(12) INTERNATIONALAPPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY(PCT)
`~~,
`(19) World Intellectual Property
`RECENTDE
`Organization
`International Bureau
`
`(43) luternational Publication Date
`10 August 2017 (10.08.2017)
`
`WIPO) PCT
`
`—\
`
`(16) International Publication Number
`WO 2017/136479 Al
`
`(31)
`
`International Patent Classification:
`CLIN S/077 (2010.03)
`CLIN$/0797 (2010.01)
`CIIN 5/074 (2010.01)
`CI2N 5/22 (2006.01)
`CI2N 8/0789 (2016.01)
`
`(21)
`
`International Application Number:
`
`PCT/US2017/0 16098
`
`(22)
`
`International Filing Date:
`
`chael; 603 {8th Street, Sania Monica, Califomia 90402
`(US). SAREEN, Dhruv, 19375 Crystal Ridge Lane, Porter
`Ranch, Califomia 91326 (US). RAJAMANTL, Uthra; 2000
`Ivar Avenue, Apt. I, Los Angeles, California 90068 (US).
`
`(74)
`
`Agents: CHEN, Stephen, W. et al.; NIXON PEABODY
`LLP, 300 South Grand Avenue, Suite 4100, Los Angeles,
`California 90071-3151 CUS}.
`
`1 February 2017 (01.02.2017)
`
`(8b
`
`(25)
`
`(26)
`
`(30)
`
`(71)
`
`(72)
`
`Filing Language:
`
`Publication Language:
`
`Prierity Data:
`62/289,52]1
`62/332,849
`62/354,040
`62/437,314
`
`1 February 2016 (01.02.2016}3
`6 May 2016 (06.05.2016)
`23 June 2016 (23.06.2016)
`21 December 2016 (21.12.2016)
`
`Enghsh
`
`English
`
`Us
`US
`Us
`US
`
`Applicant. CEDARS-SINAI MEDICAL CENTER
`[US/US], 8700 Beverly Blvd., Los Angeles, California
`90048 (US).
`
`(84)
`
`Inventors: BARRETT, Robert, 225 S. Olive St. Apt.
`1709, Los Angeles, California 90012 (US). SVENDSEN,
`Chive; 15242 Friends Street, Pacific Palisades, California
`90272 (US). TARGAN, Stephan R.; 240 22nd Street,
`Santa Monica, Califoria 90402 (US). WORKMAN, Mi-
`
`Designated States (unless otherwise indicated, for every
`kind of national protection available}: AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY,
`BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM,
`Do, DZ, EC, EE, EG, ES, FL GB, GD, GE, GH, GM, GT,
`TIN, FR, HU, 0D, IL, IN, IR, IS, JP, KE, KG, KE, KN,
`KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA,
`MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG,
`NY, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS,
`RU, RW, SA, SC, SD, SE, SG, SIS, SL, SM, ST, SV, SY,
`TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN,
`ZA, 2M, ZW.
`
`Designated States (unless otherwise indicated. for every
`kind ofregional protection available); ARIPO (BW, GH,
`GM, KE, LR, LS, MW, MZ, NA, RW, SD. SL, ST, SZ,
`TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU,
`TE, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE,
`DK, EE, ES, FL FR, GB, GR, HR, HU, TE, IS, FT, LT, LU,
`LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, Sf, SK,
`
`[Continued on next page]
`
`(54) Title: SYSTEMS AND METHODS FOR GROWTHOF INTESTINAL CELLS IN MICROFLUIDIC DEVICES
`
`Figure 40
`
`
`
`(57) Abstract: Organs-on-chips are micro-
`fluidic devices for culturing [ving cells in
`micrometer sized chambers in order to model
`physiological functions oftissues and organs.
`Engineered patterning and continnous fluid
`flow in these devices has allowed culturing
`of intestinal cells bearing physiologicallyrel-
`evant features and sustained exposure to bac-
`teria while maintaining cellular viability,
`thereby allowing study of intlammatory bowl
`diseases. However, existing intestinal ceils
`do not possess all physiologically relevant
`subtypes, do not possess the repertoive af ge-
`netic variations, or allow for study of other
`important celhiar actors such as immune
`cells. Use of iPSC-derived epithelium,
`in-
`cluding IBDpatient-specitic cells, allows for
`superior disease modeling by capturing the
`multi-faceted nature ofthe disease.
