion of the system which provides a means for fluid, cells, or gases to enter and/or exit the system. The port can be of a size and shape to accept and/or secure a connection with tubes, connections, or adaptors of a fluidic device and allow passage of fluid when attached to a fluidic device.
[0050] The systems and devices described herein can comprise multiple compartments. The compartments, depending on layout of the system and the further components of the system can be of one or more subtypes of compartment, as described herein. Two subtypes of compartments are a) subcutaneous compartments and b) peripheral compartments. These types of compartments are defined by the contents of the compartments. A subcutanteous compartment comprises preadipocytes, adipocytes, and/or fibroblasts and optionally cell culture medium and/or hydrogel. A peripheral compartment does not comprise preadipocytes and/or fibroblasts.
[0051] In some embodiments of any of the aspects, a compartment can comprise fluid and/or media inflow and/or media outflow means. The inflow and/or outflow means can be channel or a port. The inflow and/or outflow means can comprise a connection to a manual fluid flow device, e.g., a manually operated syringe and/or to an automated fluid flow device, e.g., a fluidics device and/or pump.
[0052] As used herein, the phrases “connected to,” “coupled to,” “in contact with” and “in communication with” refer to any form of interaction between two or more entities, including mechanical, electrical, magnetic, electromagnetic, fluidic, and thermal interaction. For example, in one embodiment, two compartments in a system are in fluidic communication each other. Fluidic communication means that at least a portion of the fluids in a first compartment can travel to the second compartment, and/or vice versa. Fluidic communication may be directional or reciprocal. Fluidic, non-cellular communication means that that at least a portion of the fluids in a first compartment can travel to the second compartment and/or vice versa but that cells in either compartment do not travel between compartments. When two compartments are in direct communication, the interaction occurs without the intermediary of another compartment. When two compartments are in indirect communication, the interactions occurs via an intermediary compartment.
[0053] In one aspect, provided herein is a system comprising a first subcutaneous compartment 10 comprising a mixture comprising preadipocytes, fibroblasts, cell culture medium, and hydrogel; and a first peripheral compartment 20 comprising at least one opening 30 providing fluid, non-cellular communication with the first subcutaneous compartment 10; and optionally comprising endothelial cells.
[0054] One exemplary embodiment of the system described herein is depicted in Fig. 1. In some embodiments of any of the aspects, the system further comprises a second peripheral compartment 21 comprising at least one opening 31 providing fluid, non-cellular communication with the first subcutaneous compartment 10. In some embodiments of any of the aspects, the first subcutaneous compartment 10 is medial with respect to the first peripheral compartment 20 and second peripheral compartment 21, e.g., the first subcutaneous compartment 10 is found between the first peripheral compartment 20 and second peripheral compartment 21. In some embodiments of any of the aspects, the first subcutaneous compartment 10, first peripheral compartment 20, and second peripheral compartment 21, each comprise an axis of fluid flow and the first subcutaneous compartment 10 is medial with respect to the first peripheral compartment 20 and second peripheral compartment 21 along this axis of fluid flow e.g., for at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or for the entirety of the axis of fluid flow. In some embodiments of any of the aspects, the first peripheral compartment 20 and second peripheral compartment 21 are not directly in fluid, non-cellular communication with each other. In some embodiments of any of the aspects, each of the the first subcutaneous compartment 10, first peripheral compartment 20, and second peripheral compartment 21 comprises or is a channel, cube, rectangular prism, or cylinder. [0055] One exemplary embodiment of the system described herein is depicted in Fig. 2. In some embodiments of any of the aspects, the first subcutaneous compartment 10 comprises a medial disc or sphere portion 11 that comprises at least one of the openings 30 with the first peripheral compartment 20. In some embodiments of any of the aspects, the first subcutaneous compartment 10 comprises a medial disc or sphere portion 11 that comprises at least one of the openings 31 with the second peripheral compartment 21. In some embodiments of any of the aspects, the disc or sphere portion 11 of the first subcutaneous compartment 10 is located in the medial portion of the axis of fluid flow in the first subcutaneous compartment 10. In some embodiments of any of the aspects, the disc or sphere portion 11 of the first subcutaneous compartment 10 is located in the medial portion of the longest dimensional axis of the first subcutaneous compartment 10.
[0056] In some embodiments of any of the aspects, the first subcutaneous compartment 10 comprises a first lateral channel, cube, rectangular prism, or cylinder portion 12 and a second lateral channel, cube, rectangular prism or cylinder portion 13. In some embodiments of any of the aspects, the first lateral channel, cube, rectangular prism, or cylinder portion 12 and second lateral channel, cube, rectangular prism or cylinder portion 13 are lateral with respect to the medial disc or sphere portion 11. In some embodiments, the medial disc or sphere portion 11, the first lateral channel, cube, rectangular prism, or cylinder portion 12, and second lateral channel, cube, rectangular prism or cylinder portion 13 are arranged along the axis of fluid flow in the first subcutaneous compartment 10 in the order of: 1) the first lateral channel, cube, rectangular prism, or cylinder portion 12, 2) the medial disc or sphere portion 11, and 3) second lateral channel, cube, rectangular prism or cylinder portion 13. In some embodiments, the medial disc or sphere portion 11, the first lateral channel, cube, rectangular prism, or cylinder portion 12, and second lateral channel, cube, rectangular prism or cylinder portion 13 are arranged along the longest dimensional axis of the first subcutaneous compartment 10 in the order of: 1) the first lateral channel, cube, rectangular prism, or cylinder portion 12, 2) the medial disc or sphere portion 11, and 3) second lateral channel, cube, rectangular prism or cylinder portion 13. In some embodiments of any of the aspects, the first lateral channel, cube, rectangular prism, or cylinder portion 12 and/or second lateral channel, cube, rectangular prism or cylinder portion 13 do not comprise the at least one openings 30, 31 with the first peripheral compartment 20 and optionally, second peripheral compartment 21.
[0057] In some embodiments of any of the aspects, the first and second peripheral compartments 20, 21 each comprise a medial curved channel portion 22 where they comprise the at least one openings 30, 31 with the first subcutaneous compartment 10. In some embodiments of any of the aspects, the the first and second peripheral compartments 20, 21 each comprise a first lateral channel, cube, rectangular prism, or cylinder portion 23 and a second lateral channel, cube, rectangular prism or cylinder portion 24. [0058] In some embodiments, the medial curved channel portion 22, the first lateral channel, cube, rectangular prism, or cylinder portion 23, and second lateral channel, cube, rectangular prism or cylinder portion 24 are arranged along the fluid flow axis of the first or second peripheral compartment 20, 21 in the order of: 1) the first lateral channel, cube, rectangular prism, or cylinder portion 23, 2) medial curved channel portion 22, and 3) the second lateral channel, cube, rectangular prism, or cylinder portion 24. In some embodiments, the medial curved channel portion 22, the first lateral channel, cube, rectangular prism, or cylinder portion 23, and second lateral channel, cube, rectangular prism or cylinder portion 24 are arranged along the longest dimensional axis of the first or second peripheral compartment 20, 21 in the order of: 1) the first lateral channel, cube, rectangular prism, or cylinder portion 23, 2) medial curved channel portion 22, and 3) the second lateral channel, cube, rectangular prism, or cylinder portion 24. In some embodiments of any of the aspects, the first lateral channel, cube, rectangular prism, or cylinder portion 23 and/or second lateral channel, cube, rectangular prism or cylinder portion 24 do not comprise the at least one openings 30, 31 with the subcutaneous compartment 10.
[0059] A further exemplary embodiment of the system described herein is depicted in Fig. 3. In some embodiments of any of the aspects, the system further comprises a second subcutaneous compartment 14 comprising a mixture comprising preadipocytes, fibroblasts, cell culture medium, and hydrogel; a second peripheral compartment 21 comprising at least one opening 31 providing fluid, non-cellular communication with the second subcutaneous compartment 14 and optionally comprising endothelial cells; a medial injection compartment 40 comprising at least one opening 50 providing fluid, non-cellular communication with the first subcutaneous compartment 10, and at least one opening 51 providing fluid, non-cellular communication with the second subcutaneous compartment 14. In some embodiments of any of the aspects, the first subcutaneous compartment 10 and second subcutaneous compartment 14 are lateral with respect to the medial injection compartment. In some embodiments of any of the aspects, the first peripheral compartment 20 and second peripheral compartment 21 are more lateral with respect to the medial injection compartment than the first subcutaneous compartment 10 and second subcutaneous compartment 14.
[0060] In some embodiments of any of the aspects, the medial injection compartment 40 comprises a medial disc or sphere portion 41 that comprises at least one of the openings 50, 51 with each of the first and second subcutaneous compartments 10, 14. In some embodiments of any of the aspects, the the medial injection compartment 40 comprises a first lateral channel, cube, rectangular prism, or cylinder portion 42 and a second lateral channel, cube, rectangular prism or cylinder portion 43. In some embodiments of any of the aspects, the first and/or second lateral portions 42, 43 do not comprise the at least one openings 50, 51 with the first and second subcutaneous compartments 10, 14. [0061] In some embodiments of any of the aspects, the first and second subcutaneous compartments 10, 14 each comprise a medial curved channel portion 15, 16 where they comprise at least one of the openings 50, 51 with the medial injection compartment 40. In some embodiments of any of the aspects, the first and second subcutaneous compartments 10, 14 each comprise a first lateral channel, cube, rectangular prism, or cylinder portion 17; and a second lateral channel, cube, rectangular prism or cylinder portion 18. In some embodiments, the medial curved channel portion 15, 16, the first lateral channel, cube, rectangular prism, or cylinder portion 17, and second lateral channel, cube, rectangular prism or cylinder portion 18 are arranged along the fluid flow axis of the first or second subcutaneous compartment 10, 14 in the order of: 1) the first lateral channel, cube, rectangular prism, or cylinder portion 17 2) medial curved channel portion 15, 16, and 3) the second lateral channel, cube, rectangular prism, or cylinder portion 18. In some embodiments, the medial curved channel portion 15, 16, the first lateral channel, cube, rectangular prism, or cylinder portion 17, and second lateral channel, cube, rectangular prism or cylinder portion 18 are arranged along the longest dimensional axis of the first or second subcutaneous compartment 10, 14 in the order of: 1) the first lateral channel, cube, rectangular prism, or cylinder portion 17, 2) medial curved channel portion 15, 16, and 3) the second lateral channel, cube, rectangular prism, or cylinder portion 18. In some embodiments of any of the aspects, the first lateral channel, cube, rectangular prism, or cylinder portion 17 and/or second lateral channel, cube, rectangular prism or cylinder portion 18 do not comprise the at least one openings 50, 51 with the medial injection compartment 10. In some embodiments of any of the aspects, the first lateral channel, cube, rectangular prism, or cylinder portion 17 and/or second lateral channel, cube, rectangular prism or cylinder portion 18 do not comprise the at least one openings 30, 31 with the first and/or second peripheral compartments 20, 21. [0062] In some embodiments of any of the aspects the medial curved channel portion 15 of the first subcutaneous compartment 10 comprises at least one of the openings 30 with the first peripheral compartment. In some embodiments of any of the aspects the medial curved channel portion 16 of the second subcutaneous compartment 14 comprises at least one of the openings 31 with the second peripheral compartment 21.
[0063] In some embodiments of any of the aspects, one or more of the compartments described herein can further comprise an injection port 60. An injection port can either be the only port connected to a compartment, e.g., such that directional fluid flow cannot be provided by a paired inflow and outflow means; or the injection port can be arrayed between an inflow means and an outflow means, thereby providing a means to inject material directly into the compartment instead of flowing it in via media. In some embodiments of any of the aspects, the medial injection channel, first subcutaneous compartment, and/or second subcutaneous compartment further comprises an injection port. In some embodiments of any of the aspects, the medial injection channel further comprises an injection port. In some embodiments of any of the aspects, first subcutaneous compartment further comprises an injection port. In some embodiments of any of the aspects, the second subcutaneous compartment further comprises an injection port. In some embodiments of any of the aspect, the injection port in the medial disc or sphere portion of a compartment.
[0064] Various embodiments of the systems described herein comprise at least one opening that provides fluid, non-cellular communication between two compartments. The openings can be provided in any shape (e.g., circular, ovoid, rectangular, etc), profde, or when multiple openings are present, any combination thereof. In some embodiments of any of the aspects, the at least one opening comprises an opening or slit in an interposed wall or barrier. In some embodiments of any of the aspects, the at least one opening comprises multiple openings or slits in an interposed wall or barrier.
[0065] Preferably, each of the at least one openings should have a height and width in the wall of the compartment that is smaller than the smallest dimension of the cells present in either chamber. This dimension will vary slightly depending on the types and species origin of the cells present in the system. In some embodiments of any of the aspects, the at least one opening is no greater in height or width than 500 pm, e.g., no greater in height or width than 450 pm, no greater in height or width than 400 pm, no greater in height or width than 350 pm, no greater in height or width than 300 pm, no greater in height or width than 250 pm, no greater in height or width than 200 pm, no greater in height or width than 150 pm, no greater in height or width than 100 pm, no greater in height or width than 75 pm, no greater in height or width than 50 pm, no greater in height or width than 25 pm, or no greater in height or width than 10 pm. In some embodiments of any of the aspects, the at least one opening is no greater in height or width than 500 pm. In some embodiments of any of the aspects, the at least one opening is no greater in height or width than 100 pm.
[0066] In some embodiments of any of the aspects, the at least one opening has a high fluid availability aspect ratio. That is, along the length of the compartment wall, there is more wall than openings so that at any moment in time less than half of the fluid along the wall of the compartment is available for fluid exchange through the openings. The fluid aspect ratio can be expressed as the ratio of solid wall to opening along the length of the compartment’s (or portion of the compartment’s) wall. For example, if every 50 pm along a wall, there is a 3 pm slit connecting a first compartment and a second compartment, only sees 3/50 of the first compartment’s fluid is available for exchange to the second compartment. An advantage of a high fluid availability aspect ration is that flow in a second compartment is not disturbed when perfusion occurs in a first channel. This can protect, e.g., the soft hydrogel-based subcutaneous tissue in a subcutaneous compartment from excess fluid shear.
[0067] In some embodiments of any of the aspects, the fluid availability aspect ratio is 5 or greater, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or greater. In some embodiments of any of the aspects, the fluid availability aspect ratio is 5 or greater. In some embodiments of any of the aspects, the fluid availability aspect ratio is from 50:0.5 to 50: 10. In some embodiments of any of the aspects, the fluid availability aspect ratio is from 50: 1 to 50:5. In some embodiments of any of the aspects, the fluid availability aspect ratio is from 50:2 to 50:4. In some embodiments of any of the aspects, the fluid availability aspect ratio is 50:3.
[0068] The structures of the systems described herein (e.g. the compartments, ports, and/or walls) can be formed, such as by etching, 3-D printing, machining, or micro-machining. In some embodiments, the system described herein is etching-free. The system described herein can be made of a biocompatible material(s) according to the design and application requirements. It should be noted that the designs depicted in the Figures are exemplary and the system described herein is not limited to the configurations shown in the Figures. The system and/or portions thereof can be made of a flexible material, including but not limited to, a biocompatible material such as polydimethyl siloxane (PDMS), polyurethane or polyimide. The system and/or portions thereof can also be made of non-flexible materials like glass, silicon, polysulfone, hard plastic, and the like, as well as combinations of these materials.
[0069] A biocompatible polymer refers to materials which do not have toxic or injurious effects on biological functions. Biocompatible polymers include natural or synthetic polymers. Examples of biocompatible polymers include, but are not limited to, collagen, poly(alpha esters) such as poly(lactate acid), poly(glycolic acid), polyorthoesters and polyanhydrides and their copolymers, polyglycolic acid and polyglactin, cellulose ether, cellulose, cellulosic ester, fluorinated polyethylene, phenolic, poly-4-methylpentene, polyacrylonitrile, polyamide, polyamideimide, polyacrylate, polybenzoxazole, polycarbonate, polycyanoarylether, polyester, polyestercarbonate, polyether, polyetheretherketone, polyetherimide, polyetherketone, polyethersulfone, polyethylene, polyfluoroolefin, polyimide, polyolefin, polyoxadiazole, polyphenylene oxide, polyphenylene sulfide, polypropylene, polystyrene, polysulfide, polysulfone, polytetrafluoroethylene, polythioether, polytriazole, polyurethane, polyvinyl, polyvinylidene fluoride, regenerated cellulose, silicone, ureaformaldehyde, polyglactin, or copolymers or physical blends of these materials.
[0070] A biocompatible material can also be, for example, ceramic coatings on a metallic substrate. But any type of coating material and the coating can be made of different types of materials: metals, ceramics, polymers, hydrogels or a combination of any of these materials. Biocompatible materials include, but are not limited to an oxide, a phosphate, a carbonate, a nitride or a carbonitride. Among the oxide the following ones are preferred: tantalum oxide, aluminum oxide, iridium oxide, zirconium oxide or titanium oxide. Substrates are made of materials such as metals, ceramics, polymers or a combination of any of these. Metals such as stainless steel, Nitinol, titanium, titanium alloys, or aluminum and ceramics such as zirconia, alumina, or calcium phosphate are of particular interest.
[0071] The biocompatible polymer may be shaped using methods such as, for example, solvent casting, compression molding, fdament drawing, meshing, leaching, weaving and coating. In solvent casting, a solution of one or more polymers in an appropriate solvent, such as methylene chloride, is cast as a branching pattern relief structure. After solvent evaporation, a thin film is obtained. In compression molding, a polymer is pressed at pressures up to 30,000 pounds per square inch into an appropriate pattern. Filament drawing involves drawing from the molten polymer and meshing involves forming a mesh by compressing fibers into a felt-like material. In leaching, a solution containing two materials is spread into a shape close to the final form of the RUG. Next a solvent is used to dissolve away one of the components, resulting in pore formation. (See Mikos, U.S. Pat. No. 5,514,378, hereby incorporated by reference). In nucleation, thin films in the shape of a RUG are exposed to radioactive fission products that create tracks of radiation damaged material. Next the polycarbonate sheets are etched with acid or base, turning the tracks of radiation-damaged material into pores. Finally, a laser may be used to shape and bum individual holes through many materials to form a RUG structure with uniform pore sizes. Coating refers to coating or permeating a polymeric structure with a material such as, for example liquefied copolymers (poly-DU-lactide co-glycolide 50:50 80 mg/ml methylene chloride) to alter its mechanical properties. Coating may be performed in one layer, or multiple layers until the desired mechanical properties are achieved. These shaping techniques may be employed in combination, for example, a polymeric matrix may be weaved, compression molded and glued together. Furthermore different polymeric materials shaped by different processes may be joined together to form a composite shape. The composite shape may be a laminar structure. For example, a polymeric matrix may be attached to one or more polymeric matrixes to form a multilayer polymeric matrix structure. The attachment may be performed by gluing with a liquid polymer or by suturing. In addition, the polymeric matrix may be formed as a solid block and shaped by laser or other standard machining techniques to its desired final form. Uaser shaping refers to the process of removing materials using a laser.
[0072] The subcutaneous compartment of a system described herein comprises preadipocytes, adipocytes, and/or fibroblasts. In some embodiments, a subcutaneous compartment comprises preadipocytes and fibroblasts. In some embodiments, a subcutaneous compartment comprises adipocytes and fibroblasts. In some embodiments, a subcutaneous compartment comprises preadipocytes, adipocytes and fibroblasts. In some embodiments, preadipocytes and fibroblasts are introduced into a subcutaneous compartment of a system described herein. In some embodiments, adipocytes, and fibroblasts are introduced into a subcutaneous compartment of a system described herein. In some embodiments of any of the aspects, a subcutaneous compartment further comprises cell cuulture medium and/or hydrogel. In some embodiments of any of the aspects, a subcutaneous compartment further comprises cell cuulture medium and hydrogel.
[0073] In some embodiments of any of the aspects, the adipocytes and fibroblasts form a 3D culture. Forming a 3D culture means the cells are not grown in the traditional manner of a single layer, requiring a substrate surface for the growth to occur. Rather, the adipocytes and fibroblasts to grow to confluency such that cells which are stable in their position can be found throughout the entire width/height of the compartment. It is noted that in 3D culture, there are small porous structures in the culture which permit fluid to flow freely through the culture (as opposed to fluid flowing over one surface of a culture in traditional single-layer culture).
[0074] In some embodiments of any of the aspects, the mixture comprising preadipocytes (or adipocytes), fibroblasts, cell culture medium, and hydrogel comprises more preadipocytes (or adipocytes) than fibroblasts. In some embodiments of any of the aspects, the mixture comprising preadipocytes (or adipocytes), fibroblasts, cell culture medium, and hydrogel comprises at least 2x as many preadipocytes (or adipocytes) than fibroblasts, e.g., at least 2x, 3x, 4x, 5x , 6x, 7x, 8x, 9x, lOx, 15x, 20x or more. In some embodiments of any of the aspects, the mixture comprising preadipocytes (or adipocytes), fibroblasts, cell culture medium, and hydrogel comprises at least 2x as many preadipocytes (or adipocytes) than fibroblasts. In some embodiments of any of the aspects, the mixture comprising preadipocytes (or adipocytes), fibroblasts, cell culture medium, and hydrogel comprises at least 3x as many preadipocytes (or adipocytes) than fibroblasts.
[0075] Adipocytes, also known as lipocytes or fat cells, are the primary cells found in adipose tissue. Adipocytes can be white fat cells or brown fat cells. Adipocytes differentiate from preadipocytes. Markers and phenotypes of both cell types are known in the art, e.g., during differentiation, induction of C/EBPJ3, C/EBP5, PPARy, C/EBPa, aP2, and Glut4 are observed. Preadipocytes display CD45- CD31- CD34+ CD29+ SCA1+ CD24+ surface marker profiles.
[0076] A fibroblast is a connective tissue cell that produces the ECM and collagen. Markers and phenotypes of fibroblasts are known in the art, e.g., they express the surface markers of CD90 and FAP.
[0077] As used herein, “hydrogel” refers to a network of hydrophilic polymer chains. In some embodiments of any of the aspects, the hydrogel comprises one or more extracellular matrix components, e.g., laminin, nidogen, collagen, growth factors (e.g., TGF-beta and EGF) and proteoglycans with cell adhesive peptides. In some embodiments of any of the aspects, the hydrogel comprises laminin, nidogen, collagen, growth factors (e.g., TGF-beta and EGF) and proteoglycans with cell adhesive peptides. In some embodiments of any of the aspects, the hydrogel comprises or consists essentially of MATRIGEL™ .
[0078] In some embodiments of any of the aspects, the first and/or second peripheral compartment comprises endothelial cells. In some embodiments of any of the aspects, the first peripheral compartment comprises endothelial cells. In some embodiments of any of the aspects, the first and second peripheral compartment each comprise endothelial cells. In some embodiments of any of the aspects, the first peripheral compartment comprises endothelial cells and the second peripheral compartment does not comprise cells. [0079] Endothelial cells are cells which occur on the interior surface of blood and lymphatic vessels, e.g., those in contact with blood or lymph in the lumen of a vessel. In some embodiments of any of the aspects, the endothelial cell is a lymphatic endothelial cell. In some embodiments of any of the aspects, the endothelial cells is a vascular endothelial cell. Markers and phenotypes of endothelial cells are known in the art, e.g., they can be distinguished from epithelial cells by the presence of vimentin and the absence of keratin.
[0080] In some embodiments of any of the aspects, the endothelial cells line one or more walls of the first and/or second peripheral compartments. In some embodiments of any of the aspects, the endothelial cells form a confluent single-cell monolayer on at least one wall of the first and/or second peripheral compartments. In some embodiments of any of the aspects, the endothelial cells form a confluent single-cell monolayer on any wall of the first and/or second peripheral compartment comprising an at least one opening in fluid, non-cellular communication with a subcutaneous compartment. In some embodiments of any of the aspects, the endothelial cells form a confluent single-cell monolayer on all walls of the first and/or second peripheral compartments.
[0081] The cells described herein can be from any suitable source, e.g., they can be cell lines or primary cells. In some embodiments, the cells are mammalian cells. In some embodiments of any of the aspects, the cells are murine cells. In some embodiments of any of the aspects, the cells are human cells. In some embodiments, the cells are primary cells, cultured cells, passaged cells, immortalized cells, transgenic cells, genetically modified cells, diseased cells or cells from an animal with a disease, or cells differentiated from stem cells, embryonic stem cells (ESCs), or induced pluripotent stem cells (IPSCs).
[0082] In some embodiments of any of the aspects, at least 70% of the cells present in a system are viable. In some embodiments of any of the aspects, at least 80% of the cells present in a system are viable. In some embodiments of any of the aspects, at least 90% of the cells present in a system are viable. In some embodiments of any of the aspects, at least 70% of the cells present in a system are viable for at least 24 hours. In some embodiments of any of the aspects, at least 80% of the cells present in a system are viable for at least 24 hours. In some embodiments of any of the aspects, at least 90% of the cells present in a system are viable for at least 24 hours. In some embodiments of any of the aspects, at least 70% of the cells present in a system are viable for at least 48 hours. In some embodiments of any of the aspects, at least 80% of the cells present in a system are viable for at least 48 hours. In some embodiments of any of the aspects, at least 90% of the cells present in a system are viable for at least 48 hours.
[0083] As described elsewhere herein, one or more of the compartments described herein can comprise at least 1 port, e.g., for introducing medium, agents, and/or cells. In some embodiments of any of the aspects, one or more of the compartments described herein can each comprise 2 ports. In some embodiments of any of the aspects, each of the compartments described herein comprises 2 ports. In some embodiments of any of the aspects, the 2 ports are distal to each other, e.g., such that one port can serve as an inflow means and the second port can serve as an outflow means to provide fluid flow to at least 60%, at least 70%, at least 80%, at least 90%, or more of the compartment. In some embodiments of any of the aspects, the 2 ports are lateral with respect to a medial portion of the compartment. In some embodiments of any of the aspects, the 2 ports are each located within different lateral portions of the compartment. Where 2 ports are present in a compartment, it is contemplated that they can be connected to a fluidics device, e.g., microfluidics device to provide an inflow and an outflow port in each compartment.
[0084] A “fluidic” device, composition, machine, or system is one capable of moving any amount of fluid, e.g. a fluidic device can be a microfluidic device or a device capable of moving larger volumes of fluid. A fluidic device provides one or more fluids from a fluid source to the system and preferably, removes one or more fluids after they have transited one or more compartments in the system. As used herein, the term “microfluidic” refers to a device, composition, system, or machine capable of the containing or manipulation of microliter and/or nanoliter volumes of fluids.
[0085] A fluid source can be a reservoir or other container comprising a volume of fluid such that the fluid can be caused to move from the fluid source and through the one or more compartments of the system. The fluid source can be coupled to the one or more compartments of the system by any means of conducting a fluid, e.g. tubing, piping, channels, or the like. Either positive or negative fluid pressure, or both, can be used to cause the fluid to flow through the system’s compartment(s). The fluidic device can further utilize gravity and/or pumps to cause the fluid to flow. A fluidic device can further comprise valves to control inflow and outflow to and from the device. A fluidic device can also be connected to a control system, such as a machine or computer system, to permit automated control of the valves and the fluid flow. The machine may comprise a personal computer (PC), a tablet, a Personal Digital Assistant (PDA), a cellular telephone, a web appliance, a network router, switch or bridge, or any machine capable of executing a set of instructions (sequential or otherwise) that specify actions to be taken by that machine. Further, while only a single machine is illustrated, the term "machine" shall also be taken to include any collection of machines that individually or jointly execute a set (or multiple sets) of instructions to perform any one or more of the methodologies discussed herein.
[0086] In some embodiments of any of the aspects, the fluid flow rate can be constant. In some embodiments of any of the aspects, the fluid flow rate can be varied in a regular, cyclic pattern. In some embodiments the fluid flow rate can be varied in an irregular pattern. In some embodiments of any of the aspects, the control of the fluid flow can be automated.
[0087] In some embodiments of any of the aspects, the size, shape and configuration of the system described herein can be selected so that the system can be used as a replacement for chips provided by manufacturers or suppliers for a particular fluidics or microfluidic devices. The ports can be provided in the appropriate size and shape necessary to accept the tubes and/or connectors of a particular fluidic or microfluidic device.
[0088] It should be understood that processes and techniques described herein with respect to automated control of fluid flow are not inherently related to any particular apparatus and may be implemented by any suitable combination of components. Further, various types of general purpose devices may be used in accordance with the teachings described herein. It may also prove advantageous to construct a specialized apparatus to perform the functions described herein. Those skilled in the art will appreciate that many different combinations of hardware, software, and firmware will be suitable for practicing the disclosed embodiments.
[0089] The fluid which is caused to flow through the one or more compartments of the system described herein can be any fluid appropriate for maintaining or culturing the cells present in the compartments of the device. In some embodiments, different fluids can be caused to flow through different compartments. Fluids can comprise cell culture medium, solutions, buffers, nutrients, tracer compounds, dyes, antimicrobials, or other compounds not toxic to the cells being cultured in the cell culture system described herein. One of ordinary skill in the art is well aware of suitable fluids for culturing or maintaining the cells described herein. By way of non-limiting example, fluids suitable for maintaining or culturing endothelial cells can include DMEM (ATCC) supplemented with 10% FBS. Fluids suitable for maintaining or culturing preadipocytes and/or fibroblasts can include DMEM/F12 (ATCC) supplemented with 15% FBS or fibroblast basal medium with low-serum growth kit (ATCC). Fluids suitable for maintaining or culturing any of the cells described herein can include full subcutaneous culture medium (e.g., either DMEM or DMEM/F12 with 20% FBS for mice) at 10 d/min. Fluids suitable for maintaining or culturing adipocytes and/or fibroblasts can include full subcutaneous cell culture medium (e.g., either DMEM or DMEM/F12 with 20% FBS for mice) with 10 /ig/ml insulin.
[0090] Prior to seeding endothelial cells, 200 rg/ml human fibronectin in PBS was perfused into the vessel channel. The device was then incubated for at least 45 minutes and the vessel channel was washed with endothelial culture medium at 10 d/min. Confluent endothelial cells were trypsinized from culture flasks, resuspended in endothelial culture medium at 1 X 107 cells/ml and seeded into the vessel channel at 5 d/min. The chip was then incubated for 90 minutes with top side down to promote adhesion to the top surface. Excess cells which were not adhered to channel surfaces were washed by endothelial culture medium. The seeding procedure was repeated with endothelial cells trypsinized from a second culture flask followed by incubation with a normal chip placement for at least 4 hours. Excess cells were washed by endothelial culture medium. Daily media exchange was performed using endothelial culture medium (20% FBS for mice) at 2 d/min. [0091] In some embodiments, one or more walls of a compartment described herein can be treated or coated with one or more cell adhesive materials to promote attachment of cells. Such attachment materials and treatments are known in the art. Non-limiting examples of types of attachment molecules include collagen; collagen Type I, collagen Type II; collagen Type III; collagen Type IV; collagen Type V; collagen Type VI; collagen Type VII; collagen Type VIII; collagen Type IX, collagen Type X; collagen Type XI; collagen Type XII; collagen Type XIII; collagen Type XIV; extracellular matrix, MATRIGEL™; laminin; proteoglycan; vitronectin; fibronectin; poly-D-lysine; elastin; hyaluronic acid; glycoasaminoglycans; integrin; polypeptides, oligonucleotides, DNA, and/or polysaccharide.
[0092] The systems described herein can be utilized in methods of culturing, studying, analyzing and/or maintaining the described cells, e.g, the behavior and charateristics of the cells under conditions provided by the described systems. In particular, the systems permit the in vitro study, analysis, and characterization of subcutaneous administration characteristics of a candidate subcutaneous agent.