`
`
`
`
`
`wo2017/136479AtTIINNATCISAMAATAT
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`

`

`wo 2017/136479ALTE
`CW.KM.ML.MONE.SN.Ie GA, GN, GQ,
`__
`before the expiration ofthe time Limitfor amending the
`
`claims and to be republished in the event of receipt of
`amendments (Rule 48.2(h))
`
`Published:
`
`— with international search report (Art. 22(3))
`
`

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`WO 2017/136479
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`PCT/US2017/016098
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`SYSTEMS AND METHODS FOR GROWTH OF INTESTINAL
`
`CELLS IN MICROFLUIDIC DEVICES
`
`wn
`
`The present
`
`invention relates to a combination of cell culture systems and
`
`FIELD OF THE INVENTION
`
`microfluidic fluidic systems. More specifically,
`
`in one embodiment, the invention relates
`
`microfluidic chips seeded with stem-cell-derived cells that mature and.or differentiate into
`
`intestinal cells. In one embodiment, the stems are induced pluripotent stem cells (hiPSCs) and
`
`the intestinal cells are foregut cells. In some emdodiments, such forgut chips are tested for
`
`effects of endocrine disrupting chemicals
`
`(EDCs) during critical periods
`
`in tissue
`
`development mimicking critical periods of fetal development for short and long term
`
`downstream effects. In particular, methods for use are provided for induced pluripotent stem
`
`cells (hiPSCs) to elucidate adverse effects and mechanisms of chronic low-dose EDC
`
`exposures on developing gut and hypothalamic neuropeptidergic neurons, and serves as a
`
`platform for mimicking the in utero exposure to EDCs. Morover, in yet further embodiments,
`
`iPS cells derived from obese individuals are seeded on chips for determing effects of EDCs in
`
`relation to obsesigens.
`
`The invention further relates to methods and systems for providing cells from
`
`intestinal organoids (the organoids derived from iPSCs) on microfluidic chips. In one
`
`20
`
`embodiment, additional cells are on the chip, e.g.
`
`induced neuronal cells.
`
`In some
`
`embodiments, microfluidic
`
`intestinal Organ-On-Chips mimic human gastrointestinal
`
`disorders, e.g. IBD,etc.
`
`BACKGROUND
`
`Nw oa
`
`Persistent human exposure to elevated levels of man-made endocrine disrupting
`
`chemicals (EDCs) during critical periods in fetal development may lead to long-term
`
`disruption of metabolic homeostasis in endocrine tissue progenitors,
`
`thus contributing to
`
`childhood cbesity. Specifically, endocrine control of feeding behavior
`
`involves
`
`the
`
`participation and communication between the hypothalamic arcuate nucleus and the
`
`Ge a
`
`gastrointestinal tract enteroendocrine cells, stomach in particular. The hypothalamic (HT)
`
`neuropeptidergic neurons receive endacrine signals from parts of gut including gastrin and
`
`ghrelin from stomach, peptide YYfromintestine and bring about orexigenic or anorexigenic
`
`effects. Hence, abnormalities during development due to external or environmental factors
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`WO 2017/136479
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`PCT/US2017/016098
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`such as EDCs may play a role in dysfunction of the gut-brain interactions thereby bringing
`
`about feeding disorders and obesity.
`
`There is paucity of data on the developmental effects of early exposure of EDCs on
`
`dysfunction of cells involved in feeding and hunger largely due to the implausibility of
`
`accessing humanfetal tissue at different developmental stages. To fill this void, the Inventors
`
`employed human induced pluripotent stem cells (hiPSCs) to elucidate the adverse effects and
`
`mechanisms of chronic low-dose EDC exposures on developing gut and hypothalamic
`
`neuropeptidergic neurons, and serves as a plattorm tor mimicking the 7 utero exposure to
`
`EDCs. This ts the first such application of the pluripotent stem cell technology.