[0093] In one aspect of any of the embodiments, described herein is an in vitro method of determining the subcutaneous administration characteristics of a candidate subcutaneous agent, the method comprising i) introducing a candidate subcutaneous agent into the injection compartment and/or a peripheral compartment not comprising cells, of a system described herien; and ii) measuring at least one of: the amount and/or change in the amount of subcutaneous agent in the outflow of fluid from one or more of the compartments; the amount and/or change in the amount of subcutaneous agent present in one or more of the compartments; and the amount and/or change in the amount of subcutaneous agent in the outflow of fluid from a peripheral compartment comprising endothelial cells. In some embodiments, the method comprises measuring the amount and/or change in the amount of subcutaneous agent present in each of the compartments; and the amount and/or change in the amount of subcutaneous agent in the outflow of fluid from a peripheral compartment comprising endothelial cells.
[0094] Different systems described herein are particularly suitable for modeling different in vivo administration pathways. For example, in some embodiments of any of the aspects, a peripheral compartment not comprising endothelial cells can indicate and/or model subcutaneous administration characteristics via lymphatic pathways. In some embodiments of any of the aspects, a peripheral compartment not comprising vascular endothelial cells can indicate and/or model subcutaneous administration characteristics via lymphatic pathways. In some embodiments of any of the aspects, a peripheral compartment comprising lymphatic endothelial cells and not comprising vascular endothelial cells can indicate and/or model subcutaneous administration characteristics via lymphatic pathways. [0095] Alternatively, a peripheral compartment comprising endothelial cells can indicate and/or model subcutaneous administration characteristics via vascular pathways.
[0096] The candidate agent can be introduced into an injection compartment and/or a peripheral compartment not comprising cells by adding it directly via an injection port, or by adding it to media provided by the inflow port of the compartment. In some embodiments of any of the aspects, the compartment to which the candidate agent is introduced is perfused with media during the introduction of the candidate agent, regardless of the means by which the candidate agent itself is introduced to the compartment. In some embodiments of any of the aspects, the compartment to which the candidate agent is introduced is perfused with media from at least the the introduction of the candidate agent until the measuring steps are complete, regardless of the means by which the candidate agent itself is introduced to the compartment. In some embodiments of any of the aspects, the compartment to which the candidate agent is introduced is perfused at a rate of from about 0.01 pL/min to about 10,000 pL/min. In some embodiments of any of the aspects, the compartment to which the candidate agent is introduced is perfused at a rate of from 0.01 pL/min to 10,000 pL/min. In some embodiments of any of the aspects, the compartment to which the candidate agent is introduced is perfused at a rate of from 0.1 pL/min to 1,000 pL/min. In some embodiments of any of the aspects, the compartment to which the candidate agent is introduced is perfused at a rate of from 1 pL/min to 100 pL/min. In some embodiments of any of the aspects, the compartment to which the candidate agent is introduced is perfused at a rate of from 5 pL/min to 50 pL/min. In some embodiments of any of the aspects, the compartment to which the candidate agent is introduced is perfused at a rate of from 1 pL/min to 50 pL/min. In some embodiments of any of the aspects, the compartment to which the candidate agent is introduced is perfused at a rate of from 10 pL/min to 100 pL/min. In some embodiments of any of the aspects, the compartment to which the candidate agent is introduced is perfused at a rate of from 50 pL/min to 100 pL/min.
[0097] In some embodiments, the subcutaneous compartment of the system comprises adipocytes. The adipocyte can be added to the subcutaneous compartment, or preadipocytes can be added to the subcutaneous compartment and differentiated in the compartment. Accordingly, in some embodiments, prior to the step of introducing a candidate agent, the methods described herein can comprise introducing a mixture comprising preadipocytes, fibroblasts, cell culture medium, and extracellular matrix material into the first and/or second subcutaneous compartment and then culturing the preadipocytes and fibroblasts; inducing adipocyte differentiation; and then maintaining the adipocytes and fibroblasts.
[0098] In some embodiments, one or more peripheral compartments of the system comprise endothelial cells. Endothelial cells can be introduced in a single dose or multiple dose. In some embodiments, prior to the step of introducing a candidate agent, the methods described herein can comprise introducing introducing a mixture comprising preadipocytes, fibroblasts, cell culture medium, and extracellular matrix material into the first and/or second subcutaneous compartment and then culturing the preadipocytes and fibroblasts; inducing adipocyte differentiation; maintaining the adipocytes and fibroblasts; and introducing endothelial cells into the first and/or second peripheral compartment and then culturing and maintaining the endothelial cells. In some embodiments, prior to the step of introducing a candidate agent, the methods described herein can comprise introducing introducing a mixture comprising preadipocytes, fibroblasts, cell culture medium, and extracellular matrix material into the first and/or second subcutaneous compartment and then culturing the preadipocytes and fibroblasts; inducing adipocyte differentiation; maintaining the adipocytes and fibroblasts; and twice introducing endothelial cells into the first and/or second peripheral compartment and then culturing and maintaining the endothelial cells.
[0099] Introduction of mixtures and/or cells into the system can be performed via perfusion. In some embodiments, the mixture and/or cell is introduced into the system at a low temperature, e.g., to minimize cell metabolism or modulate viscosity. In some embodiments of any of the aspects, the mixture comprising preadipocytes, fibroblasts, cell culture medium, and extracellular matrix material is at less a temperature of less than 20 C when it is introduced into the first and/or second subcutaneous compartment. In some embodiments of any of the aspects, the mixture comprising preadipocytes, fibroblasts, cell culture medium, and extracellular matrix material is at less a temperature of less than 10 C when it is introduced into the first and/or second subcutaneous compartment. In some embodiments of any of the aspects, the mixture comprising preadipocytes, fibroblasts, cell culture medium, and extracellular matrix material is at a temperature of 4 C or less when it is introduced into the first and/or second subcutaneous compartment.
[00100] As used herein, “maintaining” or “culturing” refers to continuing the viability of a population of cells. A maintained tissue will have a population of metabolically active cells. The number of these cells can be roughly stable over a period of at least 1 day or can grow.
[00101] In the context of cell ontogeny, the term "differentiated", or "differentiating" is a relative term. A "differentiated cell" is a cell that has progressed further down the developmental pathway than the cell it is being compared with. Thus, stem cells can differentiate to lineage-restricted precursor cells, which in turn can differentiate into other types of precursor cells further down the pathway, and then to an end-stage differentiated cell, which play a characteristic role in a certain tissue type, and may or may not retain the capacity to proliferate further.
[00102] The term "differentiation" as referred to herein refers to the process whereby a cell moves further down the developmental pathway and begins expressing markers and phenotypic characteristics known to be associated with a cell that are more specialized and closer to becoming terminally differentiated cells. Differentiation is a developmental process whereby cells assume a more specialized phenotype, e.g., acquire one or more characteristics or functions distinct from other cell types. In some cases, the differentiated phenotype refers to a cell phenotype that is at the mature endpoint in some developmental pathway (a so called terminally differentiated cell). In many, but not all tissues, the process of differentiation is coupled with exit from the cell cycle. In these cases, the terminally differentiated cells lose or greatly restrict their capacity to proliferate. However, in the context of this specification, the terms "differentiation" or "differentiated" refer to cells that are more specialized in their fate or function than at one time in their development. For example in the context of this application, a differentiated cell includes an adipocytes which has differentiated from a preadipocyte. A cell that is “differentiated" relative to a progenitor cell has one or more phenotypic differences relative to that progenitor cell and characteristic of a more mature or specialized cell type. Phenotypic differences include, but are not limited to morphologic differences and differences in gene expression and biological activity, including not only the presence or absence of an expressed marker, but also differences in the amount of a marker and differences in the co-expression patterns of a set of markers.
[00103] Methods of differentiating adipocytes are known in the art. As a non-limiting example, preadipocyte differentiation to adipocytes can be induced by perfusing differentiation medium daily for three days. The differentiation medium contains full subcutaneous cell culture medium (e.g., either DMEM or DMEM/F12 with 20% FBS for mice) supplemented with 5.0 rM dexamethasone, 5 mM methylisobutylxanthine and 100 . g/ml insulin. The differentiated cells can be maintained using full subcutaneous cell culture medium (e.g., either DMEM or DMEM/F12 with 20% FBS for mice) with 10 jUg/ml insulin for daily media exchange.
[00104] The cells described herein can be cultured for any amount of time prior to the introduction of a candidate agent. In some embodiments, the preadipocytes/adipocytes and/or fibroblasts are cultured for at least one of the following periods prior to introduction of a candidate agent: at least 5 days; until the preadipocytes/adipocytes and/or fibroblasts reach a high density; and until the preadipocytes/adipocytes and/or fibroblasts are confluent. As used in reference to preadipocytes/adipocytes and/or fibroblasts, high density means that the number of cells no longer increases.
[00105] In some embodiments, the endothelial cells are cultured for at least one of the following periods prior to introduction of a candidate agent: at least 3 days; until the endothelial cells reach a high density; and until the endothelial cells are confluent. As used in reference to endothelial cells, high density means at least 90% confluency.
[00106] During culture/maintenance of cells described herein, media can be perfused continuously or exchanged at intervals. Media can be perfused or exchanged by causing direct fluid flow via inflow/outflow ports in the relevant compartment, or media can be perfused or exchanged in a first compartment by causing fluid flow in a second compartment that is in fluid, non-cellular communication with the first compartment. In some embodiments, the second compartment does not comprise cells. [00107] In some embodiments of any of the aspects, culturing the preadipocytes/adipocytes and/or fibroblast and/or maintaining the adipocytes and fibroblasts comprises exchanging the culture medium in the first and/or second subcutaneous compartment at least daily. In some embodiments of any of the aspects, culturing the preadipocytes/adipocytes and/or fibroblast and/or maintaining the adipocytes and fibroblasts comprises exchanging the culture medium in the first and/or second subcutaneous compartment continuously.
[00108] In some embodiments of any of the aspects, media exchange in the injection compartment and/or a peripheral compartment not comprising cells is performed at a perfusion rate of from about 0.01 pL/min to about 10,000 pL/min. In some embodiments of any of the aspects, media exchange in the injection compartment and/or a peripheral compartment not comprising cells is performed at a perfusion rate of from 0.01 pL/min to 10,000 pL/min. In some embodiments of any of the aspects, media exchange in the injection compartment and/or a peripheral compartment not comprising cells is performed at a perfusion rate of from 0.1 pL/min to 1,000 pL/min. In some embodiments of any of the aspects, media exchange in the injection compartment and/or a peripheral compartment not comprising cells is performed at a perfusion rate of from 1 pL/min to 100 pL/min. In some embodiments of any of the aspects, media exchange in the injection compartment and/or a peripheral compartment not comprising cells is performed at a perfusion rate of from 5 pL/min to 50 pL/min. In some embodiments of any of the aspects, media exchange in the injection compartment and/or a peripheral compartment not comprising cells is performed at a perfusion rate of from 1 pL/min to 50 pL/min. In some embodiments of any of the aspects, media exchange in the injection compartment and/or a peripheral compartment not comprising cells is performed at a perfusion rate of from 10 pL/min to 100 pL/min. In some embodiments of any of the aspects, media exchange in the injection compartment and/or a peripheral compartment not comprising cells is performed at a perfusion rate of from 50 pL/min to 100 pL/min.
[00109] In some embodiments of any of the aspects, culturing or maintaining endothelial cells comprises exchanging the culture medium in the first and/or second peripheral compartment at least daily. In some embodiments of any of the aspects, culturing or maintaining endothelial cells comprises exchanging the culture medium in the first and/or second peripheral compartment continuously. In some embodiments of any of the aspects, medium exchange in the first and/or second peripheral compartment is performed at a perfusion rate of from about 0.01 pL/min to about 500 pL/min. In some embodiments of any of the aspects, medium exchange in the first and/or second peripheral compartment is performed at a perfusion rate of from 0.01 pL/min to 500 pL/min. In some embodiments of any of the aspects, medium exchange in the first and/or second peripheral compartment is performed at a perfusion rate of from 0.02 pL/min to 200 pL/min. In some embodiments of any of the aspects, medium exchange in the first and/or second peripheral compartment is performed at a perfusion rate of from 2 pL/min to 20 pL/min. In some embodiments of any of the aspects, medium exchange in the first and/or second peripheral compartment is performed at a perfusion rate of from 1 pL/min to 100 pL/min. In some embodiments of any of the aspects, medium exchange in the first and/or second peripheral compartment is performed at a perfusion rate of from 1 pL/min to 50 pL/min. In some embodiments of any of the aspects, medium exchange in the first and/or second peripheral compartment is performed at a perfusion rate of from 10 pL/min to 200 pL/min. In some embodiments of any of the aspects, medium exchange in the first and/or second peripheral compartment is performed at a perfusion rate of from 20 pL/min to 100 pL/min.
[00110] As used herein, the terms “candidate compound” or “candidate agent” refer to a compound or agent and/or compositions thereof that are to be screened and/or analyzed for their subcutaneous administration behavior/characteristics. As used herein, the terms “compound” or “agent” are used interchangeably and refer to molecules and/or compositions. The compounds/agents include, but are not limited to, chemical compounds and mixtures of chemical compounds, e.g., small organic or inorganic molecules; saccharines; oligosaccharides; polysaccharides; biological macromolecules, e.g., peptides, proteins, and peptide analogs and derivatives; peptidomimetics; nucleic acids; nucleic acid analogs and derivatives; extracts made from biological materials such as bacteria, plants, fungi, or animal cells or tissues; naturally occurring or synthetic compositions; peptides; aptamers; and antibodies and intrabodies, or fragments thereof.
[00111] Generally, compounds can be tested at any concentration. In some embodiments, compounds are tested at concentration in the range of about 0. InM to about lOOOmM. In one embodiment, the compound is tested in the range of about 0. 1 pM to about 20pM, about 0.1 pM to about lOpM, or about 0. IpM to about 5pM. In one embodiment, compounds are tested at 1 pM. Candidate agents can be introduce free in solution in the media, or in pharmaceutically relevant formulations, e.g., liposomes, nanoparticles, controlled-release formulations, etc. For the methods described herein, test compounds may be screened individually, or in groups. Group screening is particularly useful where hit rates for effective test compounds are expected to be low such that one would not expect more than one positive result for a given group.
[00112] Candidate agents can be produced recombinantly using methods well known to those of skill in the art (see Sambrook et al., Molecular Cloning: A Laboratory Manual (2 ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1989)). Methods for developing small molecule, polymeric and genome based libraries are described, for example, in Ding, et al. J Am. Chem. Soc. 124: 1594-1596 (2002) and Lynn, et al., J. Am. Chem. Soc. 123: 8155-8156 (2001). Commercially available compound libraries can be obtained from, e.g., ArQule (Woburn, MA), Panvera (Madison, WI), Ryan Scientific (Mt. Pleasant, SC), and Enzo Life Sciences (Plymouth Meeting, PA). These libraries can be screened, e.g. methods described herein. [00113] In some embodiments of any of the apects, the amount of an agent in one or more compartments and/or outflows is determined at a particular point in time. In some embodiments of any of the aspects, the amount of an agent in a compartment and/or outflow is determined at at least two particular points in time, thereby permitting the change in amount and/or rate of change of the amount to be determined.
[00114] The amount of an agent present in a compartment and/or the outflow of a compartment can be determined by any method known in the art. In some embodiments, the agent can be detectably labelled and the amount/concentration of the agent can be determined with an appropriate detector for the label type. For example, the label can be FITC and the detection can be done by fluorescence microscopy. Suitable types of labels can include a light-absorbing dye, a fluorescent dye, or a radioactive label. Detectable labels, methods of detecting them, and methods of incorporating them into reagents (e.g. antibodies and nucleic acid probes) are well known in the art. In some embodiments of any of the aspects, detectable labels can include labels that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as fluorescence, chemifluoresence, or chemiluminescence, or any other appropriate means. The detectable labels used in the methods described herein can be primary labels (where the label comprises a moiety that is directly detectable or that produces a directly detectable moiety) or secondary labels (where the detectable label binds to another moiety to produce a detectable signal, e.g., as is common in immunological labeling using secondary and tertiary antibodies). The detectable label can be linked by covalent or non-covalent means to the reagent. Alternatively, a detectable label can be linked such as by directly labeling a molecule that achieves binding to the agent via a ligand-receptor binding pair arrangement or other such specific recognition molecules. Detectable labels can include, but are not limited to radioisotopes, bioluminescent compounds, chromophores, antibodies, chemiluminescent compounds, fluorescent compounds, metal chelates, and enzymes.