`
`10
`
`Without affecting cell viability,
`
`low-dose EDCs significantly perturbed NF-«B
`
`signaling in endocrinally active iFGEs and iHTNs. Consequently, EDC treatment decreased
`
`maximal mitochondrial respiration and spare respiratory capacity in iFGEs and iHTNs upon
`
`mitochondrial stress challenges,
`
`likely via NF-KB mediated regulation of mitochondrial
`
`respiration and decreased expression of both nuclear
`
`(SCO2,
`
`7TFAAM, POLRMT) and
`
`15
`
`mitochondrially-encoded (CytB5) respiratory genes. Treatment with NF-«B inhibitor, SN50,
`
`rescued EDC-induced aberrant NF-«B signaling and improved mitochondrial respiration.
`
`This seminal work ts the first report about a human plunpotent stem cell (PSC)-based
`
`mechanistic model of endocrine disruption by environmental chemicals, describing the
`
`adverse impact of EDCs on NF-«B signaling and mitochondrial dysfunction. This paves the
`
`way for a reliable screening platform for obesogenic EDCs in the developing human
`
`endocrine system.
`
`SUMMARYOF THE INVENTION
`
`The invention provides a method of compound screening, comprising: providing a
`
`quantity of differentiated induced pluripotent stem cells (iPSCs)}; contacting the differentiated
`
`iPSCs with one or more compounds; measuring one or more properties of the differentiated
`
`iPSCs, wherein measurement of the one or more properties of the differentiated iPSCs
`
`identifies characteristics of the one or more compounds. In one embodiment, said compound
`
`screening comprises
`
`screening for endocrine disruption.
`
`In one embodiment,
`
`said
`
`characteristics of the one er more compounds comprise inducing phorphorylation of Nuclear
`
`factor kappa B (NF-kB). In one embodiment, said characteristics of the one or compounds
`
`comprise decrease in mitochondrial respiration. In one embodiment, said characteristics of
`
`the one or compounds comprise decrease in expression of one or more of Cytochrome C
`
`Oxidase
`
`Assembly
`
`Protein
`
`(SCO2),
`
`RNA
`
`Polymerase
`
`Mitochondrial
`
`ha
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`(POLRMT),Transcription Factor A, Mitochondrial
`
`(TFAM)
`
`and CYTBS.
`
`In
`
`one
`
`embodiment, said differentiated 1PSCs are foregut epithelium.
`
`In one embodiment, said
`
`differentiated iPSCs are hypothalamic neurons.
`
`The invention provides a method of differentiating induced pluripotent stem cells,
`
`comprising: providing a quantity of induced pluripotent stem cells GPSCs), and culturing in
`
`the presence of one or more factors, wherein the one or more factors are capable of
`
`differentiating the iPSCs. In one embodiment, said iPSCs are differentiated into definitive
`
`endoderm by culturing in the presence of one or more factors comprising Activin A and
`
`Wnt3A. In one embodiment, said culturing in the presence of one or more factors comprising
`
`Activin A and Wnt3A is for about 3 days. In one embodiment, said differentiated iPSCs are
`
`initially cultured under serum-free conditions, followed by addition of serum.
`
`In one
`
`embodiment, said definitive endoderm is differentiated into foregut spheroids by further
`
`culturing in the presence of one or more factors comprising CHIR99021, FGF (FGF4), LDN
`
`(small molecule), and Retinoic Acid (RA). In one embodiment, said culturing in the presence
`
`of one or more factors comprising CHIR99021, FGF (FGF4), LDN, and Retinoic Acid (RA)
`
`is for about 3 days. In one embodiment, said foregut spheroid is differentiated into foregut
`
`epithelium by culturing on a coated surface. In one embodiment, said foregut spheroid is
`
`differentiated into foregut epithelium by additional culturing in the presence of one or more
`
`factors comprising epidermal growth factor (EGF).
`
`In one embodiment, said additional
`
`20
`
`culturing in the presence of one or more factors comprising epidermal growth factor (EGF) is
`
`for about 20 days. In one embodiment, said iPSCs are initially cultured in the presence of
`
`ROCK-inhibitor Y27632.
`
`In
`
`one
`
`embodiment,
`
`said iPSCs
`
`are differentiated into
`
`neuroectoderm by culturing in the presence of one or more factors comprising LDN193189
`
`and SB431542. In one embodiment, said culturing in the presence of one or more factors
`
`comprising LDN193189 and SB431542 is for about 2 days.
`
`In one embodiment, said
`
`neuroectoderm is differentiated into ventral diencephalon by culturing in the presence of one
`
`or more factors comprising smoothened agonist SAG, purmorphamine (PMN) and IWR-
`
`endo. In one embodiment, said culturing in the presence of one or more factors comprising
`
`moothened agonist SAG, purmorphamine (PMN) and [WR-endo is for about 5-6 days. In one
`
`embodiment, said ventral diencephalon is matured by culturing in the presence of one or
`
`more factors comprising DAPT,retinoic acid (RA). In one embodiment, said culturing in the
`
`presence of one or more factors comprising DAPT, retinoic acid (RA) is for about 4-5 days.
`
`In one embodiment, said mature ventral diencephalon is further matured by culturing in the
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`presence of one or more factors comprising BDNF. In one embodiment, said culturing in the
`
`presence of one or more factors comprising BDNF is for about 20-27 days.
`
`Endocrine disrupting chemicals (EDCs) are contemplated to affect early tissue
`
`development either by causing immediate damage or causing an alteration considered
`
`harmful to an organism, such as an immediate change to one or moreof a cell function, tissue
`
`function, physiological function, developmental pathway; and/or by causing damage over
`
`longer term in a subtle or unexpected way,i.e. as deleterious during early tissue development.
`
`Example 19 discusses some ofthese tissue changes.
`
`We hypothesized that chronic low-dose exposure to endocrine disrupting chemicals
`
`10
`
`(EDCs),
`
`is deleterious during early human endocrine tissue development. Further, we
`
`hypothesized that such exposure results in hyperactive NF-«xB and HMG protein pro-
`
`inflammatorysignaling with permanent mitochondrial dysfunction.
`
`Inflammatory bowel disease (IBD), such as Crohn’s disease and ulcerative colitis,
`
`involve chronic inflammation of human intestine. Mucosal injury and villus destruction are
`
`15
`
`hallmarks of IBD believed to be caused by complex interactions between gut microbiome,
`
`intestinal mucosa, and immune components.
`
`It has been difficult
`
`to study the relative
`
`contributions of these different factors in human intestinal inflammatory diseases, due to a
`
`lack of animal or in vitro models allowing for independent control of these parameters. As a
`
`result, existing models of human intestinal inflammatory diseases rely either on culturing an
`
`intestinal epithelial cell monolayer in static culture or maintaining intact explanted human
`
`intestinal mucosa ex vivo. Given the dynamic tissue environment of the gut, these static in
`
`vitro methods cannot faithfully recapitulate the pathophysiology of human IBD. Notably,
`
`intestinal epithelial cells cultured in plates completely fail to undergo villus differentiation,
`
`produce mucus, or form the various specialized cell types of normalintestine.
`
`Organs-on-chips are microfluidic devices for culturing living cells in micrometer
`
`sized chambers in order to model physiological functions of tissues and organs. Continuous
`
`perfusion through the chambers allows
`
`incorporation of physical
`
`forces,
`
`including
`
`physiologically relevant levels of fluid shear stress, strain and compression, for study of
`
`organ-specific responses. Of great
`
`interest
`
`is adapting such fabrication techniques for
`
`development of a “gut-on-a-chip” capable of replicating the corresponding physiological
`
`environment, and dynamically incorporating those multiple components (microbiome,
`
`mucosa, immune components) in a manner mirroring IBD pathophysiology. Towards these
`
`aims, prior attempts have successfully relied on human intestinal epithelial cells (Caco-2)
`
`
`
`cultured in the presence of physiologically relevant flow and_peristalsis-likeluminal
`
`
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`WO 2017/136479
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`mechanical deformations. This approach allows formation of intestinal villi lined by all four
`
`epithelial cell
`
`lineages of the small
`
`intestine (absorptive, goblet, enteroendocrine, and
`
`Paneth), with enhanced barrier function, drug-metabolizing cytochrome P450 activity, and
`
`apical mucus secretion.