[00115] In other embodiments, the agent is labelled with a fluorescent compound. When the fluorescently labeled reagent is exposed to light of the proper wavelength, its presence can then be detected due to fluorescence. In some embodiments of any of the aspects, a detectable label can be a fluorescent dye molecule, or fluorophore including, but not limited to fluorescein, phycoerythrin, phycocyanin, o-phthaldehyde, fluorescamine, Cy3™, Cy5™, allophycocyanine, Texas Red, peridenin chlorophyll, cyanine, tandem conjugates such as phycoerythrin-Cy5™, green fluorescent protein, rhodamine, fluorescein isothiocyanate (FITC) and Oregon Green™, rhodamine and derivatives (e.g., Texas red and tetrarhodimine isothiocynate (TRITC)), biotin, phycoerythrin, AMCA, CyDyes™, 6- carboxyfhiorescein (commonly known by the abbreviations FAM and F), 6-carboxy-2',4',7',4,7- hexachlorofiuorescein (HEX), 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfiuorescein (JOE or J), N,N,N',N'-tetramethyl-6carboxyrhodamine (TAMRA or T), 6-carboxy-X-rhodamine (ROX or R), 5- carboxyrhodamine-6G (R6G5 or G5), 6-carboxyrhodamine-6G (R6G6 or G6), and rhodamine 110; cyanine dyes, e.g. Cy3, Cy5 and Cy7 dyes; coumarins, e.g umbelliferone; benzimide dyes, e.g. Hoechst 33258; phenanthridine dyes, e.g. Texas Red; ethidium dyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes; polymethine dyes, e.g. cyanine dyes such as Cy3, Cy5, etc; BODIPY dyes and quinoline dyes. In some embodiments of any of the aspects, a detectable label can be a radiolabel including, but not limited to 3H, 1251, 35S, 14C, 32P, and 33P. In some embodiments of any of the aspects, a detectable label can be an enzyme including, but not limited to horseradish peroxidase and alkaline phosphatase. An enzymatic label can produce, for example, a chemiluminescent signal, a color signal, or a fluorescent signal. Enzymes contemplated for use to detectably label an antibody reagent include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alphaglycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. In some embodiments of any of the aspects, a detectable label is a chemiluminescent label, including, but not limited to lucigenin, luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester. In some embodiments of any of the aspects, a detectable label can be a spectral colorimetric label including, but not limited to colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, and latex) beads.
[00116] In some embodiments of any of the aspects, an agent can also be labeled with a detectable tag, such as c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS, or biotin. Other detection systems can also be used, for example, a biotin-streptavidin system. In this system, the antibodies immunoreactive (i. e. specific for) with the biomarker of interest is biotinylated. Quantity of biotinylated antibody bound to the biomarker is determined using a streptavidin-peroxidase conjugate and a chromagenic substrate. Such streptavidin peroxidase detection kits are commercially available, e. g. from DAKO;
Carpinteria, CA. A reagent can also be detectably labeled using fluorescence emitting metals such as 152EU, or others of the lanthanide series. These metals can be attached to the reagent using such metal chelating groups as diethylenetriaminepentaacetic acid (DTP A) or ethylenediaminetetraacetic acid (EDTA).
[00117] Methods of detecting and/or measuring an agent can also include mass spectroscopy, immunoassays, ELISA (enzyme linked immunosorbent assay), western blot, immunoprecipitation, immunofluorescence using detection reagents such as an antibody or protein binding agents, radioimmunoassay (RIA), "sandwich" immunoassays, immunoprecipitation assays, immunodiffusion assays, agglutination assays, e.g. latex agglutination, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, e.g. FIA (fluorescence-linked immunoassay), chemiluminescence immunoassays (CLIA), electrochemiluminescence immunoassay (ECLIA, counting immunoassay (CIA), lateral flow tests or immunoassay (LFIA), magnetic immunoassay (MIA), protein A immunoassays,
PCR procedures, RT-PCR, quantitative RT-PCR Northern blot analysis, differential gene expression, RNAse protection assay, microarray based analysis, next-generation sequencing, hybridization methods, and any another method known in the art for the relevant molecular structure of the agent. [00118] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.
[00119] For convenience, certain terms employed herein, in the specification, examples and appended claims are collected here.
[00120] The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction" or “decrease" or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.
[00121] The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, a “increase” is a statistically significant increase in such level.
[00122] As used herein, a "subject" means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “individual,” “patient” and “subject” are used interchangeably herein.
[00123] Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of a disease. A subject can be male or female.
[00124] As used herein, the terms “protein" and “polypeptide" are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms "protein", and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. "Protein" and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term "peptide" is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing. The terms also refer to fragments or variants of the polypeptide that maintain at least 50% of the activity or effect.
[00125] As used herein, the term “nucleic acid” or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof. The nucleic acid can be either single -stranded or double-stranded. A single -stranded nucleic acid can be one nucleic acid strand of a denatured double- stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double -stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable DNA can include, e.g., genomic DNA or cDNA. Suitable RNA can include, e.g., mRNA.
[00126] In some embodiments, the methods described herein relate to measuring, detecting, or determining the level of at least one agent. As used herein, the term "detecting" or “measuring” refers to observing a signal from, e.g. a probe, label, or target molecule to indicate the presence of an analyte in a sample. Any method known in the art for detecting a particular label moiety can be used for detection. Exemplary detection methods include, but are not limited to, spectroscopic, fluorescent, photochemical, biochemical, immunochemical, electrical, optical or chemical methods. In some embodiments of any of the aspects, measuring can be a quantitative observation.
[00127] As used herein, “contacting" refers to any suitable means for delivering, or exposing, an agent to at least one cell. Exemplary delivery methods include, but are not limited to, direct delivery to cell culture medium, perfusion, injection, or other delivery method well known to one skilled in the art. In some embodiments, contacting comprises physical human activity, e.g., an injection; an act of dispensing, mixing, and/or decanting; and/or manipulation of a delivery device or machine.
[00128] The term “statistically significant" or “significantly" refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.
[00129] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean ±1%.
[00130] As used herein, the term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather than limitation.
[00131] The term "consisting of refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
[00132] As used herein the term "consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
[00133] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example." [00134] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
[00135] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), W. W. Norton & Company, 2016 (ISBN 0815345054, 978-0815345053); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN- 1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Lrederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties.
[00136] Other terms are defined herein within the description of the various aspects of the invention.
[00137] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely fortheir disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.
[00138] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.
[00139] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.
[00140] In some embodiments, the present technology may be defined in any of the following numbered paragraphs:
1. A system comprising: a first subcutaneous compartment comprising: i. a mixture comprising fibroblasts, cell culture medium, hydrogel, and at least one of preadipocytes and adipocytes; and a first peripheral compartment comprising: i. at least one opening providing fluid, non-cellular communication with the first subcutaneous compartment; and ii. optionally comprising endothelial cells.
2. The system of paragraph 1, further comprising a second peripheral compartment comprising at least one opening providing fluid, non-cellular communication with the first subcutaneous compartment.
3. The system of any of paragraphs 1-2, wherein the first subcutaneous compartment is medial with respect to the first and second peripheral compartments; and the first and second peripheral compartments are not directly in fluid, non-cellular communication with each other.
4. The system of any of paragraphs 1-3, wherein each compartment comprises or is a channel, cube, rectangular prism, or cylinder.
5. The system of any of paragraphs 1-4, wherein the first subcutaneous compartment comprises: a medial disc or sphere portion that comprises at least one of the openings with the first peripheral compartment and optionally, second peripheral compartment; a first lateral channel, cube, rectangular prism, or cylinder portion; and a second lateral channel, cube, rectangular prism or cylinder portion.
6. The system of paragraph 5, wherein the first and/or second lateral portions do not comprise the at least one openings with the first peripheral compartment and optionally, second peripheral compartment.
7. The system of any of paragraphs 5-6, wherein the first and second peripheral compartments comprise a medial curved channel portion where they comprise the at least one openings with the first subcutaneous compartment; a first lateral channel, cube, rectangular prism, or cylinder portion; and a second lateral channel, cube, rectangular prism or cylinder portion.
8. The system of paragraph 1, further comprising; a second subcutaneous compartment comprising: i. a mixture comprising fibroblasts, cell culture medium, hydrogel, and at least one of preadipocytes and adipocytes; and a second peripheral compartment comprising: i. at least one opening providing fluid, non-cellular communication with the second subcutaneous compartment; and ii. optionally comprising endothelial cells; a medial injection compartment comprising: i. at least one opening providing fluid, non-cellular communication with the first subcutaneous compartment; and ii. at least one opening providing fluid, non-cellular communication with the second subcutaneous compartment; wherein the first and second subcutaneous compartments are lateral to the medial injection compartment.
9. The system of paragraph 8, wherein the medial injection compartment comprises: a medial disc or sphere portion that comprises at least one of the openings with each of the first and second subcutaneous compartments; a first lateral channel, cube, rectangular prism, or cylinder portion; and a second lateral channel, cube, rectangular prism or cylinder portion. The system of paragraph 9, wherein the first and/or second lateral portions do not comprise the at least one openings with the first and second subcutaneous compartments. The system of any of paragraphs 9-10, wherein the first and second subcutaneous compartments each comprise a medial curved channel portion where they comprise at least one of the openings with the medial injection compartment; a first lateral channel, cube, rectangular prism, or cylinder portion; and a second lateral channel, cube, rectangular prism or cylinder portion. The system of paragraph 11, wherein the medial curved channel portion of the first subcutaneous compartment comprises at least one of the openings with the first peripheral compartment; and the medial curved channel portion of the second subcutaneous compartment comprises at least one of the openings with the second peripheral compartment. The system of any of the preceding paragraphs, wherein the medial injection channel, first subcutaneous compartment, and/or second subcutaneous compartment further comprises an injection port. The system of paragraph 13, wherein the injection port is in the medial disc or sphere portion. The system of any of the preceding paragraphs, wherein the at least one opening comprises multiple openings or slits in an interposed wall or barrier. The system of any of the preceding paragraphs, wherein the at least one opening is no greater in height or width than 500 pm. The system of any of the preceding paragraphs, wherein the at least one opening is no greater in height or width than 100 pm. The system of any of the preceding paragraphs, wherein the fluid availability aspect ratio is 5 or greater. The system of any of the preceding paragraphs, wherein the fluid availability aspect ratio is 50:3. The system of any of the preceding paragraphs, wherein the fibroblasts and at least one of preadipocytes and adipocytes form a 3D culture. The system of any of the preceding paragraphs, wherein the mixture comprising fibroblasts, cell culture medium, hydrogel, and at least one of preadipocytes and adipocytes comprises more preadipocytes and/or adipocytes than fibroblasts. The system of any of the preceding paragraphs, wherein the mixture comprising fibroblasts, cell culture medium, hydrogel, and at least one of preadipocytes and adipocytes comprises at least 2x as many preadipocytes and/or adipocytes as fibroblasts. The system of any of the preceding paragraphs, wherein the mixture comprising fibroblasts, cell culture medium, hydrogel, and at least one of preadipocytes and adipocytes comprises at least 3x as many preadipocytes and/or adipocytes as fibroblasts. The system of any of the preceding paragraphs, wherein the first peripheral compartment comprises endothelial cells. The system of any of the preceding paragraphs, wherein the first peripheral compartment comprises endothelial cells and the second peripheral compartment does not comprise cells. The system of any of the preceding paragraphs, wherein the first and second peripheral compartments each comprise endothelial cells. The system of any of the preceding paragraphs, wherein the endothelial cells line one or more walls of the first and/or second peripheral compartments. The system of any of the preceding paragraphs, wherein the endothelial cells form a confluent single-cell monolayer on all walls of the first and/or second peripheral compartments. The system of any of the preceding paragraphs, wherein the cells are murine or human cells. The system of any of the preceding paragraphs, wherein at least 80% of the cells are viable. The system of any of the preceding paragraphs, wherein at least 90% of the cells are viable. The system of any of the preceding paragraphs, wherein each compartment further comprises at least 2 ports. The system of paragraph 32, wherein the at least 2 ports are connected to a microfluidics system to provide an inflow and an outflow port in each compartment. The system of any of the preceding paragraphs, wherein the hydrogel comprises one or more extracellular matrix components. An in vitro method of determining the subcutaneous administration characteristics of a candidate subcutaneous agent, the method comprising: introducing a candidate subcutaneous agent into the injection compartment and/or a peripheral compartment not comprising cells, of the system of any of paragraphs 1- 34; and measuring at least one of: the amount and/or change in the amount of subcutaneous agent in the outflow of fluid from one or more of the compartments; the amount and/or change in the amount of subcutaneous agent present in one or more of the compartments; and the amount and/or change in the amount of subcutaneous agent in the outflow of fluid from a peripheral compartment comprising endothelial cells. The method of paragraph 35, wherein the following are measured: the amount and/or change in the amount of subcutaneous agent present in each of the compartments; and the amount and/or change in the amount of subcutaneous agent in the outflow of fluid from a peripheral compartment comprising endothelial cells. The method of any of paragraphs 35-36, wherein a peripheral compartment not comprising vascular endothelial cells indicates subcutaneous administration characteristics via lymphatic pathways. The method of any of paragraphs 35-36, wherein a peripheral compartment comprising lymphatic endothelial cells but not comprising vascular endothelial cells indicates subcutaneous administration characteristics via lymphatic pathways. The method of any of paragraphs 35-36, wherein a peripheral compartment comprising endothelial cells indicates subcutaneous administration characteristics via vascular pathways. The method of any of paragraphs 35-36, wherein a peripheral compartment comprising vascular endothelial cells indicates subcutaneous administration characteristics via vascular pathways. The method of any of paragraphs 35-40, wherein the candidate subcutaneous agent is introduced into the injection compartment and/or a peripheral compartment not comprising cells at a perfusion rate of from 0. 1 pL/min to 1,000 pL/min. The method of any of paragraphs 35-41, further comprising the following steps prior to the step of introducing the candidate subcutaneous agent: introducing the mixture comprising fibroblasts, cell culture medium, hydrogel, and preadipocytes into the first and/or second subcutaneous compartment and then culturing the preadipocytes and fibroblasts; inducing adipocyte differentiation and then maintaining the adipocytes and fibroblasts. The method of any of paragraphs 35-42, further comprising the following steps prior to the step of introducing the candidate subcutaneous agent: introducing the mixture comprising fibroblasts, cell culture medium, hydrogel, and at preadipocytes into the first and/or second subcutaneous compartment and then culturing the preadipocytes and fibroblasts; inducing adipocyte differentiation and then maintaining the adipocytes and fibroblasts; twice introducing endothelial cells into the first and/or second peripheral compartment and then culturing and maintaining the endothelial cells. The method of any of paragraphs 35-43, wherein the mixture comprising fibroblasts, cell culture medium, hydrogel, and at least one of preadipocytes and adipocytes is at less a temperature of less than 10 C when it is introduced into the first and/or second subcutaneous compartment.
45. The method of any of paragraphs 35-44, wherein the mixture comprising fibroblasts, cell culture medium, hydrogel, and at least one of preadipocytes and adipocytes is at less a temperature of 4 C or less when it is introduced into the first and/or second subcutaneous compartment.
46. The method of any of paragraphs 35-45, wherein culturing and/or maintaining the fibroblasts and at least one of preadipocytes and adipocytes comprises one or more of: culturing for at least 5 days; culturing until the fibroblasts and at least one of preadipocytes and adipocytes reach a high density; and culturing until the fibroblasts and at least one of preadipocytes and adipocytes are confluent.
47. The method of any of paragraphs 35-46, wherein culturing and/or maintaining the fibroblasts and at least one of preadipocytes and adipocytes comprises exchanging the culture medium in the first and/or second subcutaneous compartment at least daily.