`
`However, a chief limitation of existing approaches is that carcinoma lines such as
`
`Caco-2 cells do not possess the intestinal epithelial subtypes. As such, the impact of bacteria
`
`and/or
`
`inflammatory cytokines on various intestinal
`
`subtypes cannot be determined.
`
`Additionally, Caco-2 cells do not possess the repertoire of genetic variations now understood
`
`to be associated with IBD, thereby limiting opportunity to further evaluate response of IBD
`
`genetic factors. Finally, existing models fail to incorporate other cell types, such as immune
`
`cells (e.g., macrophages, neutrophils, and dendritic ceils) to investigate their role in disease
`
`pathology. Thus, there is a great need in the art to establish improved gut organ chip models
`
`that faithfully incorporate these multi-faceted elements.
`
`To test this, the gastrointestinal organoids (GIOs) and hypothalamic neurons GHTNs)
`
`seeded on “organ-on-chip” microfluidic device are exposed to chronic low-dose treatments
`
`(TDI range) of EDC pollutants/mixtures.
`
`As an example, in some embodiments, iPSC lines derived from obese individuals
`
`were used in testing on microfluidic chips for responses to compounds, including but not
`
`limited to endocrine disrupting chemicals CEDCs), i.e. obesogens, e.g. as chronic low-dose
`
`20
`
`treatments (TDI range) of EDC pollutants/mixtures (e.g. tributyltin (TBT), perfluorooctanoic
`
`acid (PFOA), butylated hydroxytoluene (BHT), and bis(2-ethylhexyl) phthalate (DEHP), etc.
`
`Testing is contemplated to include determining signs of detrimental effects of exposure to
`
`putative endocrine disrupting chemicals in developing cells i.e. THTNs and iFGEs, with an
`
`example of analysis including but not limited to dysregulated secreted protein groups will be
`
`identified by quantitative proteomics. Exemplary results are described in Example 32.
`
`The invention provides a method of manufacturing a microfluidic apparatus
`
`comprising a population of intestinal cells with an organized structure, comprising:
`
`disaggregating human intestinal organoids (H1Os) into single cells; and adding the single
`
`cells to the apparatus. In one embodiment, said single cells are purified based on CD326+
`
`Ma D
`
`expression before addition to the apparatus. In one embodiment, said adding the single cells
`
`to the apparatus comprises resuspension in a media comprising one or more of: ROCK
`
`inhibitor, SB202190 and A&3-01.
`
`In one embodiment, said human intestinal organoids
`
`(HIOs) are cultured in the presence of ROCK inhibitor prior to disaggregation.
`
`In one
`
`embodiment, said human intestinal organoids (HIOs) are derived from induced pluripotent
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`WO 2017/136479
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`stem cells GPSCs). In one embodiment, said iPSCs are reprogrammed lymphoblastoid B-cell
`
`derived induced pluripotent stem cells (LCL-iPSCs). In one embodiment, said iPSCs are
`
`reprogrammed cells obtained from a subject afflicted with an inflammatory bowel disease
`
`and/or condition.
`
`The invention provides a method of manufacturing a microfluidic apparatus
`
`comprising a population of intestinal cells with an organized structure, comprising:
`
`disageregating human intestinal organoids (H1Os) into single cells; and adding the single
`
`cells to the apparatus. In one embodiment, said single cells are purified based on CD326+
`
`expression before addition to the apparatus. In one embodiment, said adding the single cells
`
`to the apparatus comprises resuspension in a media comprising one or more of: ROCK
`
`inhibitor, SB202190 and A83-01.
`
`In one embodiment, said human intestinal organoids
`
`(HIOs) are cultured in the presence of ROCK inhibitor prior to disaggregation.