48. The method of any of paragraphs 35-47, wherein culturing and/or maintaining the fibroblasts and at least one of preadipocytes and adipocytes comprises exchanging the culture medium in the first and/or second subcutaneous compartment continuously.
49. The method of any of paragraphs 35-48, wherein medium exchange in the injection compartment and/or a peripheral compartment not comprising cells is performed at a perfusion rate of from 0.1 pL/min to 1000 pL/min.
50. The method of any of paragraphs 35-49, wherein culturing and/or maintaining the endothelial cells comprises one or more of: culturing for at least 3 days; culturing until the endothelial cells reach a high density; and culturing until the endothelial cells are confluent.
51. The method of any of paragraphs 35-50, wherein culturing and/or maintaining the endothelial cells comprises exchanging the culture medium in the first and/or second peripheral compartment at least daily.
52. The method of any of paragraphs 35-51, wherein culturing and/or maintaining the endothelial cells comprises exchanging the culture medium in the first and/or second peripheral compartment continuously.
53. The method of any of paragraphs 35-52, wherein medium exchange in the first and/or second peripheral compartment is performed at a perfusion rate of from 0.02 pL/min to 200 pL/min.
[00141] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting. EXAMPLES
Example 1: A Subcutaneous Tissue Chip for Assessing Biologies Transport
[00142] Transport of macromolecules from the subcutaneous tissues to systemic circulation via lymphatic or blood vessels plays a vital role in the pharmacokinetics of subcutaneously injected drugs. Currently there is an urgent need of reliable preclinical models to study transport processes in the subcutaneous environment. Described herein is a microfluidic model utilizing 3D subcutaneous cultures and 2D vascular or lymphatic barriers. To the best of our knowledge, this is the first in vitro cellular model for the subcutaneous drug administration route. Consistent with the in vivo data, the inventors demonstrated the effects of molecular weight, charge and size influencing transport rates. Lymphatic transport played a dominant role in the clearance of macromolecules, and the overall rate of absorption can be predicted from the subcutaneous diffusion. The use of a microfluidic chip with precisely controlled fluid conditions and analytical tools to assess the spatial-temporal concentration profile also highlight the novelty of our method as a standardized assay with wide applicability. [00143] Subcutaneous injection is an attractive route of drug administration over intravenous administration. Drugs which have limited oral availability can be conveniently and quickly selfadministered to result in high patient compliance and reduced cost of treatment1-3. Despite the expanding market of subcutaneously-administered drugs, especially biomacromolecules such as monoclonal antibodies (mAbs), there is still a lack of validated preclinical models to support their development1. Animal models4-8, though widely used, show poor correlation with human data due to anatomical and physiological differences between species1. Within species, factors such as site-to-site variations and experimental setup also contribute to inconsistencies1. On the other hand, to the best of our knowledge, the most advanced in vitro model so far is an acellular system made of a buffer solution and a membrane with a single measurable quantity8. This simple setup only offers limited information regarding the complex transport process involving the interaction between subcutaneous microstructure and drug properties such as molecular weight, shape, charge and formulation viscosity1. This evidences a pressing need for a reliable in vitro subcutaneous model to tackle the bottleneck in preclinical research between a fundamental understanding of the transport process and predicting the pharmacokinetics for clinical translations.
[00144] An ideal in vitro model for subcutaneous transport should possess at least the following components: (i) adipocytes as the major cell type and fibroblasts forming the connective tissue (II) the interstitial space containing the extracellular matrix (ECM) within which the drug molecules traverse, and (iii) capillaries and lymphatics embedded in the subcutaneous space to enter systemic circulation1- 3. With the development of organs-on-chips, these key components can be constructed on a microfluidic device using a combination of 2D and 3D cellular cultures9-10. The majority of similar systems utilizes custommade devices which provide the most flexibility in chip design and setup. Despite an immense popularity in academic research9-21, this approach, however, has not been widely used in preclinical studies due to several major drawbacks. The design and fabrication processes impact both the research timeline and costs. The first fabrication step using photolithography requires specialized equipment and training which are not available to many biological labs5 . Even with published protocols6 , defects due to chip fabrication result in a lack of quality control which may introduce variations and uncertainties comparable to animal models. The use of commercially- available chips facilitates wide adoption of microfluidic -based assays, similar to established in vitro models such as Transwell plates. This approach is still in its infancy because the tradeoff of a fixed geometry is a stricter requirement for processing conditions, especially in the case of 3D cell cultures which are more technically demanding 10. Therefore, the knowledge of various flow conditions in a given chip geometry is not only important for creating a functional tissue environment, but also essential for developing a standardized analysis method to quantitatively compare the transport data with in vivo measurements.
[00145] Understanding flow conditions under geometric constraints is critical to chip selection and creating a functional tissue environment. Described herein is a method to evaluate subcutaneous transport on a commercial chip demonstrating three novel aspects. First, the inventors used a simplistic chip design and setup coupled with precisely controlled flow conditions to mimic subcutaneous transport. Secondly, the inventors derived analytical expressions for multiple rate constants describing the spatio-temporal concentration profile without using time-consuming numerical simulations. Lastly, the inventors validated the model by systematically investigating the effects of molecular weight (MW), charge and shape in comparison to existing in vivo data, and provided new insights to the pathway to enter systemic circulation and the overall rate of absorption that the current in vitro model2 cannot capture.
[00146] Results
[00147] Constructing the subcutaneous tissue in an idealized geometry. The inventors selected a commercial chip with a three-compartment design for this study which is made of poly dimethylsiloxane (PDMS) on a glass slide (Fig. 4A, Fig. 9). Each channel is 100 rm in depth. The top and bottom compartments are 200 rm wide and the central compartment is 500 rm wide (Fig. 4B). This chip geometry can mimic the subcutaneous transport pathway involving drug injection, transport through the ECM and uptake by blood and lymphatic capillariesl (Fig. 10): the top channel serves as the injection channel containing the liquid solution of interest (Fig. 4B); the central channel serves as the subcutaneous channel containing a mixture of adipose cells and fibroblasts organized in a 3D structure; the bottom channel represents vessels which are either lymphatics or capillaries with endothelial cells grown in a 2D monolayer lining the channel walls.
[00148] The three compartments are connected by narrow slits (3 pm wide X 100 pm deep X 50 pm long with 50 pm spacings, Fig. 4C) which blocks cell migration across channels and enables sequential diffusion of macromolecules from the injection channel. Based on our analysis of the flow condition (Example 2 Discussion), these slits play an important role of controlling the rate of mass transport across the channels without using microvalves. Due to the high aspect ratio design, flow in the neighboring channel is not disturbed when perfusion occurs in one channel (Figs. 11A-11C), thus protecting the soft hydrogel-based subcutaneous tissue from excess fluid shear12 . These narrow slits also enable media exchange via slow diffusion. The inventors measured the permeability of FITC- dextran (MW = 40k) across the slits in PBS-filled chips (Fig. 4D), which agrees with theoretical estimates based on a porous media. The slit size is three to four orders of magnitude larger than the macromolecules and steric hindrance is negligible.
[00149] The inventors developed a protocol to seed three types of cells from either mice or human into the chip in sequence and maintained the cell culture for at least 10 days (Fig. 4E). In this study, the inventors focused on mice cells due to the availability of in vivo data for validation. Fibroblasts and preadipocytes were mixed with cold MATRIGEL™ liquid and quickly perfused into the subcutaneous channel. The device was then incubated at 37°C for the mixture to polymerize. Based on our rheological measurements (Fig. 4F, Figs. 12A-12E), mixing MATRIGEL™with cell culture medium did not create significant changes in its temperature dependent polymerization property11 .The resulting gel mixture at 37°C had loss (G”) and storage (G’) moduli typical for ECM22 and were similar to pure MATRIGEL™23 properties. There are two constraints on the flow rate for seeding. The perfusion process needs to be fast enough to prevent cell settling and premature polymerization24 . On the other hand, a high fluid shear can result in an inhomogeneous distribution of cells across the channel in addition to disrupting cells and MATRIGEL™13 18 . This nonuniform distribution of cellular suspensions is often overlooked in microfluidic processing and is based on hydrodynamic effects of the non-Newtonian suspending fluid and cell deformability (Fig. 13, Discussion). Using an intermediate perfusion rate, the inventors achieved a homogeneous initial cell distribution which was verified using live cell staining and confocal microscopy (Fig. 4G). Preadipocytes and fibroblasts grew inside the subcutaneous channel and reached high density after 5 days (Fig. 4H, Fig. 14). The inventors then induced adipocyte differentiation inside the chip to achieve the final subcutaneous tissue environment.
[00150] To mimic the blood capillaries, the inventors seeded endothelial cells into the vessel channel at a high density twice to ensure sufficient attachment of endothelial cells onto the channel walls (Fig. 41, Fig. 15). To mimic the lymphatics which have an open and loose structure2 , the inventors left the vessel channel empty such that molecules can diffuse freely.
[00151] To maintain the subcutaneous cell culture (3D) and the endothelial cell culture (2D), the inventors performed daily medium exchange utilizing various flow rates based on the chip geometry and tissue environment (Fig. 24).
[00152] Characterization of cellular functions and properties. In a confluent subcutaneous channel, the inventors observed a dense 3D cellular network (Fig. 5A) where individual cells are more much more difficult to distinguish compared with confluent 2D cell cultures in a flask (Figs. 16A- 16B). This striking difference has rarely been discussed before because most 3D systems have much lower cell densities. Although it is not possible to calculate the cell density from the images obtained, the cell density significantly increased compared to seeding (2 X 106 /ml) and became closer to the cell density in normal tissues ( 108 ~ IO9 /ml)39. In addition, both preadipocytes and fibroblasts displayed more elongated shapes with fewer protrusions in 3D, consistent with previous findings25,26 . The inventors also observed a small fraction of cells that did not spread and remained rounded only in 3D. This morphology was previously found to be related to the fibroblasts and is unique to 3D MATRIGEL™26 . This morphology occurs much less frequently in our 3D culture and is probably due to the mixing of cell culture medium with MATRIGEL™ attenuating this effect.
[00153] Given the dense tissue structure, preadipocytes and fibroblasts are difficult to differentiate especially due to their similar morphologies. Based on their 2D morphologies and 3D morphologies when cultured independently (Figs. 16C-16D), preadipocytes are slightly smaller, less elongated and have fewer protrusions. The inventors found that fibroblasts grew and expanded faster than preadipocytes both on the chip as well as in flasks. Therefore, the inventors seeded more preadipocytes to prevent the confluent subcutaneous co-culture from being disproportionate.
[00154] In the vessel channel, the inventors observed a 2D endothelial morphology (Fig. 41) similar to those in flask (Fig. 16E) as previously reported. The endothelial spacing was small and asymmetric filopodia extensions indicating polarization towards endothelial migration were rare13. The inventors found human subcutaneous and endothelial cells cultured on the chip to be bigger than mice cells but have similar shapes (Fig. 16F). This size difference may influence the rate of transport to be discussed later.
[00155] The inventors monitored adipocyte differentiation in the confluent subcutaneous channel, which may influence subcutaneous transport both physically and biologically. After inducing differentiation, the inventors observed a morphological change of cells from elongated to a more rounded shape as previously reported27 (Fig.5B). The presence of intracellular lipid droplets was confirmed using Oil Red O staining (Fig. 5C). The inventors measured a significant increase in lipid content after differentiation (Fig. 5D). Since adipocyte can be derived from both preadipocytes and fibroblasts28, the inventors independently monitored their differentiation processes on a plate assay and quantified the amount of staining. The inventors found that preadipocyte differentiation plays the dominant role in the co-cultured subcutaneous environment Fig. 17).
[00156] To make sure that the cells were viable for the entire culture period until the end of transport measurement, the inventors used live/dead staining at the end of the transport study (Fig. 5E). The inventors observed more than 90% viability for both subcutaneous cells and endothelial cells (Fig. 5F). This confirms the lasting period of our cell culture protocol is comparable to other organ- on-chip systems on custom-made devices14,17. The injection dose of fluorescently-labeled macromolecules for transport measurement does not cause toxic effects and the live cell imaging system is valid for multiple hours.
[00157] The inventors examined the tissue structure in the subcutaneous and vascular channels using actin and nucleus staining (Fig. 5G). The inventors confirmed the 3D character of the subcutaneous tissue spanning the entire channel depth (z). In the vessel channel, endothelial cells formed a complete lumen with a confluent monolayer lining the inside of the channel walls, mimicking blood capillaries. This 2D monolayer structure allows perfusion through the channel. The 2D and 3D cellular structures (Fig. 5H) pose distinct types of barriers for the transport mechanism. [00158] Measurement of transport properties and the effect of MW. The inventors measured subcutaneous transport by perfusing fluorescently-labeled macromolecules into the injection channel. The fluorescent intensity shows a linear correlation with the solute concentration for quantification (R2 = 0.9966, Fig. 18). The inventors acquired a time series of images under live conditions (Fig. 6A) for at least three hours. To fully utilize the spatial -temporal concentration profile measured, the inventors derived analytical expressions describing the concentration changes in each channel using three rate constants: the permeability from the injection channel to the subcutaneous channel If. the diffusivity in the subcutaneous channel D2 and the permeability from the subcutaneous channel into the vessel channel R23 (lymphatic or vascular). Obtaining these expressions is not trivial considering the chip geometry but is much simpler than numerical simulations for data analysis12 13 . For each experiment, the inventors fitted three rate constants and a good fit was obtained between expected values and experimental values for all three constants (Fig. 6B, Figs. 19A-19B).
[00159] The inventors first investigated the effect of MW on transport properties using FITC- dextran. Since diffusion is the major transport mechanism on our chip, the inventors observed a reduction in all three measured rate constants with increasing MW (Fig. 6C-6E). The inventors measured a more than 80% reduction in R compared to the PBS control (FITC-dextran, MW = 40k), indicating that the major transport barrier for entering the subcutaneous channel is the tissue environment instead of the narrow slits. The scaling between MW and the rate constants is related to the microstructure of the transport barrier. The interstitium consists of a fibrous collagen network supporting a gel phase1 . As a result, diffusion of macromolecules within the interstitium may be physically retarded due to steric hindrance1-3.
[00160] The 2D endothelial barrier limits paracellular diffusion due to the presence of gap junctions between the endothelial cells, thus reducing the permeability of macromolecules29 . The pore size in ECM is estimated to be ~10 to 100 nm2 , larger than the size of endothelial gap junctions (1 nm)29. Therefore, less hindrance was expected in the subcutaneous channel, consistent with the slower decay observed for W compared to R23.
[00161] The inventors verified that the measured rate constants are comparable to those from the literature studies. Interstitial diffusion is known to be slow compared to free diffusion in water, making it an attractive property for a sustained drug release. The measured D2 values agree with the in vivo data for diffusion in normal tissues and show similar MW scaling7,30 (Fig. 6F). On the other hand, in in vitro acellular models, measured diffusivities show large variations25,31-34. The variations can be largely explained by structural differences in the fiber network due to gel composition and concentration21 as shown from SEM images33 and is also influenced by the gel degradation process34 . Therefore, these modelsare expected to show differences in MW scaling19 . With the addition of subcutaneous cells, the inventors observed a 50% reduction in diffusivity for FITC-dextran (MW = 40k) compared to the MATRIGEL™ control, similar to other in vitro 3D cultures25,34 . Therefore, it is necessary to include cellular components in in vitro models to provide a more physiological transport environment for several reasons. First, they reduce the interstitial space available for diffusion, which may contribute to interspecies (Figs. 16E-16F) and intertissue differences. Secondly, fibroblasts can produce a network of glycosaminoglycans, collagen and elastin1, which reorganizes the existing structure of ECM and its mechanical properties25,34. Finally, nonspecific interactions can also occur in the vicinity of cells, further delaying the transport process25. In vivo measurement of R23 is on the order of 10-3 rni/sl4,15,34-36, comparable to our estimates. A key challenge in in vitro endothelial culture is to maintain an intact lumen with highly confluent cells, resulting in permeabilities which are an order of magnitude higher14,37 . Our endothelial channel thus demonstrates the desired barrier properties.
[00162] The influence of molecular charge and shape on subcutaneous transport. The inventors investigated the charge effects on subcutaneous transport using FITC-CM-dextran and FITC-DEAE- dextran with matching MW (40k) but opposite surface charges (Fig. 7A-7C). The inventors found that the cationic FITC-DEAE-dextran shows the fastest /?i 2 - the slowest D but no significant differences in R23 compared to the anionic FITC-CM-dextran. Various ECM components carry opposite charges, and overall the interstitial space is negatively-charged1 . Due to electrostatic repulsion, negatively- charged molecules enter the subcutaneous space more slowly (R12). Once in the subcutaneous tissue, negatively charged molecules diffuse faster as reflected in the measured Devalue, consistent with existing findings16. When crossing the endothelial barrier, it is unclear whether the negative charge on the endothelial surface or the ECM governs R23 based on our measurements. Existing findings also report opposite trends29,36 . While the negative charge leads to less repulsions and may facilitate the transport, it may repel cationic molecules from entering the gap junctions.
[00163] The inventors also investigated the effect of molecular shape using fluorescently labeled IgG which have MW comparable to 150k dextran. The inventors observed a significant reduction in rate constants for IgG (Fig. 7D-7F), consistent with previous findings6 . IgG usually have spherical shapes in comparison to the linear dextran. The hydrodynamic radius of IgG (5 nm) is thus much bigger than those for dextran (0.8 nm). As discussed before, the inventors observed hindered diffusion in both the subcutaneous tissue as well in the endothelial barrier. The increase in hydrodynamic radius can significantly reduce its diffusivity. Although serum IgG carry a slightly negative charge under physiological conditions38 which should facilitate their transport as mentioned earlier, overall the shape effect dominates.
[00164] The rate of subcutaneous diffusion measured in our model for IgG agrees with in vivo data for rats (1 rm2 /s)5. Many biomacromolecules have similar MW but exhibit varying shapes and charges dependent on the solution environment. In addition, these two factors are interdependent because the globular shape of proteins can render nonuniform charge distributions and results in enhanced charge interactions with the ECM6 16 . By mimicking the physiological environment of the subcutaneous tissue, our in vitro chip model captures the overall effect of charge and shape dictating the rate of transport. Another important consideration for proteins is their catabolic degradation leading to presystemic elimination and reduced bioavailability, unlike dextran molecules which remain stable throughout the transport process. It is conceivable that the competition between the timescale of transport and the timescale of degradation influences bioavailability, as antibody bioavailability shows a charge-dependent trend. Therefore, a fast diffusion in vitro, such as in the existing model8 , may fail to capture the effect of protein degradation which complicates the prediction of pharmacokinetics.
[00165] Prediction of circulation pathways and in vivo rate of absorption. In addition to the results to validate our model, the inventors also studied effects which have so far only been investigated in vivo in limited studies. Using our chip design, the inventors are able to distinguish lymphatic and vascular pathways of entering systematic circulation, based on the value of R23. Lymphatic transport, represented by an absence of endothelial cells in the vessel channel in our model, contributes to over 80% transport for all macromolecules the inventors tested (Fig. 8A). The inventors also observed an increasing fraction of lymphatic transport as the molecule becomes bigger. As discussed earlier, endothelial gap junctions result in greater hindrance, and thus making lymphatic vessels the preferred pathway for large molecules. In vivo measurement of lymphatic absorption is rare due to technical difficulties associated with the surgical preparation in animal models3. Based on the limited amount of data in sheep, over 80% transport occurs through the lymphatics after subcutaneous administration for proteins with MW greater than 30kDa, agreeing with our predictions. In vitro measurement comparing a monolayer of vascular endothelial cells and lymphatic endothelial cells showed no significant difference in permeation under static conditions15. The difference can be reconciled by a less confluent vascular monolayer in in vitro experiments and the mechanism of lymphatic junctions opening under interstitial pressure3 . Narrow slits in our model therefore resembles the open structure of lymphatic pores. Our findings also indicate that in the case of biomacromolecules, the current chip design can be simplified by removing the endothelial barrier in the vessel channel. [00166] Compared to the existing one-step in vitro assays, our in vitro model can predict the rate constant associated with each transport barrier following subcutaneous injection independently due to its multicompartment geometry and the spatial -temporal concentration profile. As a result, the inventors can predict the overall rate of absorption as a direct comparison to in vivo measurements. Among the three rate constants the inventors calculated, D , is the rate limiting step for the overall rate of absorption. The inventors plotted the diffusive timescale versus the distance from the injection site to the lymphatics (Fig. 8B). This length scale depends on the species and the injection site. The Df, measured on our device with a 500 pm diffusive distance can therefore be adjusted for predicting the overall rate of absorption instead of selecting different channel geometries. Using a diffusive distance of 1mm, the inventors estimated a rate of absorption close to 100 hours, comparable to the estimate from pharmacokinetic modeling in rats39.
[00167] Discussion
[00168] Most in vitro tissue culture systems predict faster rates of transport due to the failure to encompass all interactions in the complex physiological system14 . Tissue transport is governed by both physical and biological influences. The in vitro model the inventors developed captures biological influences by constructing a physiological tissue environment. Physical influences such as flow conditions and tissue dimensions were either accounted for in the device setup or analyzed post measurement. As seen from our measurement, the slow diffusivity in the subcutaneous tissue, though preferred for a sustained drug release, requires modifications to existing 2D culturing and staining protocols and extended experimental timeframe to reach equilibrium (Example 3). The inventors highlighted the importance of flow conditions to build a complex tissue environment under geometric constraints9 . The use of a commercial chip with easily-obtainable equipment setup as well as analytical expressions for data analysis are attractive properties of this system as a standardized assay for high-throughput testing in a preclinical setting and can be combined with pharmacokinetic modeling.
[00169] References
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2.Porter, C. & Charman, S. Lymphatic Transport of Proteins After Subcutaneous Administration. Journal of Pharmaceutical Sciences 89, 297-310 (2000).
3. McLennan, D., Porter, C. & Charman, S. Subcutaneous drug delivery and the role of the lymphatics. Drug Discovery Today: Technologies 2, 89-96 (2005).
4. Clauss, M. A., & Jain, R. K. (1990). Interstitial transport of rabbit and sheep antibodies in normal and neoplastic tissues. Cancer research, 50(12), 3487-3492. 5.Flessner, M., Lofthouse, J. & Zakaria, E. In vivo diffusion of immunoglobulin G in muscle: effects of binding, solute exclusion, and lymphatic removal. American Journal of Physiology-Heart and Circulatory Physiology 273, H2783-H2793 (1997).
6.Reddy, S., Berk, D., Jain, R. & Swartz, M. A sensitive in vivo model for quantifying interstitial convective transport of injected macromolecules and nanoparticles. Journal of Applied Physiology 101, 1162-1169 (2006).
7. Swabb, E. A., Wei, J., & Gullino, P. M. (1974). Diffusion and convection in normal and neoplastic tissues. Cancer research, 34(10), 2814-2822.
8.Bown, H. et al. In vitro model for predicting bioavailability of subcutaneously injected monoclonal antibodies. Journal of Controlled Release 273, 13-20 (2018).
9.Bhatia, S. & Ingber, D. Microfluidic organs-on-chips. Nature Biotechnology 32, 760-772 (2014).
10. Shin, Y. et al. Microfluidic assay for simultaneous culture of multiple cell types on surfaces or within hydrogels. Nature Protocols 7, 1247-1259 (2012).
1 l.Lii, J. et al. Real-Time Microfluidic System for Studying Mammalian Cells in 3D Microenvironments. Analytical Chemistry 80, 3640-3647 (2008).
12.Hung, P. et al. A novel high aspect ratio microfluidic design to provide a stable and uniform microenvironment for cell growth in a high throughput mammalian cell culture array. Lab on a Chip 5, 44 (2005).
13.Shamloo, A., Ma, N., Poo, M., Sohn, L. & Heilshom, S. Endothelial cell polarization and chemotaxis in a microfluidic device. Lab on a Chip 8, 1292 (2008).
14.Adriani, G., Ma, D., Pavesi, A., Kamm, R. & Goh, E. A 3D neurovascular microfluidic model consisting of neurons, astrocytes and cerebral endothelial cells as a blood-brain barrier. Lab on a Chip 17, 448-459 (2017).
15. Sato, M. et al. Microcirculation-on-a-Chip: A Microfluidic Platform for Assaying Blood- and Lymphatic-Vessel Permeability. PLOS ONE 10, e0137301 (2015).
16. Arends, F. et al. A microfluidics approach to study the accumulation of molecules at basal lamina interfaces. Lab on a Chip 15, 3326-3334 (2015).
17.Loskill, P. et al. WAT-on-a-chip: a physiologically relevant microfluidic system incorporating white adipose tissue. Lab on a Chip 17, 1645-1654 (2017).
18.Bruzewicz, D., McGuigan, A. & Whitesides, G. Fabrication of a modular tissue construct in a microfluidic chip. Lab on a Chip 8, 663 (2008).
19. Choi, N. et al. Microfluidic scaffolds for tissue engineering. Nature Materials 6, 908-915 (2007). 20. Sung, J., Kam, C. & Shuler, M. A microfluidic device for a pharmacokinetic-pharmacodynamic (PK-PD) model on a chip. Lab on a Chip 10, 446 (2010).
21. Saadi, W. et al. Generation of stable concentration gradients in 2D and 3D environments using a microfluidic ladder chamber. Biomedical Microdevices 9, 627-635 (2007). 22. Tanner, K. High Frequency Active Microrheology Reveals Mismatch in 3D Tumor Intracellular and Extracellular Matrix Viscoelasticity. Biophysical Journal 116, 8a (2019).
23. Kane, K. et al. Determination of the rheological properties of Matrigel for optimum seeding conditions in microfluidic cell cultures. AIP Advances 8, 125332 (2018).
24. Kim, L., Toh, Y., Voidman, J. & Yu, H. A practical guide to microfluidic perfusion culture of adherent mammalian cells. Lab on a Chip 7, 681 (2007).
25.Kihara, T., Ito, J. & Miyake, J. Measurement of Biomolecular Diffusion in Extracellular Matrix Condensed by Fibroblasts Using Fluorescence Correlation Spectroscopy. PLoS ONE 8, e82382 (2013).
26.Hakkinen, K., Harunaga, J., Doyle, A. & Yamada, K. Direct Comparisons of the Morphology, Migration, Cell Adhesions, and Actin Cytoskeleton of Fibroblasts in Four Different Three- Dimensional Extracellular Matrices. Tissue Engineering Part A 17, 713-724 (2011).
27.Lai, N., Sims, J., Jeon, N. & Lee, K. Adipocyte Induction of Preadipocyte Differentiation in a Gradient Chamber. Tissue Engineering Part C: Methods 18, 958-967 (2012).
28.Takeda, Y., Harada, Y., Yoshikawa, T. & Dai, P. Direct conversion of human fibroblasts to brown adipocytes by small chemical compounds. Scientific Reports 7, (2017).
29.Feine, I., Pinkas, I., Salomon, Y. & Scherz, A. Local Oxidative Stress Expansion through Endothelial Cells - A Key Role for Gap Junction Intercellular Communication. PLoS ONE 7, e41633 (2012).
30.Pluen, A. et al. Role of tumor-host interactions in interstitial diffusion of macromolecules: Cranial vs. subcutaneous tumors. Proceedings of the National Academy of Sciences 98, 4628-4633 (2001).
31.Galgoczy, R. et al. A spectrophotometer-based diffusivity assay reveals that diffusion hindrance of small molecules in extracellular matrix gels used in 3D cultures is dominated by viscous effects. Colloids and Surfaces B: Biointerfaces 120, 200-207 (2014).
32.Anguiano, M. et al. Characterization of three-dimensional cancer cell migration in mixed collagenMatrigel scaffolds using microfluidics and image analysis. PLOS ONE 12, e0171417 (2017).
33.Chhetri, R. et al. Probing biological nanotopology via diffusion of weakly constrained plasmonic nanorods with optical coherence tomography. Proceedings of the National Academy of Sciences 111, E4289-E4297 (2014).
34.Yuan, W., Lv, Y., Zeng, M. & Fu, B. Non-invasive measurement of solute permeability in cerebral microvessels of the rat. Microvascular Research 77, 166-173 (2009).
35. Fu, B. & Shen, S. Acute VEGF effect on solute permeability of mammalian microvessels in vivo. Microvascular Research 68, 51-62 (2004).
36.Bischoff, I. et al. Pitfalls in assessing microvascular endothelial barrier function: impedance-based devices versus the classic macromolecular tracer assay. Scientific Reports 6, (2016). 37. Au, P. et al. Differential in vivo potential of endothelial progenitor cells from human umbilical cord blood and adult peripheral blood to form functional long -lasting vessels. Blood 111, 1302-1305 (2008).
38.Yang, D., Kroe-Barrett, R., Singh, S. & Laue, T. IgG Charge: Practical and Biological Implications. Antibodies 8, 24 (2019).
39. Dahlberg, A. et al. The Lymphatic System Plays a Major Role in the Intravenous and Subcutaneous Pharmacokinetics of Trastuzumab in Rats. Molecular Pharmaceutics 11, 496-504 (2014).
Example 2: Methods
[00170] Microfluidic device and processing. Microfluidic chips with three-compartment designs were purchased (catalog # 108011, Synvivo, Inc.). The basic device setup and manipulation follows manufacturer instructions (downloadable at synvivobio.com). In brief, each of the three inlet and three outlet ports was connected to a Tygon ND-100-80 non-DEHP medical tubing (Saint Gobain). During incubation, these tubings were closed using clamps (catalog # 202003, Synvivo, Inc.). During liquid exchange, clamps were removed. Each channel was individually controlled by connecting the corresponding inlet tubing to a 24” Jensen global dispensing needle (TestEquity LLC) on a 1ml luer lock syringe (BD biosciences). Flow rates were controlled by mounting the syringe on a programmable syringe pump (Harvard Apparatus). The outlet tubing was connected to a waste collecting tube.
[00171] Liquid exchange procedures were performed under the microscope with a 4X objective to monitor cell growth and bubble formation in real time. To prevent contamination, the inlet tubing was replaced during each liquid exchange procedure. A drop of sterile water was placed around the inlet port before the removal of old tubing to seal the port. A new tubing containing fresh liquid was then inserted into the inlet port. To prevent bubble formation, the new tubing formed a protruding surface at the tip before insertion. Unless noted otherwise, each liquid exchange step was performed as a brief perfusion in the specified channel and lasted for no more than five minutes to ensure cell viability outside the incubator. Most liquid exchange steps were stopped when three drops of liquid were collected from the outlet, which ensures a complete replacement of existing liquid in the channel. For simplicity, all perfusion procedures in the injection channel were performed at 10 d/min. All perfusion procedures in the vascular channel were performed at 2 d/min unless specified otherwise (Fig. 24).
[00172] Estimation of slit permeability. The diffusivity of dextran (MW = 40k) in water is D = 4.4 xlO-7 cm2 /s. Approximating narrows slits as a porous media, the permeability across the slits can be written as:
Figure imgf000052_0001
The porosity a is estimated based on the size and spacing of the narrow slits:
Figure imgf000052_0002
Ax is the travel distance of the slits which equals 50 rm. The hindrance factor H is calculated based on the ratio A of dextran molecule’s hydrodynamic radius (4.5 A) relative to the slit size using the
Figure imgf000052_0003
[00173] Rheological measurement. AR-G2 rheometer (TA instruments) with a 40-mm-diameter, 2° cone and plate geometry was used to measure the rheological properties of pure MATRIGEL™ and MATRIGEL™: medium mixture. The mixture sample was prepared by mixing 50% thawed MATRIGEL™ and 50% pre-cooled DMEM/F12 on an ice bath. For frequency sweeps, the shear storage (G') and loss (G") moduli were measured at an oscillatory strain of 0. 1, which is below the maximum strain amplitude in the linear response regime12 . For flow sweeps, the viscosity was measured with increasing and decreasing shear rates. For each type of measurement, the temperature effects were investigated in two ways. For a fixed temperature, the inventors set the rheometer temperature to 37 C followed by an equilibration for 2 minutes.