`
`In one
`
`embodiment, said human intestinal organoids (HIOs) are derived from induced pluripotent
`
`stem cells GPSCs). In one embodiment, said derivation of human intestinal organoids (HIOs)
`
`from induced pluripotent stem cells GPSCs) comprises: generation of definitive endoderm by
`
`culturing? nduced pluripotent stem cells (@PSCs) in the presence of Activin A and Wnt Family
`
`Member 3A (Wnt3A); differentiation into hindgut by culturing definitive endoderm in the
`
`presence of FGF and either Wnt3A or CHIR99021; collection of epithelial spheres or
`
`epithelial tubes; suspension of epithelial spheres or epithelial tubes in Matrigel; and culturing
`
`20
`
`in the presence of CHIR99021, noggin and EGF In one embodiment,
`
`said apparatus
`
`comprises an organized structure comprising villi. In one embodiment, said villi are lined by
`
`one or more epithelial cell lineages selected from the group consisting of: absorptive, goblet,
`
`enteroendocrine, and Paneth cells. In one embodiment, said organized structure possesses
`
`barrier function, cytochrome P450 activity, and/or apical mucus secretion.
`
`The invention provides a microfluidic apparatus comprising: a population of intestinal
`
`cells, wherein the population comprises an organized structure. In one embodiment, said
`
`organized structure comprises villi. In one embodiment, said villi are lined by one or more
`
`epithelial
`
`cell
`
`lineages
`
`selected from the group consisting of:
`
`absorptive, goblet,
`
`enteroendocrine, and Paneth cells. In one embodiment, said organized structure possesses
`
`Ma D
`
`barrier
`
`function, cytochrome P450 activity, and/or apical mucus
`
`secretion.
`
`In one
`
`embodiment, said intestinal cells are derived from human intestinal organoids (HIOs)
`
`disaggregated into single cells and purified based on CD326+ expression.
`
`In one
`
`embodiment, said human intestinal organoids (HJOs) are derived from iPSCs by a method
`
`comprising: generation of definitive endoderm byculturing iPSCs in the presence of Activin
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`A and Wnt3A,; differentiation into hindgut by culturing definitive endoderm in the presence
`
`of FGF and either Wnt3A or CHIR99021,; collection of epithelial spheres or epithelial tubes;
`
`suspension of epithelial spheres or epithelial tubes in Matrigel; and culturing in the presence
`
`of CHIR99021, noggin and EGF.
`
`The use of microfluidic intestinal chips described herein improves/increases
`
`maturation of iPS derived intestinal cells. More specifically, use of such chips improves
`
`maturation efficiency, e.g.
`
`iPS cell differentiation into foregut increases numbers of ceils
`
`such as synaptophysin (SYP) positive cells, and improves quality of intestinal epithelium,te.
`
`an epithelial layer folds into finger-like projections lined with epithelial cells of which same
`
`are separated by pit-like areas mimicking villus-like structures lined with epithelium and pit-
`
`like areas, for mimicking human intestinal microvillus when seeded with iPSC derived
`
`intestinal cells. Further, these villus structures are continuously growing as basal cells divide
`
`and move up the sides of the villi. Moreover, the folds of epithelium comprise non-epithelial
`
`intestinal cells.
`
`Moreover, the chip provides an environment where a “complete” set of relevant non-
`
`epithelial cell types can develop. These non-epithelial intestinal cells include but are not
`
`limited to goblet cells, Paneth cells, endocrine cells, etc.
`
`The invention provides On-chip differentiation/maturation of cells and tissues, including
`
`but not limited to intestinal
`
`tissue, epithelium. During the development of the present
`
`20
`
`inventions, the inventors discovered that a flow condition promotes the maturation and/or
`
`differentiation of intestinal cells forming finger-ltke/villi-like projections. Further,
`
`it was
`
`discovered that flow of media promotes the formation of tight cell-to-cell junctions, which in
`
`some embodiments these tight cell-to-cell junctions are detected by TEER measurements,
`
`and/or cell-to-cell junctions are detected by cell permeability assays.
`
`Onerestriction on the use of intestinal enteroids (and cells) derived from humaniPS cell
`
`lines is that these cells need to be used during a certain time period for producing viable and
`
`reproducible microfluidic intestinal chips. However, during the development of the present
`
`inventions, methods and conditions were developed for using multiple aliquots (i.e. duplicate
`
`samples) of the same humanintestinal enteroid cells in experiments separated by long time
`
`Ma D
`
`periods from the first experiment using these cells. Alternatively, intestinal enteroid cells
`
`derived from human iPS cell lines may be stored long term before use in a microfluidic chip.
`
`As shown herein,
`
`the inventors discovered that human intestinal Caco-2 cell lines as
`
`representative intestinal epithelial cells grown in chips were found to show responses to
`
`compounds that were significantly different when compared to responses of intestinal
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`WO 2017/136479
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`epithelial grown on microfluidic intestinal organ-on-chips. Therefore, use of stem cell
`
`derived intestinal cells in these chips are improvements over the use of Caco cells (e.g. the
`
`stem cell derived cells have a proper response to interferon gamma, cellular production of
`
`antimicrobials}. In particular, the wide range of Caco-2 cell lines used over the last twenty
`
`years are subpopulations and/or clones of cells that were originally obtained from a human
`
`colon adenocarcinoma. In part because of their capability to spontaneously differentiate to
`
`form monolayers having similar characteristics to enterocyte/epithelial layers, Caco-2 cell
`
`lines are extensively used as a model of the intestinal barrier and intestinal epithelial cell
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`function. However, during development of the present inventions microfluidic intestinal chips
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`10
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`showed responses to compounds that are more similar to human intestinal epithelial
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`responses, considered "proper" responses, than Caco-2 cell lines (e.g. proper responses to
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`interferon gamma, cellular production of antimicrobials, etc.). Therefore,
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`the use of
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`microfluidic intestinal organ-on-chips described herein, are an improvement over using Caco-
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`2 cell lines. Moreover, primary intestinal cells also show a more natural phenotype than
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`15
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`Caco2 cells when growing on microfluidic chips.
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`The use of microfluidic intestinal chips described herein show that diseases may be
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`modeled using microfluidic chips described herein.
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`In particular, microfluidic chips
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`comprising iPSC derived intestinal cells, are contemplated for use as disease models,
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`in
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`particular for intestinal diseases such as gastrointestinal disorders, inflammatoryintestinal
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`disorders, gastrointestinal cancer cells, gastrointestinal cancer development, gastrointestinal
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`tumors, polyps, cells derived from gastrointestinal tissue, etc. In some embodiments, cells for
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`use in producing iPS cells may be obtained from patients having a range of Inflammatory
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`bowel diseases (IBD) involving chronic inflammation of a smal! patch in the digestive tract
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`up to large regions, e.g. colitis, ulcerative colitis, Crohn's disease, etc. Thus, white blood cells
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`from IBD patients may be used for producing iPS cells for personalized chips. For
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`comparisons, white blood cells from IBD patients may be used for producing iPS cells. In
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`some embodiments, cell components, microbial components, etc. may be directly obtained
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`from a healthy person, a patient showing symptoms of and IBD, fluid samples and biopsies.
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`The use of microfluidic chips and systems described herein, a personalized therapy
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`can be tested in the chip before being used in the patient. It is well known in the field that not
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`every patient diagnosed with the same disease responds in the same manner to a treatment.
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`Thus, testing a therapy in the chip using the cells of the very same person that will be treated,
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`allows determination (e.g. prediction) of how that patient will respond. Similarly, diagnostic
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`tests can be done in orderto identify the nature of the disease and then determine a proper
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`therapy, e.g. for reducing or eliminating symptoms, or curing the disorder or the disease.
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`Microfluidic intestinal chips described herein are contemplated for use in personalized
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`medicine (e.g. individual patient derived) for developing treatments, including but not limited
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`to disorders, diseases and cancer, (e.g. individual patient derived). Such use includes but not
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`limited in use in personalized components ie. iPS-derived cell types such as immunecells or
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`bacteria from stool samples.