[00174] Measurements were taken immediately after loading the cold sample and repeated for 1 hour until the sample was fully polymerized41 . For an increasing temperature, the rheometer was cooled to 2C and equilibrated for 2 minutes before samples were loaded. Measurements were repeated at increasing temperatures until 37 C with 1 -minute equilibration between each temperature.
[00175] Microfluidic cell culture. Mice subcutaneous fibroblasts (L, ATCC), mice subcutaneous preadipocytes (X9, ATCC), mice aortic endothelial cells (MAOEC, iXCells), human subcutaneous preadipocytes (PCS-210-010, ATCC), human dermal fibroblasts (NHDF, C-12302, PromoCell), human endothelial cells (EA.hy926, ATCC) were obtained from commercial suppliers. To maintain cells in 150-cm2 tissue culture flasks, the inventors used DMEM (ATCC) supplemented with 10% FBS for endothelial cells, DMEM/F12 (ATCC) supplemented with 15% FBS for mice preadipocytes and fibroblasts, fibroblast basal medium with low-serum growth kit (ATCC) for human preadipocytes and fibroblasts. Unless noted otherwise, all incubation steps for tissue culture flasks and chips were performed at 37°C, 5% CO2 and 85-90% humidity. For seeding into chips, passage numbers between 6 and 15 were used for each cell line. Prior to seeding cells into microfluidic channels, serum-free medium (DMEM/F12 for mice and fibroblast basal medium) was injected slowly by hand into each channel. Bubbles were removed by connecting one inlet port to a Pneumatic Primer (catalog # 205001, Synvivo, Inc.) at 5 psi for at least 40 minutes. Bubble-free chips were cooled at 2~4 0 C prior to use. Matrigel (catalog # 356230, Coming) was thawed on ice bath for at least 90 minutes.
[00176] When cell cultures reach confluency, preadipocytes and fibroblasts were trypsinized, resuspended in serum-free medium at 4 X 106 cells/ml and cooled on ice for 1 minute. Preadipocyte suspension, fibroblast suspension and Matrigel were mixed on ice at 3 : 1: 4 volumetric ratio using a pre-cooled pipette. This mixture was quickly injected to the central subcutaneous channel at 5 d/min and incubated for 90 minutes. To avoid cell sedimentation, each chip was placed leaning against the incubator wall with either the inlet or outlet side lifted.
[00177] After a complete polymerization of MATRIGEL™, the injection and vessel channels were perfused with full subcutaneous culture medium (e.g., either DMEM or DMEM/F12 with 20% FBS for mice) at 10 d/min. This media exchange was repeated daily for at least 5 days.
[00178] After cells reach confluency in the subcutaneous channel, adipocyte differentiation was induced by perfusing differentiation medium into the injection channel daily for three days. The differentiation medium contains full subcutaneous cell culture medium (e.g., either DMEM or DMEM/F12 with 20% FBS for mice) supplemented with 5.0 pM dexamethasone, 5 mM methylisobutylxanthine and 100 p g/ml insulin. These supplements were used at higher concentrations than established protocols42 to account for their low partitioning into the subcutaneous channel. The differentiated cells were maintained using full subcutaneous cell culture medium (e.g., either DMEM or DMEM/F12 with 20% FBS for mice) with 10 rg/ml insulin for daily media exchange.
[00179] Prior to seeding endothelial cells, 200 rg/ml human fibronectin in PBS was perfused into the vessel channel. The device was then incubated for at least 45 minutes and the vessel channel was washed with endothelial culture medium at 10 d/min. Confluent endothelial cells were trypsinized from culture flasks, resuspended in endothelial culture medium at 1 X 107 cells/ml and seeded into the vessel channel at 5 d/min. The chip was then incubated for 90 minutes with top side down to promote adhesion to the top surface. Excess cells which were not adhered to channel surfaces were washed by endothelial culture medium. The seeding procedure was repeated with endothelial cells trypsinized from a second culture flask followed by incubation with a normal chip placement for at least 4 hours. Excess cells were washed by endothelial culture medium. Daily media exchange was performed using endothelial culture medium (20% FBS for mice) at 2 d/min.
[00180] Cell staining on a chip
[00181] Live/dead. Cell viability was assessed using the live/dead cell vitality assay kit containing C12 Resazurin/SYTOX Green (ThermoFisher). After transport measurements, fresh media was perfused to the injection channel and the endothelial channel to remove fluorescence from the injected solution. The device was incubated for 1 hour followed by a wash with fresh media. 100 nM of Sytox green and 5 uM of C12-resazurin were injected into the subcutaneous channel. 10 nM of Sytox green and 500 nM of C12-resazurin were injected into the endothelial channel. The device was incubated again for 1 hour followed by a wash with fresh media. After 30-minute incubation, the device was washed again with fresh media for imaging.
[00182] Actin/nucleus. To stain the actin cytoskeleton and the nucleus, DPBS was perfused to the injection channel and the endothelial channel to remove fluorescent dyes from transport measurements. The device was incubated for 1 hour followed by a wash with DPBS. For fixation, 16% PFA was perfused into the injection channel and 4% PFA was perfused into the vascular channel. The device was incubated for 1 hour followed by a wash with DPBS. After 30-minute incubation, 1.0% Triton X-100 in DPBS was perfused into the injection channel and 0.2% Triton X- 100 in DPBS was perfused into the endothelial channel. The device was incubated for 1 hour to permeabilize fixed cells. After washing with DPBS for 30 min, ActinRed 555 ReadyProbes (2 drops per 200 ul of DPBS, ThermoFisher) and Hoechst 33342 (2 drops per 200 ul of DPBS, ThermoFisher) were perfused to the injection channel. ActinGreen 488 ReadyProbe (2 drops per 1 ml of DPBS, ThermoFisher) and Hoechst 33342 (2 drops per 1 ml of DPBS) were perfused to the endothelial channel. The device was incubated for at least 3 hours followed by a wash with DPBS, a 30-minute incubation and a second wash with DPBS.
[00183] Oil Red O. For visualization of lipids inside adipocytes, the subcutaneous cells were fixed and permeabilized using the injection channel for perfusion as previously described. Oil Red O solution was perfused into the injection channel and incubated for at least 2 hours. The stock solution (500 mg/L in isopropanol) was diluted to 300 mg/L with distilled water and filtered using a syringe filter immediately before use. The device was washed with DPBS, incubated for 1 hour and washed again with DPBS.
[00184] Adipocyte differentiation on a plate assay. To compare the effect of adipocyte differentiation between fibroblasts and preadipocytes at matching cell densities, the inventors seeded them separately on 6-well plates. Mice cells were maintained to confluency following the same procedure as in tissue culture flasks. Adipocyte differentiation was induced using cell culture medium supplemented with 0.5 p M Dexamethasone, 0.5 mM methylisobutylxanthine and 10 rg/ml insulin for three days. Differentiated cells were maintained using cell culture medium supplemented with 10 jUg/ml insulin for two days. Cells were then fixed using 4% PFA and permeabilized with 0.2% Triton X-100 in DPBS. Cells were incubated with Oil Red O solution for 1 hour and washed before imaging. The stock Oil Red O solution (300 mg/L in isopropanol) was diluted to 180 mg/L with distilled water and filtered before use.
[00185] Confocal microscopy. Z-stacks of images were taken using an inverted confocal microscope (Zeiss LSM 710) for chips with stained cells. For fixed cells (Oil Red O or actin), z-stacks were taken at 1 pm intervals, close to the limit of confocal depth of a 32X water immersion objective. For live dead staining, the z-stacks were taken at 5 um intervals, close to the limit of the confocal depth of a 1 OX water immersion objective to minimize the acquisition time.
[00186] Image processing and three-dimensional z-stack reconstruction were done using ImageJ™ software (Figs. 20A-20D). Images were first rotated using bilinear interpolation to align with the channel directions. They were then cropped to separate files for subcutaneous and vessel channels for quantitative analysis. For rendering purposes, channels were combined with individually adjusted brightness to account for lower fluorescence intensity in the subcutaneous channel. For Oil Red O and live/dead staining, the inventors combined z-stack images using ZProjection with maximum intensity. For quantification analysis, fluorescence intensities in each channel were converted to binary values (black and white). A threshold area was used in the analyze particle function to detect individual cells or lipid droplets. Lipid content was determined as the total stained area. Live and dead cell counts were determined from the particle counts. Cell viability may be underestimated due to overlapping live cells unable to be detected. For rendering purposes, dead stains were processed using a median filter with a radius of 2.0. The confocal 3D rendering of actin and nucleus staining was performed with interpolation using ImageJ 3D Project. The fluorescence intensity across channel depth in each channel was plotted using plot Z-profile.
[00187] Transport measurement. FITC-dextran (MW 10k, 40k and 150k), FITC-DEAE-dextran (40k), FITCCM-dextran (40k) were obtained from Sigma Aldrich. Mouse FITC-IgG was obtained from Southern Biotechnology Associates. Acquisition of images fortransport measurement were performed using an inverted microscope (Axio Observer Zl, Zeiss) with an incubator chamber (Zeiss) to maintain 37°C, 5% CO2 and controlled humidity for live cell imaging. Image acquisition was configured by establishing a calibration curve of fluorescence intensity vs. concentration on the chip using a 5X objective. At the beginning of each experiment, 1 mg/ml solution in DPBS was flown into the injection channel. For cellular devices, images were acquired at 1-minute intervals for 3 to 6 hours. For acellular devices used for control experiments, images were acquired at 30-second intervals for 2 hours.
[00188] Acquired images were analyzed using a customary MATLAB script (Fig. 21) to detect the channel borders and store fluorescent intensities as three matrices representing each channel. Each matrix contains three dimensions representing width(x), length (y) and time. Narrow slit regions were excluded from the analysis.
[00189] Diffusivity calculation. The inventors did not consider diffusion across the channel depth (z) due to the limitation of our microscope as well as its small dimension compared to channel length (y) and width (x). Due to the close spacing of narrow slits (50 pm) compared to the channel width (200-500 pm), variations in y is much smaller than in x (Figs. 22A-22B). The inventors approximated the diffusivity of macromolecules inside the chip as a one-dimensional diffusion problem by averaging fluorescence intensities along y. The inventors further simplified the problem by realizing that variations along the x is small in the injection and vessel channels due to both an increased diffusivity in an aqueous solution as compared to the subcutaneous tissue and a reduction in channel width (Figs. 22C-22D). The inventors therefore approximated the injection and vessel channels as well-stirred compartments with only temporal variations in concentration represented by Ci(t) and (t) respectively. The concentration profde in the subcutaneous channel is denoted Cz(x, t). The inventors assumed Ci » C2 » C3 throughout the experiment, which is validated by comparing their fluorescence intensities at the end of each experiment (Fig. 22E). This assumption allowed us to calculate R12, D and R23 individually to obtain analytical solutions and bypass simulation approaches which are more time-consuming and less user-friendly. After converting all fluorescence intensities to concentrations, the inventors performed data fitting in MATLAB using Isqcurvefit.
[00190] Based on Fick’s first law of diffusion, the apparent permeability from the injection channel to the subcutaneous channel can be written as:
Figure imgf000056_0001
where, AC12 is the concentration difference between Ci and C2 (x = 0). Q12 is the cumulative amount of permeant transported at time t and calculated as
Figure imgf000056_0002
This expression is valid when a steady state flux is formed such that Q12 grows linearly with t and AC12 is constant, which holds true based on our assumption of Ci » C2 » C3. The inventors therefore approximated AC12 « Ci and estimated R12 when Q12 grew linearly with t (Fig. 19A). The apparent permeability R23 is the combined effect of permeation across narrow slits Rsiit followed by partitioning and absorption onto the subcutaneous channel. Therefore, the actual permeability into the subcutaneous channel R°i2 can be written as:
1
^12
Figure imgf000056_0003
to assess the relative contribution of slits and subcutaneous barrier to the permeability.
[00191] For diffusion in the subcutaneous space, the governing equation is:
Figure imgf000056_0004
The inventors considered the case where the concentration at x < 0 is maintained constant over time, starting at initial time t = 0. The solution to this equation is
Figure imgf000057_0001
The inventors adjusted this solution for diffusion in the chip in the following form:
Figure imgf000057_0002
In this expression, there are five unknown parameters to be determined. D2 is the diffusivity in the subcutaneous space. At is the time lag between the start of diffusion and the beginning of image acquisition, e is the distance to the fixed concentration source and T is a reflective distance such that at
T
Figure imgf000057_0003
, zero flux is established for a finite boundary instead of a semi-infinite regime in the original solution. In the absence of narrow slits connecting adjacent channels, T and e can be determined from the channel geometry. The narrow slits complicate the problem and therefore T and e become fitting parameters. Co is the concentration in the subcutaneous channel at equilibrium. To test the validity of our analytical expression, the inventors also considered fitting with a different expression
Figure imgf000057_0004
. This expression considers point sources with a constant concentration instead of a semi-infinite domain.
The actual condition in the injection channel lies between these two scenarios. The diffusivities calculated from two values differ by no more than 5% and the fit using Equation (3) gave a slightly better estimate in terms of the sum of least squares.
[00192] The geometry between the subcutaneous channel and the vessel channel is similar to the entrance from the injection channel to the subcutaneous channel. However, both the subcutaneous and
Figure imgf000057_0005
the vessel channels have increasing concentrations over time. A plot of
Figure imgf000057_0006
shows a linear correlation (Fig. 19B), where
Figure imgf000058_0001
. The inventors therefore determined AY-i as:
Figure imgf000058_0002
Similar to R12, this apparent permeability can be decomposed into the actual permeability and the slit contribution:
Figure imgf000058_0003
. In the presence of endothelial cells, R°23 can be further decomposed to obtain the contribution from the endothelial barrier.
[00193] Statistical analysis. Data in all plots are expressed as mean ± s.e.. Statistical analysis was done in MATLAB, and statistical significance was attributed to values of P < 0.05 as determined by unpaired Student’s t-test, as described in the figure legends. Different significance levels (P values) are indicated in each figure with asterisks: *P < 0.05, **P < 0.01.
[00194] References
40. Qi, Q. & Mitragotri, S. Mechanistic study of transdermal delivery of macromolecules assisted by ionic liquids. Journal of Controlled Release 311-312, 162-169 (2019).
41. Zuidema, J., Rivet, C., Gilbert, R. & Morrison, F. A protocol for rheological characterization of hydrogels fortissue engineering strategies. Journal of Biomedical Materials Research Part B: Applied Biomaterials 102, 1063-1073 (2013).
42. Kang, X., Xie, Y. & Kniss, D. Adipose Tissue Model Using Three-Dimensional Cultivation of Preadipocytes Seeded onto Fibrous Polymer Scaffolds. Tissue Engineering 11, 458-468 (2005).
Example 3: The effect of flow conditions on microfluidic processing
[00195] Although the transport measurement was performed at static conditions, all nutrient exchange and cell seeding procedures of microfluidic processing occur through perfusion. Therefore, flow conditions under the channel geometry need to be assessed to optimize the processing conditions24 . In Table 1, the inventors summarized flow rates used in this study. Key factors governing the flow consideration are discussed below.
[00196] The influence of slit size on perfusion conditions.
[00197] A finite element simulation of the flow profile on a multi -compartment microfluidic chip with interconnecting narrow slits reveal that when pressure driven flow occurs in one channel to drive perfusion, the neighboring channel experiences a disturbance flow no greater than 5% the inlet velocity due to the high aspect ratio between the slit and channel dimensions 12 . The inventors approximated the flow through slits as multiple resistances connected in sequence and parallel43 (Fig.
1 IB) and solved for the flow rates using Excel. Similar to the above finding, the inventors found a significant reduction of flow rate in the neighboring channel, which is beneficial for controlling perfusion conditions in each channel independently.
[00198] The inventors considered the influence of flow conditions on nutrient transfer during both perfusion and static incubation. During perfusion, the Peclet number describing ratio of advection rate to diffusion rate is written as:
Figure imgf000059_0001
. Using 100 zm channel depth and 100 zm /s for the diffusivity of glucose, the inventors estimated the Peclet number to be at least 200000 for the slowest perfusion rate used (2 d/min). Therefore, the perfusion process is convection dominated and the entire liquid content in the perfusion channel was replaced.
[00199] During static incubation, the mass transport is diffusion-dominated. To ensure sufficient nutrient transport, the inventors considered the ratio of nutrient volume to contact area for both the subcutaneous and vascular spaces43 . In the subcutaneous space, nutrient exchange occurs through narrow slits, which yields a ratio of
Figure imgf000059_0002
In the vascular space, perfusion occurs inside the channel, which yields a ratio of
Figure imgf000059_0003
In a tissue culture flask, this ratio is typically
Figure imgf000059_0004
Therefore, in the subcutaneous space, we are not limited by the volume of media used. However, we are limited by the slow diffusion inside the subcutaneous space, which poses a challenge for cell staining. To achieve sufficient staining, the inventors increased the concentration for solutions used in each staining step and increased the duration of incubation procedures compared to 2D staining protocols in a flask. In the vascular space, we are limited by the channel geometry. To maintain cell viability, the inventors performed media exchange daily instead of every 2 to 3 days in a flask. In addition, excess fluids reside in the inlet and outlet ports and tubings. This approach can be replaced by a continuous perfusion across the vascular channel enabled by a programmable syringe pump. The flow rate needs to be slow enough to prevent excess shear24 . The current flow rate of 2 d/min is appropriate for a continuous feeding as well. [00200] Cell migration during subcutaneous seeding
[00201] During subcutaneous seeding, a mixture of cells, medium and MATRIGEL™ was perfused into the channel. Although we 11 -mixed at the inlet, cells migrate in the cross-flow direction, resulting in a nonhomogeneous concentration distribution profile. This profile can influence cell growth and expansion, result in a cell density different from the actual tissue environment and affect the measurement of transport rates. Cell migration can arise due to various mechanisms. Reynolds number is very small for microfluidic applications due to the small channel size and thus inertia and inertia-induced migration can be neglected44,45 . Cell migration can occur due to curvature in the flow profile, cell deformability as well as viscoelasticity in the suspending liquid. The inventors focused on the effect of cell deformability under varying flow conditions and estimated the resulting distribution profile (Fig. 13).
[00202] Based on previous theoretical models44,46 , the inventors consider the cells as a dilute suspension of deformable particles (radius a, shear modulus G) with total volume fraction =10%. Their motion in a microchannel or in the microvasculature can be approximated as flowing in a slit bounded by two walls at z = 0 and z = 2H and unbounded in x and y. The inventors consider pressure driven flow in the channel and thus the local shear rate can be described as: y(z) = e flow viscous effect relative to the deformability of cells can be described by :
Figure imgf000060_0001
The number density of cells n is assumed to be only a function of z. In the dilute limit, the inventors only consider binary hydrodynamic interactions which result in shear-induced diffusion45,47 jdiffu ion - Due to the cell deformability, they migrate away from the wall under stokes flow with a velocity viift(z), generating a lift flux jm 48.
[00203] The inventors made the equation above dimensionless using the cell radius a and the wall shear rate ywaii to arrive at the following equation as shown in Figure 31:
Figure imgf000060_0002
According to previous work46 , the lift flux can be written as:
Figure imgf000061_0001
This expression is a function of the Capillary number Ca and the wall confinement . It accounts for migration due to both flow curvature (Ka) and walls (Kw). The unknown coefficient depends on the complex interplay between material properties of cells and the local flow conditions and requires computer simulations to determine. It is therefore beyond the scope of this study.
[00204] The shear-induced diffusional flux /diffusion is proportional to the product of the number densities of two particles multiplied by the z velocity difference between them and is an integral over all possible spatial configurations47 . According to previous work46 , this flux can be simplified to the following expression:
Figure imgf000061_0002
Using a finite volume scheme, the inventors solved for the steady state concentration distribution of cells (p(z) under two scenarios: Ca = 1, 100. A large Capillary number indicates a high flow rate. To match the experimental conditions, the inventors set C= 25 for both cases. The inventors set Ki = 0.16,
KW = 0.068, Ka = 0.0041, KC = KC’ = 0.0984, Kd = Kd'= 0.3445. The inhomogeneity in the concentration profile increases with Ca (Fig. 13). Therefore, it is important that the inventors maintain a low flow rate during cell seeding.
[00205] References
42.Walker, G., Monteiro-Riviere, N., Rouse, J. & O'Neill, A. A linear dilution microfluidic device for cytotoxicity assays. Lab Chip 7, 226-232 (2007).
43.Young, E. & Beebe, D. Fundamentals of microfluidic cell culture in controlled microenvironments. Chemical Society Reviews 39, 1036 (2010).
44. Qi, Q. et al. In Vitro Measurement and Modeling of Platelet Adhesion on VWF-Coated Surfaces in Channel Flow. Biophysical Journal 116, 1136-1151 (2019).
45. Qi, Q. & Shaqfeh, E. Theory to predict particle migration and margination in the pressuredriven channel flow ofblood. Physical Review Fluids 2, (2017).
46.Henriquez Rivera, R., Zhang, X. & Graham, M. Mechanistic theory of margination and flow-induced segregation in confined multicomponent suspensions: Simple shear and Poiseuille flows. Physical Review Fluids 1, (2016).
47. Leighton, D. & Acrivos, A. The shear-induced migration of particles in concentrated suspensions. Journal of Fluid Mechanics 181, 415 (1987).
48. Zhao, H., Spann, A. & Shaqfeh, E. The dynamics of a vesicle in a wall-bound shear flow. Physics of Fluids 23, 121901 (2011).

Claims

What is claimed herein is:
1. A system comprising: a first subcutaneous compartment comprising: i. a mixture comprising fibroblasts, cell culture medium, hydrogel, and at least one of preadipocytes and adipocytes; and a first peripheral compartment comprising: i. at least one opening providing fluid, non-cellular communication with the first subcutaneous compartment; and ii. optionally comprising endothelial cells.
2. The system of claim 1, further comprising a second peripheral compartment comprising at least one opening providing fluid, non-cellular communication with the first subcutaneous compartment.
3. The system of any of claims 1-2, wherein the first subcutaneous compartment is medial with respect to the first and second peripheral compartments; and the first and second peripheral compartments are not directly in fluid, non-cellular communication with each other.
4. The system of any of claims 1-3, wherein each compartment comprises or is a channel, cube, rectangular prism, or cylinder.
5. The system of any of claims 1-4, wherein the first subcutaneous compartment comprises: a medial disc or sphere portion that comprises at least one of the openings with the first peripheral compartment and optionally, second peripheral compartment; a first lateral channel, cube, rectangular prism, or cylinder portion; and a second lateral channel, cube, rectangular prism or cylinder portion.
6. The system of claim 5, wherein the first and/or second lateral portions do not comprise the at least one openings with the first peripheral compartment and optionally, second peripheral compartment.
7. The system of any of claims 5-6, wherein the first and second peripheral compartments comprise a medial curved channel portion where they comprise the at least one openings with the first subcutaneous compartment; a first lateral channel, cube, rectangular prism, or cylinder portion; and a second lateral channel, cube, rectangular prism or cylinder portion.
8. The system of claim 1, further comprising; a second subcutaneous compartment comprising: i. a mixture comprising fibroblasts, cell culture medium, hydrogel, and at least one of preadipocytes and adipocytes; and a second peripheral compartment comprising: ii. at least one opening providing fluid, non-cellular communication with the second subcutaneous compartment; and iii. optionally comprising endothelial cells; a medial injection compartment comprising: iv. at least one opening providing fluid, non-cellular communication with the first subcutaneous compartment; and v. at least one opening providing fluid, non-cellular communication with the second subcutaneous compartment; wherein the first and second subcutaneous compartments are lateral to the medial injection compartment.
9. The system of claim 8, wherein the medial injection compartment comprises: a medial disc or sphere portion that comprises at least one of the openings with each of the first and second subcutaneous compartments; a first lateral channel, cube, rectangular prism, or cylinder portion; and a second lateral channel, cube, rectangular prism or cylinder portion.
10. The system of claim 9, wherein the first and/or second lateral portions do not comprise the at least one openings with the first and second subcutaneous compartments.
11. The system of any of claims 9-10, wherein the first and second subcutaneous compartments each comprise a medial curved channel portion where they comprise at least one of the openings with the medial injection compartment; a first lateral channel, cube, rectangular prism, or cylinder portion; and a second lateral channel, cube, rectangular prism or cylinder portion.
12. The system of claim 11, wherein the medial curved channel portion of the first subcutaneous compartment comprises at least one of the openings with the first peripheral compartment; and the medial curved channel portion of the second subcutaneous compartment comprises at least one of the openings with the second peripheral compartment.
13. The system of any of the preceding claims, wherein the medial injection channel, first subcutaneous compartment, and/or second subcutaneous compartment further comprises an injection port.
14. The system of claim 13, wherein the injection port is in the medial disc or sphere portion.
15. The system of any of the preceding claims, wherein the at least one opening comprises multiple openings or slits in an interposed wall or barrier. The system of any of the preceding claims, wherein the at least one opening is no greater in height or width than 500 pm. The system of any of the preceding claims, wherein the at least one opening is no greater in height or width than 100 pm. The system of any of the preceding claims, wherein the fluid availability aspect ratio is 5 or greater. The system of any of the preceding claims, wherein the fluid availability aspect ratio is 50:3. The system of any of the preceding claims, wherein the fibroblasts and at least one of preadipocytes and adipocytes form a 3D culture. The system of any of the preceding claims, wherein the mixture comprising fibroblasts, cell culture medium, hydrogel, and at least one of preadipocytes and adipocytes comprises more preadipocytes and/or adipocytes than fibroblasts. The system of any of the preceding claims, wherein the mixture comprising fibroblasts, cell culture medium, hydrogel, and at least one of preadipocytes and adipocytes comprises at least 2x as many preadipocytes and/or adipocytes as fibroblasts. The system of any of the preceding claims, wherein the mixture comprising fibroblasts, cell culture medium, hydrogel, and at least one of preadipocytes and adipocytes comprises at least 3x as many preadipocytes and/or adipocytes as fibroblasts. The system of any of the preceding claims, wherein the first peripheral compartment comprises endothelial cells. The system of any of the preceding claims, wherein the first peripheral compartment comprises endothelial cells and the second peripheral compartment does not comprise cells. The system of any of the preceding claims, wherein the first and second peripheral compartments each comprise endothelial cells. The system of any of the preceding claims, wherein the endothelial cells line one or more walls of the first and/or second peripheral compartments. The system of any of the preceding claims, wherein the endothelial cells form a confluent single-cell monolayer on all walls of the first and/or second peripheral compartments. The system of any of the preceding claims, wherein the cells are murine or human cells. The system of any of the preceding claims, wherein at least 80% of the cells are viable. The system of any of the preceding claims, wherein at least 90% of the cells are viable. The system of any of the preceding claims, wherein each compartment further comprises at least 2 ports. The system of claim 32, wherein the at least 2 ports are connected to a microfluidics system to provide an inflow and an outflow port in each compartment. The system of any of the preceding claims, wherein the hydrogel comprises one or more extracellular matrix components. An in vitro method of determining the subcutaneous administration characteristics of a candidate subcutaneous agent, the method comprising: introducing a candidate subcutaneous agent into the injection compartment and/or a peripheral compartment not comprising cells, of the system of any of claims 1-34; and measuring at least one of: the amount and/or change in the amount of subcutaneous agent in the outflow of fluid from one or more of the compartments; the amount and/or change in the amount of subcutaneous agent present in one or more of the compartments; and the amount and/or change in the amount of subcutaneous agent in the outflow of fluid from a peripheral compartment comprising endothelial cells. The method of claim 35, wherein the following are measured: the amount and/or change in the amount of subcutaneous agent present in each of the compartments; and the amount and/or change in the amount of subcutaneous agent in the outflow of fluid from a peripheral compartment comprising endothelial cells. The method of any of claims 35-36, wherein a peripheral compartment not comprising vascular endothelial cells indicates subcutaneous administration characteristics via lymphatic pathways. The method of any of claims 35-36, wherein a peripheral compartment comprising lymphatic endothelial cells but not comprising vascular endothelial cells indicates subcutaneous administration characteristics via lymphatic pathways. The method of any of claims 35-36, wherein a peripheral compartment comprising endothelial cells indicates subcutaneous administration characteristics via vascular pathways. The method of any of claims 35-36, wherein a peripheral compartment comprising vascular endothelial cells indicates subcutaneous administration characteristics via vascular pathways. The method of any of claims 35-40, wherein the candidate subcutaneous agent is introduced into the injection compartment and/or a peripheral compartment not comprising cells at a perfusion rate of from 0.1 pL/min to 1,000 pL/min. The method of any of claims 35-41, further comprising the following steps prior to the step of introducing the candidate subcutaneous agent: introducing the mixture comprising fibroblasts, cell culture medium, hydrogel, and preadipocytes into the first and/or second subcutaneous compartment and then culturing the preadipocytes and fibroblasts; inducing adipocyte differentiation and then maintaining the adipocytes and fibroblasts. The method of any of claims 35-42, further comprising the following steps prior to the step of introducing the candidate subcutaneous agent: introducing the mixture comprising fibroblasts, cell culture medium, hydrogel, and at preadipocytes into the first and/or second subcutaneous compartment and then culturing the preadipocytes and fibroblasts; inducing adipocyte differentiation and then maintaining the adipocytes and fibroblasts; twice introducing endothelial cells into the first and/or second peripheral compartment and then culturing and maintaining the endothelial cells. The method of any of claims 35-43, wherein the mixture comprising fibroblasts, cell culture medium, hydrogel, and at least one of preadipocytes and adipocytes is at less a temperature of less than 10 C when it is introduced into the first and/or second subcutaneous compartment. The method of any of claims 35-44, wherein the mixture comprising fibroblasts, cell culture medium, hydrogel, and at least one of preadipocytes and adipocytes is at less a temperature of 4 C or less when it is introduced into the first and/or second subcutaneous compartment. The method of any of claims 35-45, wherein culturing and/or maintaining the fibroblasts and at least one of preadipocytes and adipocytes comprises one or more of: culturing for at least 5 days; culturing until the fibroblasts and at least one of preadipocytes and adipocytes reach a high density; and culturing until the fibroblasts and at least one of preadipocytes and adipocytes are confluent. The method of any of claims 35-46, wherein culturing and/or maintaining the fibroblasts and at least one of preadipocytes and adipocytes comprises exchanging the culture medium in the first and/or second subcutaneous compartment at least daily. The method of any of claims 35-47, wherein culturing and/or maintaining the fibroblasts and at least one of preadipocytes and adipocytes comprises exchanging the culture medium in the first and/or second subcutaneous compartment continuously. The method of any of claims 35-48, wherein medium exchange in the injection compartment and/or a peripheral compartment not comprising cells is performed at a perfusion rate of from 0.1 pL/min to 1000 pL/min. The method of any of claims 35-49, wherein culturing and/or maintaining the endothelial cells comprises one or more of: culturing for at least 3 days; culturing until the endothelial cells reach a high density; and culturing until the endothelial cells are confluent. The method of any of claims 35-50, wherein culturing and/or maintaining the endothelial cells comprises exchanging the culture medium in the first and/or second peripheral compartment at least daily. The method of any of claims 35-51, wherein culturing and/or maintaining the endothelial cells comprises exchanging the culture medium in the first and/or second peripheral compartment continuously. The method of any of claims 35-52, wherein medium exchange in the first and/or second peripheral compartment is performed at a perfusion rate of from 0.02 pL/min to 200 pL/min.
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EP4379035A1 (en) * 2022-11-30 2024-06-05 Fundació Institut de Bioenginyeria de Catalunya (IBEC) Cell culture system and cell culture method
WO2024115270A1 (en) 2022-11-30 2024-06-06 Fundació Institut De Bioenginyeria De Catalunya Cell culture system and cell culture method

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