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`Further, personalized chips may be used for tissue analysis, e.g. capability to develop
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`normal intestinal structures and cells from iPCs, responses of iPSC derived intestinal cells to
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`compound testing, e.g. cytokines, drug testing, treatment, etc. Such chips are not limited to
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`one type of patient derived cell and are contemplated for use in growing personalized chips
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`with other personalized components, including but not limited to a particular iPS-derived cell
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`type for use in deriving intestinal cells, such as white blood cells, and other types of cells that
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`are contemplated for use in microfluidic intestinal chips, such as immune cells, including
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`resident, e.g. obtained or derived from tissue biopsies, cell collection from fluids, isolated
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`from tumors, obtained from populations of circulating white blood cells from patient blood
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`samples, genetically modified patient's cells for testing responses or testing for use in
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`treatments; or other types of patient samples, such as microorganisms, e.g. bacteria, fung),
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`viruses, isolated from stool samples that may be added to the patients iPSC derived intestinal
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`20
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`cells on a personalized microfluidic organ-on-chip. In fact, an individualize intestinal chip
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`mayfurther comprise biological components for testing that are not derived from the patient,
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`such as microorganisms, genetically modified cells, including microorganisms, for use in
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`testing treatments.
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`While personalization was discussed above, the personalized therapy developed for
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`one patient, can be used to treat another patient. As one example, the treatment developed for
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`one patient may then be used to treat another patient, e.g. a patient considered having a
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`similar genetic match, such as an identical twin, sibling, parent, grandparent, relative, etc.
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`Microfluidic intestinal organ-on-chips described herein are contemplated for use in
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`isogenic experiments where a cell or tissue is altered (e.g. express a newgene and/or protein,
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`remove a gene or protein, e.g. reduce expression of that gene or protein) and then compare
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`that altered cell or tissue with a control cell or tissue of the same genotype or phenotype that
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`is not altered.
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`Tsogenic cell lines refer to a population of cells that are selected or engineered to
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`model the genetics of a specific patient population, 4 vifre. Isogenic human disease models
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`include isogenic cell lines that have genetically matched ‘normal cells' to provide an isogenic
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`system for use in researching disease biology and testing therapeutic agents.
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`Thus in one embodiment, iPSCs of matching genetics, i.e. clones, are separated into at
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`least two samples, wherein one sample is used for a control, compared to one or more of the
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`samples that is genetically engineered to alter expression of one or more genes of interest,
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`€.g.
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`increase gene expression by overexpressing gene(s), i.e. by using transient or constitutive
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`expression vectors, knock-in gene expression, specific or nonspecific; or lower the amount of
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`gene expressed, as is underexpressed, i.e. by using silencing constructs or gene knock-outs
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`Gn transient or constitutive expression vectors); or gene editing,
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`i.e. clustered regularly
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`16
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`interspaced short palindromic repeats (CRISPR) mediated gene editing, etc.However, it is
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`not
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`intended to limit how an isogenic experiment
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`is done, with nonlimiting examples
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`provided herein, so long as there is a matched control.
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`Thus in one embodiment, a gene ofinterest in inserted into the genome of an iPS cell
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`or derived organoid cell, for comparison to duplicate samples ofcells that are not modified by
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`this insertion. In some embodiments, instead of changing expression levels, a gene 1s mutated
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`in a cell for comparison to duplicate cell samples not having that mutation.
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`In some
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`embodiments, cells are altered or mutated prior to seeding microfluidic chips.
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`In other
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`embodiments, cells are altered or mutated after seeding into microfluidic chips.
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`In some
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`embodiments, instead of altering a gene, an expressed protein from DNA inserted into the
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`20
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`genome of a cell
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`is altered, e.g. such as for gene therapy.
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`In some embodiments, an
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`expression DNA vector or RNA for expressing a protein is introduced into the cell, e.g. such
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`as for gene therapy.
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`In one embodiment, sources of iPSC derived intestinal cells containing an endogenous
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`mutation in one or more genes of interest are selected for use in deriving intestinal cells for
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`seeding organ-on-chips. For comparison, e.g. control, matching sources of iPSCs may be
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`selected with similar or the same genetic background that do not have the same mutations in
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`the one or more genes of interest.
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`Microfluidic intestinal organ-on-chips described herein are contemplated for use in
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`modeling obesity related disorders including but not limited to obese individuals without
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`additional symptoms and obese individuals further showing symptoms including prediabetic,
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`diabetic, i.e. Type I and Type IT diabetes, etc. For example, during the development of the
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`present inventions, iPSC lines were generated from individuals with normal body mass index
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`(BMI <25) and individuals considered super obese (SO) with BMI>50,